In oduc ion o RNAi
and miRNA pa hways
Pe S oboda
KAROLINUM PRESS
PRAGUE 2020
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KAROLINUM PRESS
Ka olinum P ess is a publishing depa men o he Cha les Uni e si y
www.ka olinum.cz
© 2020 by Pe S oboda
Fi s edi ion
ISBN 978-80-246-4372-4 (pd )
h ps://doi.o g/10.14712/9788024643724
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CONTENTS
5 P e ace
7 In oduc ion
29 RNAi and miRNA pa hways in mammals I – molecula mechanisms
81 RNAi and miRNA pa hways in mammals II – biological oles
119 RNAi and miRNA pa hways in bi ds
133 RNAiandmiRNApa hwaysin ish
143 RNAi and miRNA pa hways in a h opods
177 RNAi and miRNA pa hways in Annelids and Molluscs
191 RNAi and miRNA pa hways in nema odes
215 RNAi and miRNA pa hways in plan s I – molecula mechanisms
255 RNAi and miRNA pa hways in plan s II – mobili y o small RNAs
275 Speci ici yo  a ge inginRNAiandmiRNApa hways
313 Ex acellula small RNAs and hei ans e be ween species and kingdoms
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h ps://doi.o g/10.14712/9788024643724.1 5
PREFACE
Small RNA pa hways o RNA silencing is a g oup o pa hways, which u ilize small (20–
30n )RNAsasguides o sequence-speci ic ep ession.Thiscollec iono  ex sonsmall
RNA pa hways o igina es om a epo I p epa ed o he Eu opean Food and Sa e y
Au ho i y (EFSA) in 2016 and 2017. EFSA eques ed li e a u e su ey as i was conce ned
because o se e al wo ks epo ing ha small RNAs, which na u ally exis in plan s, can
en e in oamammalianbodyanda ec geneexp ession.Themainissuewas ha RNA
in e e ence (one o a small RNA pa hways), ep esen ed a p omising way o de eloping
sequence-speci icpes icides.I plan smallRNAscoulden e in oamammaliano ganism
and egula e genes, RNAi-based pes icides could ep esen a po en ial heal h haza d.
Wi h help o my colleagues, who se up a sys ema ic li e a u e sea ch, I w o e an ex en-
si e sys ema ic li e a u e e iew o which I inspec ed ~10,000 i les and abs ac s du ing
oneyea o w i ing.Theo iginalEFSA epo hadanumbe o  echnicalsec ionsdesc ibing
sea chme hodologyandwascompilingin o ma ion ompublishedwo ks o speci ic
asksde inedbyEFSA.Once he epo wascomple edandp esen ed oEFSA,Ibecame
in e es edincon e ing hescien i icpa o  he epo in oamo ecohe en o e iewo 
RNA silencing (pa icula ly o RNAi and mic oRNA pa hways) ac oss di e en animal
g oupsandplan s.I hough i wouldbebe e i  hescien i icpa wouldbe e ised,and
p o ided as a cohe en collec ion o chap e s o s udying RNAi and ela ed pa hways.
I subsequen ly con ac ed EFSA and discussed possible op ions o p oducing a se o
chap e s based on he epo , which could se e as a s udy ma e ial o my lec u ing. EFSA
ep esen a i es ag eed ha I could p oduce a se o s udy ma e ials om he epo ha
would be published by a uni e si y publishing house unde condi ions ha EFSA would
be c edi ed and he book would no be sold – as a solu ion, he ma e ial is p o ided as an
openaccesscollec iono chap e s.Tosa is y hesecondEFSA equi emen ,Iacknowl-
edge he eby ha he con en s ha e been p oduced unde a con ac wi h EFSA (OC/EFSA/
GMO/2015/01-CT01)and ha  heopinionsexp esseda e hoseo  hecon ac o onlyand
dono  ep esen EFSA’so icialposi ion.
Rega ding he con e sion, he o iginal ex was eo ganized in o wel e chap e s, which
we e e o ma ed and e ised in o de o emo e copy igh ed ma e ial om hi d pa ies
and p o ide a in oduc o y pa s o s and alone chap e s. Nine o he chap e s ocus on
smallRNApa hwaysinanimals(mammals,bi ds, ish,a h opods,nema odes,molluscs,
andannelids)andplan s.The emaining h eechap e sincludeagene alin oduc ionand
e iews o impo an phenomena – o - a ge ing and ex acellula small RNAs. I hope ha
his collec ion will se e as a use ul sou ce o many.
Pe S oboda
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h ps://doi.o g/10.14712/9788024643724.2 7
INTRODUCTION
In oduc ion
Keywo ds:dsRNA,siRNA,miRNA,Dice ,TARBP2,PACT,A gonau e
PETRSVOBODA
Ins i u e o Molecula Gene ics, Academy o Sciences o he Czech Republic,
Videnska1083,14220P ague4,CzechRepublic
Co espondence o: Pe S oboda, Ins i u e o Molecula Gene ics ASCR,
Videnska1083,14220P ague4,CzechRepublic, el.#+420241063147,
e-mail: [email p o ec ed].
ABSTRACT
RNAsilencingdeno esag oupo pa hways,whichu ilizesmallRNAsassequence-speci icguides o  ep essing
geneexp ession.Two ela edRNAsilencingpa hwaysexis inanimalsandplan s:RNAin e e ence(RNAi)and
mic oRNA (miRNA) pa hway. While he miRNA pa hway egula es endogenous p o ein-coding gene exp ession,
RNAi se es as a o m o inna e immuni y a ge ing i uses and mobile elemen s, al hough i occasionally also
acqui ed unc ioninp o ein-codinggene egula ion.Theaimo  he ollowing ex is op o ideanelemen a y
in oduc ionin oRNAiandmiRNApa hways o ase ieso  axon-speci icand ea u e-speci ic e iews,which
ollow.Theideais ob ingupcommongene alp inciplesallowing he eade  obe e na iga e h oughcommon
andde i edmechanismsand unc ionso RNAsilencing ha a ep esen edin axon-o ien ed e iews.Theen i e
e iew se ies was de i ed om an expe epo o he Eu opean Food and Sa e y Agency, which was eo ganized
obemo eaccessible o  hescien i iccommuni y.
In oduc ion o mechanis ic p inciples and oles o RNA silencing
The olumeo  heRNAsilencinganddouble-s andedRNA(dsRNA)- ela edpublished
da a is s unning. In 2016, me and my colleagues did li e a u e assessmen o he Eu opean
FoodandSa e yAgency,whichiden i iedo e 200000publica ions(Pacese al.,2017)
and which se ed as a ounda ion o his a icle se ies. While selec ed axons a e e iewed
sepa a ely,I hough  hecollec ionwouldbene i  omin oducing heco emolecula 
mechanisms o RNAi and miRNA pa hways (admi edly animal-cen ic).
As men ioned in he abs ac , RNA silencing ( e iewed in Ke ing, 2011) designa es
ep ession guided by small RNA molecules (20–30 nucleo ides long) and includes di e se
silencing mechanisms including RNA deg ada ion, ansla ional ep ession, induc ion o
ep essi e ch oma in, and e en DNA dele ions. RNA silencing esea ch e ol ed om pa al-
lels udiesinse e aldi e en modelsys ems,p ima ily lowe ingplan modelsandanimal
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models including Caeno habdi is elegans, D osophila melanogas e ,zeb a ish,mouse,and
humans. Some o m o RNA silencing exis s in almos e e y euka yo e. He e, he p ima y
ocus will be on he RNA in e e ence (RNAi) and mic oRNA (miRNA) pa hways (Fig. 1
and2).The e mRNAihasbeeno iginallyused o sequence-speci icmRNAdeg ada ion
inducedbylongdsRNA(Fi ee al.,1998).Thismechanism,whichemployssmallRNAs
p oduced om long dsRNA, is he canonical RNAi. Howe e , he e m RNAi is also used
as a common name o a b oad ange o RNA silencing pa hways (Ke ing, 2011). He e,
I will use he e m RNAi s ic ly in i s o iginal conno a ion. miRNAs a e genome-encoded
sho RNAs ha egula e gene exp ession by ansla ional ep ession and/o deg ada ion o
cogna e mRNAs.
His o ically, he i s disco e edRNAsilencingpa hwaywasplan co-supp ession,
whichappea edassequence-speci icsilencingo endogenousgenesinducedby ans-
geneexp ession(Napolie al.,1990).ThemiRNApa hwaywas i s  oundin1993in
Figu e 1 RNAi pa hway o e iew
Canonical RNAi is igge ed by some o m o long dsRNA. dsRNA can o igina e om a ious sou ces
including i uses and hei eplica ion in e media es o base pai ing i RNAs ansc ibed in he genome
(ei he as an in amolecula duplex (hai pin dsRNA), o by base pai ing RNAs ansc ibed in cis (con-
e gen ansc ip ion) o in ans ( om in e spe sed elemen s, pseudogenes e c.). The co e mechanism
o RNAi has h ee s eps: dicing – clea age o long dsRNA in o siRNA duplexes by RNase III Dice ,
loading – whe e one s and o siRNA duplex is selec ed and loaded on o an A gonau e p o ein om
AGO sub amily o ming he RNA-induced silencing complex (RISC), and slicing – whe e siRNA guides
RISC o cogna e RNAs. Upon making a pe ec duplex wi h a cogna e RNA, AGO p o eins pe o ms en-
donucleoly ic clea age o he cogna e RNA in he middle o he base-pai ed sequence. In some species,
RNAi also in ol es an RNA-dependen RNA polyme ase (RdRp), which may gene a e ini ial subs a es
o pa icipa e in ampli ica ion o he esponse by con e ing cogna e RNAs in o dsRNA.
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he nema ode Caeno habdi is elegans(Leee al.,1993).Theideao aconse edmiRNA
pa hway eme ged upon disco e y o Le -7 miRNA in 2000, which was is conse ed om
Caeno habdi is elegans o mammals (Pasquinelli e al., 2000). In he mean ime, RNAi
was ound in Caeno habdi is as well (Fi e e al., 1998). A ound he yea 2000, i became
appa en ha ea lie obse a ions, which included he a o emen ioned plan co-supp ession,
quelling in ungi, and animal RNAi and miRNA pa hways (Lee e al., 1993; Napoli e al.,
1990; Romano and Macino, 1992; an de K ol e al., 1990), belong o one g oup o ela ed
molecula mechanisms commonly called RNA silencing.
Theco ep incipleo RNAsilencing( ep essionmedia edbya ibonucleop o eincom-
plex guided by a small RNA) was deciphe ed du ing 1998–2004 using a combina ion o
gene ic and biochemical app oaches. Key s eps in unde s anding how RNA silencing wo ks
we e biochemical s udies in D osophilaemb yolysa es(Tuschle al.,1999;Zamo ee al.,
2000) and gene ic s udies in Caeno habdi is elegans and plan s (e.g. (Bohme e al., 1998;
Dalmay e al., 2000; Faga d e al., 2000; G ishok e al., 2000; Lynn e al., 1999; Mou ain
e al.,2000;Sma done al.,2000;Taba ae al.,1999)).Thelas disco e y,whicha gua-
bly closed he e a o deciphe ing he key p inciples o RNA silencing, was he s uc u al
Figu e 2 Canonical animal miRNA pa hway o e iew
miRNAs a e genome-encoded. Thei syn hesis s a s wi h Pol II-media ed ansc ip ion o long p ima y
miRNA ansc ip s (p i-miRNAs), which ca y one o mo e local sho hai pins, which a e eleased as
p ecu so miRNAs (p e-miRNAs) by he ac i i y o he nuclea “Mic op ocesso complex”. P e-miRNAs
a e anspo ed in o he cy oplasm ia Expo in 5. In he cy oplasm, Dice clea es a p e-miRNA and
one s and o he duplex is loaded on o an AGO p o ein, which o ms he co e o he e ec o complex
(RISC o miRISC). The e ec o complex con ains addi ional p o eins, which media e ansla ional e-
p ession and RNA deg ada ion. The key b idge be ween AGO and p o eins media ing deadenyla ion
and decapping is GW182 p o ein. Ta ge ed mRNAs usually localize o P-bodies, which a e cy oplasmic
oci associa ed wi h RNA me abolism
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Table 2 O e iew o A gonau e p o eins and associa ed RNAs in key model o ganisms
The able was compiled o m he ollowing li e a u e (Ba is a e al., 2008; Buckley e al., 2012; Das e al.,
2008; Du an-Figue oa and Vielle-Calzada, 2010; Fische e al., 2011; Fo s emann e al., 2007; Iwasaki
e al., 2015; Liu e al., 2009; Tijs e man e al., 2002a; Tijs e man e al., 2002b; Vasale e al., 2010;
Vou ekas e al., 2012; Wang and Reinke, 2008; Yigi e al., 2006; Zhang e al., 2016; Zheng e al., 2007).
Slice ac i i y “+” indica es ha a gi en A gonau e p o ein has po en ial o ac as a slice , no ha slicing
is i s p ima y mode o ac ion. In some case, slicing po en ial has been in e ed om he sequence, i.e.
i is no suppo ed wi h expe imen al e idence.
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s ep, in which RdRPs gene a e seconda y siRNAs and (2) sys emic RNAi whe e an RNAi
esponse can sp ead ac oss cellula bounda ies.
Because dsRNA o en o igina es om i uses, he ole o RNAi has been iewed as
a o m o na i e immuni y. While his ole is expe imen ally suppo ed in some models,
RNAi may also ha e o he oles in main aining genome in eg i y, and con ol o gene
exp ession.
RNA i uses gene a e dsRNA du ing hei eplica ion cycle in hos cells. DNA i uses
o en p oduce complemen a y sense and an isense ansc ip s, which can o m dsRNA upon
annealing.Thus,dsRNAisacommonma ke o  i alin ec ionandi is ecognizedby
di e en mechanismsmedia inganinna eimmune esponse.Theidea ha RNAsilencing
may unc ion as a o m o inna e immuni y is suppo ed by se e al lines o e idence, which
we e i s  oundinplan sandla e alsoinin e eb a es( e iewedinMa quesandCa hew,
2007; Xie and Guo, 2006): 1) siRNAs de i ed om i al sequences we e ound in in ec ed
o ganism (Hamil on and Baulcombe, 1999), 2) inhibi ion o RNA silencing esul ed in
inc eased i al eplica ion (Mou ain e al., 2000), and 3) some i uses p oduce supp esso s
o RNAsilencing(Voinne e al.,1999).
The oleo RNAi a iesamongdi e en o ganisms.Ve eb a es eplaced hean i i al
de ense sys em p o ided by RNAi by an a ay o inna e immune senso s o dsRNA mol-
ecules,whoseac i a ioncon e gesonasequence-independen in e e on esponse.Thus,
he canonical RNAi is gene ally no a ubiqui ous p ima y mechanism in esponse o dsRNA
in e eb a esal houghi isobse edinspeci iccases.Inspecies,whichs illuseRNAias
he p ima y an i i al immuni y pa hway (such as plan s, nema odes, a h opods), i is e-
quen ly obse ed ha i uses o e come he RNAi esponse wi h a ious p o ein inhibi o s.
Whe he he mo e complex in e e on sys em in e eb a es p o ides a s onge de ense
ba ie is unclea as he in e e on pa hway is jus a pa o a highly complex immune sys-
em. One in e es ing aspec o RNAi and in e e on esponse e olu ion is he ewi ing o he
RIG-I helicase amily, which is associa ed wi h RNAi in Caeno habdi is elegans and in e -
e on esponse in mammals. Unde s anding he ole o he RNAi module in immuni y o
molluscs and annelids equi es u he esea ch. Molluscs a e a pa icula ly in e es ing case,
because hei genome ca ies homologs o he genes in ol ed in he in e e on esponse,
and s udying hem migh p o ide an insigh in o how he in e e on esponse has eplaced
RNAi, as he main an i i al esponse.
Sys emic and en i onmen al RNAi
RNAi can ei he ac in a cell au onomous manne , i.e. a ec ing only cells di ec ly exposed
odsRNA,o canp opaga eac osscellbounda ies.Twomodeso non-cellau onomous
RNAi a e ecognized: (1) en i onmen al RNAi in ol es p ocesses whe e dsRNA is aken
up by a cell om he en i onmen . (2) sys emic RNAi includes p ocesses whe e a silencing
signal sp eads om a cell ac oss cellula bounda ies in o o he cells. Bo h modes can be
combinedandsys emicRNAican ollowen i onmen alRNAi.Twopa hways o dsRNA
up akewe edesc ibed:(1)aspeci ic ansmemb anechannel-media edup akeand(2)an
al e na i e endocy osis-media ed up ake ( e iewed in Hu enne and Smagghe, 2010; Whang-
bo and Hun e , 2008).
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Thenon-cellau onomousRNAiwasobse edal eadydu ing he i s RNAiexpe i-
men s in Caeno habdi is elegans (Fi e e al., 1998). When animals we e mic oinjec ed wi h
dsRNA in o head, ail, in es ine o gonad a m, o e en jus soaked in dsRNA solu ion o ed
bybac e iaexp essingdsRNA, hese ea men sinducedaspeci icnullpheno ypein he
whole animal and e en in i s p ogeny, demons a ing a su p ising abili y o dsRNA o c oss
cellula bounda ies(Fi ee al.,1998;Taba ae al.,1998;TimmonsandFi e,1998).Non-
cell au onomous RNAi has been disco e ed also in pa asi ic nema odes (Geldho e al.,
2007), hyd a (Che a e al., 2006), plana ia (Newma k e al., 2003; O ii e al., 2003), insec s
(Tomoyasue al.,2008;XuandHan,2008),o plan s(Himbe e al.,2003).
miRNA pa hway
unlikesiRNAs,miRNAsa egenome-encodedsho RNAswi hde inedsequences ha  eg-
ula e gene exp ession by media ing ansla ional ep ession and/o deg ada ion o cogna e
mRNAs. miRNAs play impo an oles in many p ocesses and a e one o he mos common
small RNAs ound in animal and plan cells. miRNAs ha e been implica ed in coun less
cellula and de elopmen al p ocesses; in some cases a e changes in hei exp ession linked
o pa hological condi ions. Bioin o ma ics es ima es sugges ha miRNAs migh di ec ly
a ge o e 60% o mammalian genes (F iedman e al., 2009); miRNA-dependen egula ion
in in e eb a es and plan s a e less ex ensi e.
Thousandso miRNAsha ebeenanno a ed.Thecen almiRNAda abasemiRBase
(h p://www.mi base.o g,(Kozoma aandG i i hs-Jones,2014)includes2654human,
1978 mu ine, 469 D osophila melanogas e , 437 Caeno habdi is elegans, and 428 A abi-
dopsis haliana ma u e miRNAs ( elease 22.1). Rema kably, he e a e only a ew miRNAs
conse ed be ween D osophila and mammals and i is no clea i he e a e any conse ed
miRNA genes be ween plan s and animals. Animal miRNAs seem o eme ge om andom
o ma ion o D osha/Dice subs a es (discussed in de ail in (S oboda and Ca a, 2006).
Newly e ol ing miRNAs likely o m a conside able po ion o anno a ed miRNAs, espe-
cially in species whe e miRNAs we e in ensely s udied by nex gene a ion sequencing
(NGS),whichcaniden i ylow-abundancemiRNAs.Thenewlyeme gingmiRNAsei he 
acqui esigni ican  ep essi e unc ionsandbecome e aineddu inge olu iono  hey
become los . Fu he mo e, a ge epe oi e o indi idual miRNAs can e ol e as since
a single poin mu a ion can weaken an exis ing egula ion o c ea e a new one.
Animal miRNAs biogenesis s a s wi h long p ima y ansc ip s (p i-miRNAs), which
a ep ocessedby henuclea “Mic op ocesso ”complex,in osho hai pinin e media es
(p e-miRNAs). P e-miRNAs a e anspo ed o he cy oplasm whe e hey a e u he p o-
cessed by Dice in o a small RNA duplex, om which is one RNA s and loaded on o an
A gonau e p o ein whe e i guides ecogni ion and ep ession o cogna e mRNAs (Fig. 2).
TheAGO-con aininge ec o complexhasbeengi endi e en names;he ei willbe
e e ed oasmiRNA-InducedSilencingComplex(miRISC).Themechanismo ac iono 
an AGO-con aining e ec o complex a ies and may include ei he ansla ional ep ession
and/o RNAi-like endonucleoly ic clea age. Func ional base pai ing o animal miRNAs wi h
hei mRNA a ge sappea s oin ol eli lebeyond he“seed” egioncomp isingnucle-
o ides 2 o 8 o he miRNA (B ennecke e al., 2005; Son heime , 2005). Pai ing be ween
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miRNAs and mRNAs in plan s is ypically much mo e ex ensi e and esul s in di ec endo-
nucleoly ic clea age.
Impe ec miRNA:mRNA base pai ing in animals gene ally esul s in ansla ional
ep ession(Doenche al.,2003;Hu agne andZamo e,2002),whichiscoupledwi h
mRNAdeg ada ion(Baggae al.,2005;Lime al.,2005).Themolecula mechanismo 
mRNA deg ada ion induced by impe ec base pai ing di e s om he RNA-like clea age
desc ibed abo e (Schmi e e al., 2006) and in ol es mRNA deadenyla ion and decapping
ac i i ies (Chen e al., 2014; Dju ano ic e al., 2012; Nishiha a e al., 2013; Rouya e al.,
2014). RNA deg ada ion migh ac ually be he dominan componen o cogna e gene ep es-
sion (Eichho n e al., 2014). Rep essed mRNAs, miRNAs, and AGO p o eins localize o
cy oplasmic oci known as P-bodies (Liu e al., 2005; Pillai e al., 2005), which con ain
mRNA deg ading enzymes such as he decapping complex, deadenylases, and he exonu-
clease XRN1 ( e iewed in Decke and Pa ke , 2012).
The ea eonlymino di e encesinmiRNApa hwaysac ossanimals.Themainoneis
gene ic sepa a ion be ween miRNA and RNAi pa hways in a h opods, which u ilize miR-
NA-dedica ed Dice , dsRBP, and AGO while o he animals use one Dice o p oduce miR-
NAsandsiRNAs.The eisaclea di e encebe weenanimalsandplan s.Plan semploy
a single RNase III, one o hei Dice pa alogs, o p ocess p i-miRNA in o p e-miRNA and
henin omiRNAduplexin henucleus.ThesemiRNAsa e2’-O-me hyla eda  hei 3’
e mini.Thismodi ica ionisabsen inanimalmiRNAs(bu  oundinpiRNAsmallRNAs
in he ge mline). In addi ion, animals employ wo dis inc RNase III enzymes – D osha in
he Mic op ocesso complex in he nucleus, which eleases p e-miRNA om p i-miRNA,
and Dice , which p oduces miRNA duplex in he cy oplasm.
O he ele an pa hways in Me azoa
Adenosine deamina ion
A- o-I edi ing is media ed by Adenosine Deaminases Ac ing on RNA (ADAR) enzymes,
which con ain dsRBD domains and ecognize bo h in e - and in amolecula dsRNAs
longe han 20–30 bp (Nishiku a e al., 1991). ADARs con e adenosines o inosines,
which ansla ion and e e se ansc ip ion in e p e as guanosines. ADARs we e ound in
animals (including ea lies b anching g oups) bu no plan s, yeas s o p o ozoa (G ice and
Degnan, 2015; Nishiku a, 2010). I was p edic ed ha mo e han 85% o p e-mRNAs could
be edi ed, p edominan ly in he non-coding egions (A hanasiadis e al., 2004).
RNAedi ingcannega i elyin luenceRNAiinse e always.Fi s ,ADARscancompe e
wi h RNAi o dsRNA subs a es including siRNAs. A change o a single base in a sequence
may esul ei he in des abiliza ion o dsRNA s uc u e (inosine-u idine pai ) o in i s s abi-
liza ion(inosine-cy idinepai )(Nishiku a,2010).This ansi ionin helocalandglobals a-
bili yo dsRNAs uc u ecanin luence u he p ocessingo dsRNA,suchas heselec ion
o  hee ec i emiRNAs and(Ba el,2004;DuandZamo e,2005;Meis e andTuschl,
2004). While mode a e deamina ion (one I-U pai pe siRNA) does no p e en Dice p o-
cessing osiRNAs(Zamo ee al.,2000),hype edi ing(~50%o deamina edadenosines)
In oduc ion_ o_RNAi.indd 19In oduc ion_ o_RNAi.indd 19 09.07.20 8:3409.07.20 8:34
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20
can make dsRNA esis an o Dice p ocessing (Scadden and Smi h, 2001). Hype edi ed
dsRNAisalsodeg adedbyTudo -SN(TSN)nuclease(Scadden,2005).ADARmu an sin
Caeno habdi is elegans exhibi de ec i e chemo axis while he pheno ype can be escued
byRNAi-de iciency(TonkinandBass,2003).Inmammaliancells,ADAR1limi ssiRNA
e iciency(Yange al.,2005).Edi ingcana ec  a ge  ecogni ion;amisma chbe ween
siRNAand a ge mRNAcan educeRNAie icacy(ScaddenandSmi h,2001)o modi y
a ge speci ici y(Kawaha ae al.,2007b).Se e alp i-miRNAs(e.g.miR-142)unde go
edi ing, which inhibi s miRNA biogenesis o causes e en deg ada ion o p i-miRNA by
TSN(Kawaha ae al.,2007a;Nishiku a,2010;Scadden,2005;Yange al.,2006).
In e e on pa hway
Mammalian soma ic cells can espond o dsRNA in a sequence-independen manne . A pio-
nee ing wo k by Hun e e al. showed ha di e en ypes o dsRNA can block ansla ion
in e iculocy elysa es(Hun e e al.,1975).Analysiso  hephenomenoniden i iedp o ein
kinase R (PKR) ha is ac i a ed upon binding o dsRNA and blocks ansla ion by phos-
pho yla ing healphasubuni o euka yo icini ia ion ac o 2(eIF2α)(Meu se al.,1990).
Ac i a ion o PKR ep esen s a pa o a complex esponse o o eign molecules known as
he in e e on esponse ( e iewed in Sadle and Williams, 2007), which includes ac i a ion
o  heNFκB ansc ip ion ac o andmanyin e e on-s imula edgenes(ISGs)(Geisse al.,
2001). In addi ion o PKR, se e al o he p o eins ecognizing dsRNA induce he in e e on
esponse, including helicases RIG-I and MDA5, which sense cy oplasmic dsRNA and ac i-
a e in e e on exp ession, and he 2’,5’-oligoadenyla e syn he ase (OAS) , which p oduces
2’,5’-linked oligoadenyla es ha induce gene al deg ada ion o RNAs by ac i a ing la en
RNaseL,andspeci icToll-like ecep o s(TLRs)( e iewedinGan ie andWilliams,2007;
Sadle and Williams, 2007).
The eisane olu iona yconnec ionbe weenRNAiand hein e e on esponse.Mam-
malian RNA helicases Ddx58, Dhx58 and I ih1, which a e in ol ed in immune esponse,
a e he closes homologs o helicases in ol ed in p ocessing o long dsRNA du ing RNAi
in Caeno habdi is elegans. No ably, DDX58, also known as RIG-I, is an es ablished com-
ponen o  hein e e on esponse olongdsRNA(Yoneyamae al.,2004).Thissugges s
ha he in e e on esponse, which has a common igge and e ol ed a e he RNAi pa h-
way, adop ed se e al componen s om he la e pa hway. No ably, he e is also connec-
ion be ween in e e on pa hway and A- o-I edi ing; analysis o mu an mice showed mice
sugges ed ha Ada 1 a ge s dsRNA and p e en s MDA5-media ed in e e on esponse
(Liddicoa e al., 2015).
Acknowledgemen
Iwouldlike o hankmycolleaguesJanPaces,Milosla Nic,andTomasNo o ny o help
wi hcollec ingli e a u e o  he e iew.The e iewcon en wasp oducedunde acon-
ac OC/EFSA/GMO/2015/01-CT01wi hEu opeanFoodSa e yAu ho i y(EFSA); he
opinionsexp esseda e hoseo  hecon ac o onlyanddono  ep esen EFSA’so icial
In oduc ion_ o_RNAi.indd 20In oduc ion_ o_RNAi.indd 20 09.07.20 8:3409.07.20 8:34
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21
posi ion. Publica ion o he e iew was unded by LO1220 and LM2015063 by he Minis y
o Educa ion, You h and Spo s.
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Me hyl-CpG-bindingp o einMECP2(Chenge al.,2014;Tsujimu ae al.,2015),which
is known o s ably bind me hyla ed DNA. Acco ding o one epo , MECP2 p omo es he
pos ansc ip ional p ocessing o pa icula miRNAs including miR-199a, which s imula es
mTORsignalling( hekeypa hway egula ingcellme abolism,g ow h,andsu i al)by
a ge inginhibi o so mTORsignalling(Tsujimu ae al.,2015).Incon as ,Chenge al
epo ed ha MECP2 binds di ec ly o DGCR8 and in e e es wi h he assembly o he
Mic op ocesso complex, hus a ec ing gene exp ession pos ansc ip ionally ia elie ing
ep ession o miRNA a ge s (Cheng e al., 2014).
Mic op ocesso complex localiza ion and unc ion(s)
TheMic op ocesso complexshowsappa en nuclea compa men aliza ion.While an-
sien ly exp essed p i-miRNAs accumula e in nuclea oci wi h splicing ac o SC35 and
Mic op ocesso componen s, D osha and DGCR8. (Pawlicki and S ei z, 2008), hese oci
do no appea o be majo si es o p i-miRNA p ocessing, which seems o be coupled o
ansc ip ion(PawlickiandS ei z,2009).Thisisconsis en wi hli e-imaging,which
e ealed ha a la ge ac ion o Mic op ocesso esides wi h unspliced p i-miRNAs in close
p oximi y o hei genes.Thisanalysisalsop o idedadi ec  isuale idence ha DGCR8
and D osha a e a ge ed o p i-miRNAs as a p e o med complex (Belleme e al., 2012).
Impo an ly,li e a u e e iewiden i iedalso epo sdesc ibingaddi ional oleso  he
Mic op ocesso complex and i s componen s beyond miRNA biogenesis al hough Mic o-
p ocesso exp ession seems o be uned acco ding o p i-miRNA subs a es (Ba ad e al.,
2012). Non-canonical oles o Mic op ocesso (o D osha) include: 1) mRNA clea age
(Chonge al.,2010),exempli iedbyD osha-dependen clea ageo Hoxd4 RNA (Phua
e al., 2011) o des abiliza ion o Neu og2 mRNA, which suppo s neu al s em cell main-
enance by blocking accumula ion o di e en ia ion and de e mina ion ac o s (Knuckles
e al., 2012), 2) p ocessing o long non-coding RNAs es ic ed o he nucleus (Ganesan
and Rao, 2008), 3), ibosomal RNA biogenesis (Liang and C ooke, 2011), and 4) clea age
o i al RNA (Shapi o e al., 2014). While immunop ecipi a ion o he Mic op ocesso
complex ollowed by nex -gene a ion sequencing showed ha p ecu so s o canonical
miRNAs and miRNA-like hai pins a e he majo subs a es o he Mic op ocesso com-
plex (Seong e al., 2014), high- h oughpu sequencing and c oss-linking immunop e-
cipi a ion(HITS-CLIP)analysiso RNAsbound oDGCR8sugges  ha miRNAsmay
no be he mos abundan a ge s. DGCR8-bound RNAs also comp ised se e al hund ed
mRNAs, small nucleola RNAs (snoRNAs), and long noncoding RNAs (Macias e al.,
2012). In e es ingly, DGCR8-media ed clea age o snoRNAs was independen o D osha,
indica ing pa icipa ion o DGCR8 in o he RNA p ocessing complexes (Macias e al.,
2012). One o such complexes is he exosome (an hRRP6-con aining nucleola o m),
whe e DGCR8 is essen ial o i s ec ui men o snoRNAs and o he human elome -
aseRNAcomponen (hTR/TERC)(Maciase al.,2015).Thus,DGCR8ac sasanadap-
o ec ui ing he exosome complex o s uc u ed RNAs and inducing hei deg ada ion.
(Macias e al., 2015).
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Mic op ocesso complex c oss alk wi h o he pa hways
In e ms o a c oss alk wi h o he pa hways, i has been es ablished ha some miRNA
p ecu so s a e edi ed by ADARs (Alon e al., 2012; Ga cia-Lopez e al., 2013; Peng e al.,
2012;Tomasellie al.,2015;Veselye al.,2014;Veselye al.,2012;Yange al.,2006)
appa en ly as ea ly as p i-miRNAs (Bahn e al., 2015; Chen e al., 2015). Acco ding o one
model, ADAR1 in e ac s wi h D osha and DGCR8 in he nucleus and possibly ou com-
pe esDGCR8inp ima ymiRNAbinding, husenhancingma u emiRNAexp ession.This
appea s dependen on ADAR1 edi ing ac i i y, a leas o a subse o a ge s (Bahn e al.,
2015). Acco ding o he selec i e elimina ion model, miRNAs, such as miR-151, a e edi ed
andelimina edbyTudo -SN(a ibonucleasespeci ic oinosine-con ainingdsRNAsand
a epo ed componen o RISC) du ing mouse p eimplan a ion de elopmen (Ga cia-Lopez
e al., 2013). Simila ly, p i-miR-142 edi ing esul s in supp ession o i s p ocessing by
D oshawhile heedi edp i-miR-142isdeg adedbyTudo -SN.Consequen ly,ma u emiR-
NA-142 exp ession subs an ially inc eases in ADAR1-/- o ADAR2-/- mice (Yang e al.,
2006).Acco ding o hes imula ionmodel,exempli iedbymiR-497,abundan edi inge en 
p omo esp ocessingbyD oshao  heco espondingp i-miRNA(Veselye al.,2014).
Dice – cy oplasmic p oduc ion o miRNA om p e-miRNA
A p e-miRNA p oduced by he Mic op ocesso complex is anspo ed o he cy oplasm ia
Expo in5inaRanGTP-dependen manne .Thenex p e-miRNAp ocessings episDice 
media ed clea age, which akes place he cy oplasm.
S uc u e o Dice
The ullleng hmammalianDice hasno beenc ys allized.Thecu en unde s andingo 
he mammalian Dice s uc u e has hus been in e ed om se e al di e en sou ces, which
can be di ided in o ou g oups:
(I) Biochemical s udies o ecombinan Dice and indi idual domains (Ma e al.,
2008;Pa ke al.,2011;P o os e al.,2002;Zhange al.,2002;Zhange al.,2004).
(II) The c ys al s uc u e o Gia dia in es inalis Dice (se ing as a compa a i e sca -
old) (MacRae e al., 2007; MacRae e al., 2006b).
(III) C ys allog aphic s udies on mammalian Dice agmen s (Du e al., 2008; Wilson
e al., 2015) o on indi idual domains(Mae al.,2004;Takeshi ae al.,2007;Tian
e al., 2014; Wilson e al., 2015)
(IV) C yo-EM s udies o human Dice and i s complexes wi h o he p o eins (Lau
e al.,2012;Laue al.,2009;Taylo e al.,2013;Wange al.,2009;Wilsone al.,
2015).
Dice is an siRNA-p oducing RNase III enzyme conse ed ac oss euka yo es (Be ns ein
e al., 2001). Mammalian Dice p o eins a e ~220 kDa mul idomain p o eins, which a e
composed o domains o de ed om he N- o he C- e minus as ollows: N- e minal DExD
and helicase supe amily C- e minal domains, a domain o unknown unc ion DUF283,
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aPAZdomain,RNaseIIIaandRNaseIIIbdomains,and heC- e minaldsRBD(Fig.2)
(Nicholson and Nicholson, 2002). In con as o he simples RNase III amily membe s
(exempli iedbyE.coliRNaseIII),whichca yonlyoneRNaseIIIdomainanddime ize
when clea ing dsRNA (Johanson e al., 2013; Lamon agne e al., 2001). Dice p o eins
ca y woRNaseIIIdomains,which o manin amolecula dime (Zhange al.,2004).
Gia dia Dice s uc u e e ealed spa ial o ganiza ion o he co e pa o euka yo ic Dice
p o einsandexplainedhowDice gene a essmallRNAso speci icleng hs(MacRaee al.,
2006a).Thisc ys als uc u e hense edasa amewo k o deciphe ing hes uc u eo 
o he Dice p o eins,includingmammalianDice s.The on  iewo  heGia dia Dice
s uc u e esemblesanaxe.Thebladeis o medo anin amolecula duplexo  woRNase
III domains, which a e connec ed by a b idging domain cons i u ing he back end o he
blade.Thepla o mdomainisadjacen  o heRNaseIIIadomainandmakesup heuppe 
pa o  hehandle.ThePAZdomainisconnec edbyalonghelix o heRNaseIIIadomain
and o ms he base o he handle (MacRae e al., 2006b). Al oge he , he Gia dia Dice is
o medo  h ee igid egions,whicha elinkedby lexiblehinges.One egionis o med
by RNase III domains and he b idging domain, he second by he pla o m domain and
heconnec o helix,and he hi dby hePAZdomain.These h eepa scanswing ela i e
o each o he and possibly ensu e accommoda ion o Dice o he s uc u e o i s subs a e
(MacRaee al.,2006a).Thiscon o ma ional lexibili ylikelyenablesbindingo dsRNAs
wi h non-canonical base pai ing as well as impe ec duplexes o p e-miRNAs (MacRae
e al., 2006a). In addi ion, dsRNA binding is p esumably s abilized by se e al posi i ely
cha gedpa cheson hesu aceo Gia diaDice be ween hep ocessingcen e and hePAZ
domain, which a e in con ac wi h dsRNA (MacRae e al., 2006a; MacRae e al., 2007).
Mammalian Dice s a e much la ge and con ain domains absen in he Gia dia Dice
bu  ollow hesameo ganiza ionaland unc ionalp inciples(Fig.2).Thec ys als uc-
u e o Gia diaDice con i medanea lie biochemicalanalysisp edic ing ha  he wo
RNase III domains o he human Dice o m an in amolecula dime esul ing in a single
p ocessingcen e placeda aspeci icdis ance om hePAZdomain(Zhange al.,2004).
Figu e 2 Domain a chi ec u es o Dice s om Gia dia and humans.
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As uc u alcomponen de ining hisdis anceisanαhelix(connec o helix),whichdi ec ly
linksPAZandRNaseIIIdomains(MacRaee al.,2006b).Thus, hekey unc ionalaspec 
ha eme ged om Dice ’s s uc u al analysis was ha i unc ions as a molecula ule ,
measu ing heleng ho  hesubs a e om hePAZdomain oRNaseIIIdomainswhe e
each domain clea es one s and. Impo an ly, he mammalian Dice (and me azoan Dice s
ingene al)di e  omGia dia’sin womainaspec s.The i s is hegene al opology
e lec ing he ac  ha  he ypicalmammalianDice p oduc issho e (21–23n ).Thesec-
ond one is ha he mammalian Dice con ains addi ional unc ional domains impo an o
subs a e ecogni ion and p ocessing.
As indica ed abo e, he a chi ec u e o he human Dice and posi ions o i s domains and
in e ac ing pa ne s ha e been in e ed by c yo-EM o he ull leng h p o ein and i s mu an s
(Laue al.,2012;Laue al.,2009;Taylo e al.,2013;Wange al.,2009;Wilsone al.,
2015).Theo e allshapeo  hehumanDice  esembles hele e L; heshapeis u he 
di idedin oahead,abodyandabase(Fig.2).ThePAZdomainisadjacen  o hepla o m
domainin heheado  hep o einwhile heRNaseIIIbisloca edin hebody.Thus, hehead
o he human Dice is a opological equi alen o he base o he handle in Gia dia’s Dice .
Thehelicasedomaincons i u es hebase,whichhasnoequi alen inGia dia’sDice .The
posi iono  hep ocessingcen e  ela i e o hePAZdomainsdi e sbe weenhumanand
Gia dia Dice s, which explains he ac ha he human Dice p oduces siRNA abou ou
nucleo ides sho e han he Gia dia Dice , which co esponds o ~ one- hi d o a dsRNA
helical u n(Laue al.,2012).The e o e, hep ocessingcen e has oaccess heclea age
si e o dsRNA om he di e en angle ela i e o he dsRNA helical end in compa ison wi h
Gia dia Dice (Lau e al., 2012).
Fo unde s anding subs a e selec ion and p ocessing, wo a eas o Dice ’s s uc u e
dese especiala en ion: hePAZand heN- e minaldomains,whicha edesc ibedbelow.
The ollowing ex  ep esen sexhaus i eli e a u esu ey ocusedon hes uc u aland
unc ional aspec s o he wo domains.
The PAZ domain
ThePAZdomain oundinDice andA gonau ep o einsisadsRNA- e minusbinding
module(Mae al.,2004;MacRaee al.,2006b).ThePAZdomainhasa3’o e hangbinding
pocke bu only hePAZdomaino Dice hasanex aloopen ichedinbasicaminoacids,
changingelec os a icpo en ialandmolecula su aceo  hepocke .Thesechangesmay
in luenceRNAbindingbyDice andhanding-o  hesubs a e oo he p o einscomplexes
(MacRaee al.,2006b).ThePAZdomaino me azoanDice salso ecognizesphospho yl-
a ed 5’ end o a p e-miRNA. A mu a ion o he 5’ binding pocke leads o dys egula ion
o miRNA biogenesis in i o(Pa ke al.,2011).The5’bindingpocke isconse edin
D osophila DCR-1 and human Dice bu no in Gia dia Dice (Pa k e al., 2011). Impo -
an ly, he 5’ binding pocke appea s conse ed in Dice p o eins unc ioning in miRNA
biogenesis (human Dice , D osophila DCR-1) bu no in Dice p o eins dedica ed o long
dsRNA p ocessing (Gia dia, Schizosaccha omyces, D osophila DCR-2). Acco dingly,
simul aneous ixingo 3’and5’endseme gesasa ea u eimpo an  o  ideli yo miRNA
biogenesis bu no o siRNAs (Pa k e al., 2011).
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The N- e minal helicase domain
TheN- e minuso me azoanDice sha bou sacomplexhelicases uc u e,whichisadja-
cen o RNase III ca aly ic domains (Lau e al., 2012). Al hough he helicase mus come
in ocon ac wi h hesubs a e,i s unc ionalsigni icanceiss illonlypa iallyunde s ood.
Howe e , i is clea ha he N- e minal helicase egion is he key o he subs a e p e e -
ence. In mammals (and in mos me azoan phyla), a single gene encodes Dice , which has
o p ocess bo h: miRNA p ecu so s in o miRNAs as well as long double-s anded RNAs
in o small in e e ing RNAs (siRNAs). Di e en axons appa en ly di e in how much
hey employ bo h ypes o Dice ac i i ies; he mammalian Dice is mainly dedica ed o he
miRNA pa hway while i s na u al p oduc ion is e y limi ed.
TheN- e minalhelicasebelongs o heRIG-I-likehelicase amily(Zoue al.,2009)and
consis s o a p oximal DExD/H domain and an adjacen helicase supe amily c- e minal
domain(Fig.2).Acon en ionalhelicasedomainhasanATPaseac i i y.Indeed,in e e-
b a eDice sbindandhyd olyzeATP(Be ns eine al.,2001;Ke inge al.,2001;Nykanen
e al.,2001;Zamo ee al.,2000).Howe e ,despi e heN- e minalhelicasewi hconse ed
mo i simpo an  o ATPbindingandhyd olysisisp esen inmammalianDice s, he e
isnoe idenceo ATP equi emen  o  hehumanDice ac i i y(P o os e al.,2002;
Zhange al.,2002).ThehumanDice has hesamep ocessinge iciencyin hep esence
o absenceo ATP.Mo eo e , he a eo clea ageisno in luencedbyaddi iono o he 
nucleo ides,non-clea ableATPanalogueso amu a ionin heWalke Amo i o ATPase/
helicasedomain(P o os e al.,2002;Zhange al.,2002).No ably, heseexpe imen swe e
pe o med using a long dsRNA subs a e wi h blun ends, whose p ocessing by in e e-
b a esDice sisATP-dependen (Be ns eine al.,2001;Ke inge al.,2001;Nykanene al.,
2001;Zamo ee al.,2000;Zhange al.,2002).Rema kably,dele iono  hehelicasedomain
esul s in high clea age a e o long dsRNAs by human Dice in i o (Ma e al., 2008) as
well as in i oinmu ineandhumancells(Flem e al.,2013;Kennedye al.,2015).Thus,
he N- e minal helicase in mammalian Dice s has a di e en ole in subs a e ecogni-
ion and p ocessing han he helicase in in e eb a e Dice s al hough he o e all shapes o
human and D osophila Dice p o eins a e simila (Lau e al., 2012).
Thec ys als uc u eo  heN- e minalhelicasehasno beenob ained.Thus,basedon
he c yo-EM-based modelling, he N- e minal helicase is composed o h ee globula sub-
domains (HEL1, HEL2, HEL2i) whe e he DExD/H domain co esponds o HEL1 and he
helicase supe amily c e minal domain o HEL2 and HEL2i. All h ee pa s o he helicase
o m a clamp nea he RNase III domain ac i e si e. In e es ingly, he N- e minal helicase
was ound in wo dis inc con o ma ions, wi h espec o he body o he enzyme (Lau e al.,
2012), simila o he RIG-I helicase which was used as a empla e o modelling (Kowal-
inski e al., 2011).
Analysiso subs a e-speci ics uc u al ea angemen sp oposed ha humanDice exis s
in h ees a esdependingonp esenceand ypeo subs a e(Taylo e al.,2013).unbound
Dice exis ingin“canonicals a e” ea angesuponsubs a ebinding ha in ol es hePAZ
domainaswellas hehelicasedomain.Subs a e-boundDice exis sei he inan“open”o 
closed”s a e.Theopens a eisclea age-compe en andi is ypical o p e-miRNAbinding.
I is cha ac e ized by binding o a p e-miRNA along he pla o m, bending o he helicase
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domain,andaccesso RNaseIIIaandIIIbsi es o hesubs a e(Taylo e al.,2013).The
closed s a e has been obse ed o a 35 bp A- o m RNA duplex, which ep esen s a siRNA
p ecu so .In hiss a e, hesubs a eis appedbe ween hePAZandhelicasedomainsaway
om heca aly icsi es(Taylo e al.,2013).Thisp o idesas uc u alexplana ion o p e-
ious obse a ions ha Dice poo ly p ocesses longe pe ec duplexes in i o and in i o
(Kim e al., 2005; Nejepinska e al., 2012b).
Taken oge he ,i isappa en  ha miRNAbiogenesishasbeen hep e e ed ole o Dice 
du ing e eb a ee olu ion.ThehelicasedomaininmammalianDice sp o idesas uc u al
basis o subs a especi ici y,namelydis inguishingp e-miRNAsas hep e e edsubs a e.
In addi ion, a na u al Dice iso o m has been ound in mouse oocy es, which lacks he N- e -
minalhelicasedomain,cane icien lygene a esiRNAs omlongdsRNAs,andissu icien 
o enhancingRNAiincul u edcells.Thisiso o misaconsequenceo a oden -speci ic
e o ansposon inse ion and is p esen in Mu idae amily(Flem e al.,2013).Thisdemon-
s a es ha , while he mammalian Dice p ima ily dedica ed o he miRNA pa hway, a small
change in a mammalian Dice gene can es o e RNAi ac i i y.
Subs a es and hei p ocessing by mammalian Dice p o eins
The i s in i o s udies o ecombinan human Dice showed ha subs a e clea age is
dependen onMg2+bu no onATPp esence(P o os e al.,2002;Zhange al.,2002).
Subsequen ly, i was epo ed ha Dice can clea e long dsRNAs and p e-miRNAs wi h
di e en e iciency,whichs ems omsubs a e’ss uc u alp ope ies(Chak a a hye al.,
2010;Fenge al.,2012;Flo es-Jassoe al.,2009;Mae al.,2008).The e o e,clea ageo 
miRNA p ecu so s and long dsRNAs will be discussed in sepa a e sec ions.
Canonical miRNA subs a es
Canonical miRNAs o ~22 n in leng h (Fig. 3) a e he dominan Dice p oduc s in mam-
malian cells. Dice mu agenesis showed ha inac i a ion o he RNase IIIA domain esul s
in comple e loss o 3p-de i ed ma u e miRNAs, bu only pa ial educ ion in 5p-de i ed
ma u e miRNAs (Gu an e al., 2012). Con e sely, inac i a ion o he RNase IIIB domain
by mu a ion o D1709, a esidue mu a ed in some cance s, p oduced comple e loss o 5p-de-
i ed ma u e miRNAs, bu only pa ial educ ion in 3p-de i ed ma u e miRNAs (Gu an
e al.,2012).Mu a iono  hePAZdomaincausedglobal educ iono miRNAp ocessing,
while mu a ion o he Walke A mo i in he helicase domain o Dice did no al e miRNA
p ocessing(Gu ane al.,2012).These esul sa econsis en wi h heabo emen ioned
s uc u al ea u es o Dice .
P e-miRNAsa e hemos e icien lyclea edDice subs a esin i o. In con as o
long dsRNA, a canonical p e-miRNA is clea ed only once and eleases a single small RNA
duplex. Human Dice alone clea es p e-miRNAs much as e han p e-siRNA subs a es
unde bo h single and mul iple u no e condi ions; wi h mo e han 100- old di e ence in
maximalclea age a es(Vmax)unde mul iple u no e condi ions(Chak a a hye al.,
2010).Thisindica es ha  hemammalianDice isop imized o miRNAbiogenesisand
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se e alspeci ics uc u aladap a ionsdiscussedbelowsuppo  hisno ion.Dice seems o
in e ac di ec ly wi h he e minal loop egion o a p e-miRNA (Feng e al., 2012; Gu e al.,
2012b) while a la ge p e-miRNA e minal loop u he enhances p e-miRNA clea age (Feng
e al., 2012). A la ge-scale in i o analysis and mu agenesis s udy o 161 human p e-miR-
NAs showed ha human Dice ole a es ema kable s uc u al a ia ion in p e-miRNA sub-
s a es(Fenge al.,2012).ThedsRNAs uc u ein hes em egionand he2-n 3’-o e hang
s uc u e in a p e-miRNA con ibu e o binding and clea age by Dice (Feng e al., 2012).
Acha ac e is ic ea u eo  hep e-miRNAhai pin,whichisaccessedby hePAZdomain
o Dice , is a 2 n 3’ o e hang gene a ed by he nuclea Mic op ocesso complex (G ego y
e al., 2004). P e-miRNAs wi h he 2 n 3’ o e hang a he 3’ e minus a e bound by Dice
wi hhighe a ini y hanp e-miRNAswi hdi e en ends(Fenge al.,2012).Mo eo e ,
he 2 n 3’end o e hang leads o a highe subs a e p ocessing, which was shown on bo h,
p e-miRNAsandpe ec duplexes(Fenge al.,2012;Pa ke al.,2011;Zhange al.,2004).
Such p e e ence is likely con e ed by/due o simul aneous binding o p e-miRNA end by
bo h5’and3’bindingpocke sin hePAZdomain(Pa ke al.,2011).Impo an ly, ideli yo 
miRNA biogenesis is c i ical o miRNA unc ionali y because a single nucleo ide shi a he
5’endo amiRNAwould ede inei s a ge  epe oi e.Incon as ,RNAi,which ypically
in ol es pe ec complemen a i y be ween a small RNA and i s a ge , would be essen ially
insensi i e o a p ecise clea age posi ioning as long as i would no a ec A gonau e loading.
Thus, hesimul aneous ecogni iono bo hs andsa  he2n 3’o e hang e minusbyDice 
can be seen as an adap a ion d i en by miRNA biogenesis (Pa k e al., 2011).
Theseconds uc u aladap a iono mammalianDice suppo ingmiRNAbiogenesisis
he N- e minal helicase, which o ms a clamp-like s uc u e adjacen o RNase III domains,
hence i is posi ioned o bind he s em loop o a p e-miRNA (Lau e al., 2012). While he
loss o he en i e N- e minal helicase only sligh ly inc eases p e-miRNA p ocessing ac i i y
0
100
200
300
400
500
18 19 20 21 22 23 24 25 26 27 28
numbe o miRNAs
miRNA leng h
miRNA size dis ibu ion in Mus musculus
Figu e 3 Mammalian miRNA size dis ibu ion
Dis ibu ion o ma u e mu ine miRNA leng hs acco ding o miRNA anno a ions in miRBase ( elease 21)
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MAMMALS I
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in i o (Ma e al., 2008), p e-miRNA-p ocessing by ecombinan Dice in i o is much
as e han ha o a pe ec duplex (Chak a a hy e al., 2010; Ma e al., 2008). In i o,
a na u ally occu ing N- e minally unca ed Dice iso o m can escue miRNA biogenesis
in Dice -/-emb yonics emcells(ESCs)(Flem e al.,2013).Thissugges s ha  heN- e mi-
nal helicase domain in mammalian Dice s is no impo an o miRNA biogenesis pe se; i
a he p o ides cons ains o subs a e selec i i y a ou ing p e-miRNAs.
Thisisconsis en wi h hemodelwhe ep e-miRNAbindingisassocia edwi h heclea -
age-compe en open con o ma ion. In he open s a e, a p e-miRNA is bound along he
pla o m, he helicase domain is ben , and RNase IIIa and IIIb si es ha e access o he
subs a e(Taylo e al.,2013).I hasbeenp oposed ha  heloopo ap e-miRNAmayp e-
en adop ion o he closed con o ma ion by Dice by in e ac ing wi h HEL1 and HEL2i
domains and possibly s abilizing he open con o ma ion o Dice (Feng e al., 2012; Lau
e al.,2012;Mae al.,2012).Thisalsoindica es ha  heN- e minalhelicasehadacqui ed
dis inc oles in Dice unc ion in RNA silencing du ing e olu ion. In mammalian cells, he
N- e minal helicase has a ga ekeepe unc ion whe e p e-miRNA loops appea o be a key
keeping he ga e open.
Dice -dependen non-canonical miRNA subs a es
Apa om canonical miRNA subs a es men ioned abo e, Dice is p ocessing addi ional
miRNA-like subs a es, which a e independen o he Mic op ocesso complex (desc ibed in
a sepa a e sec ion below). Some non-canonical miRNAs a e p oduced by Dice in a Mic o-
p ocesso -independen ashion, including mi ons, which u ilize he splicing machine y
o bypass he Mic op ocesso complex. Mi ons a e subs an ially longe han Mic op o-
cesso -gene a ed p e-miRNAs and exhibi 3’ u idyla ion and 5’ he e ogenei y (Wen e al.,
2015). A ecen analysis yielded ~500 no el mouse and human in ons ha gene a e Dic-
e -dependen smallRNAduplexes(Wene al.,2015).These ep esen nea ly1000loci
dis ibu ed in ou splicing-media ed biogenesis subclasses, wi h 5’- ailed mi ons being
he dominan sub ype (Wen e al., 2015). Ano he example o non-canonical miRNAs ound
in he li e a u e a e Mic op ocesso -independen miRNAs which we e o iginally desc ibed
as small in e e ing RNAs de i ed om a unique hai pin o med om sho in e spe sed
nuclea elemen s (SINEs) (Babia z e al., 2008; Cas ellano and S ebbing, 2013).
While a ypical p e-miRNA is a hai pin RNA wi h 2-n 3’ o e hangs, p oduc ion o
a ma u e miRNA om an endogenous hai pin RNA wi h 5’ o e hangs has also been epo -
ed; mouse p e-mi -1982 is a mi on wi h an 11 n ail a he 5’ end (Babia z e al., 2008).
A possible mechanism o p ocessing such empla es has been p o ided by an in i o s udy
which showed ha Dice can p oduce such miRNAs in a wo-s ep clea age, which eleases
dsRNAsa e  he i s clea ageandbinds hemagainin hein e sedi ec ion o asecond
clea age (Ando e al., 2011a).
Long dsRNA subs a es
In addi ion o p e-miRNA, Dice can p ocess long dsRNAs coming om di e en sou c-
es. Exogenous sou ces o dsRNA include i al dsRNAs and imply unc ion o RNAi in
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MAMMALS I
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euka yo ican i i alimmune esponse(VanceandVauche e ,2001;Wange al.,2006;
Wilkins e al., 2005). Endogenous dsRNAs ha e a iable leng h and e mini, and a e gen-
e a ed by ansc ip ion o in e ed epea s, by con e gen ansc ip ion o by pai ing o
complemen a y RNAs in ans. Impo an ly, mammals lack an o holog o RNA-depend-
en -RNA polyme ase (RdRP), which is a conse ed componen o RNAi- ela ed mecha-
nisms in plan s, ungi and in e eb a es (see he sepa a e RdRP sec ion). Endogenous RNAi
in mouse oocy es, he bes documen ed mammalian endogenous RNAi example, wo ks
independen ly o RdRP ac i i y (S ein e al., 2003).
ThehumanDice bindslongdsRNAbu no siRNAsin i o (P o os e al., 2002). Long
dsRNA binding is independen bo h on Mg2+andATP.ThehumanDice p e e en ially
bindsandclea eslongdsRNA om heend,due oine icien bindingo in e nal egions
o dsRNA(Zhange al.,2002).Incompa ison op e-miRNAp ocessing,humanDice 
exhibi s lowe clea age ac i i y on pe ec dsRNA subs a es (Ma e al., 2008). An expla-
na ion was p oposed ha a closed con o ma ion o he N- e minal helicase domain dis u bs
he RNase III ca aly ic co e and inhibi s clea age o pe ec dsRNAs (Lau e al., 2012). As
i was men ioned, in i o dele ion o he N- e minal helicase domain inc eases clea age
ac i i y o human ecombinan Dice (~65- old). Au ho s hypo hesize ha DExD/H-box
domain mainly inhibi s he unc ionali y o he Dice ac i e si e, bu no RNA binding (Ma
e al.,2008).Thismodelissuppo edbyp e iouslymen ioneds uc u alda a,whe eDice 
isinacloseds a ewi ha35bpA- o mRNAduplex appedbe weenPAZandhelicase
domainsaway om heca aly iccen e (Taylo e al.,2013).
Thecomplexi yo  hedi e en ialsubs a ep ocessingbyDice isillus a edbyaDic-
e mu an ca ying an in- ame 43-amino-acid inse ion immedia ely adjacen o he
DExHbox.ThisDice exhibi sde ec sin hep ocessingo mos ,bu no all,endogenous
p e-miRNAsin oma u emiRNAbu enhancedp ocessinge iciencyandconcomi an 
RNA in e e ence when he modynamically s able, long-hai pin RNAs a e used (Soi e
e al.,2008).This esul impliesanimpo an  unc ion o  hehelicasedomainin hep o-
cessing o he modynamically uns able hai pin s uc u es (Soi e e al., 2008).
Dice -media ed clea age o dsRNA can be s imula ed in i obyTARBP2.Howe e ,i 
isno clea i TARBP2s imula ioncouldbesu icien  oinduceendogenousRNAiin i o
(Chak a a hy e al., 2010). So a , he e idence o endogenous RNAi (including a emp s
o induce RNAi wi h exogenous subs a es) is sca ce ( e iewed in de ail in Nejepinska
e al.,2012a;S oboda,2014).Theonly issue ype,whe eabundan endogenoussiRNAs
a e p esen and whe e long dsRNA eadily induces RNAi a e mouse oocy es, which exp ess
anoocy e-speci icDice iso o mlackingapa o  heN- e minalhelicasedomain(Flem 
e al., 2013), hus mimicking some o he Dice mu an s es ed in i o (Ma e al., 2008).
Taken oge he ,longdsRNA, he ypicalendogenousRNAisubs a e,ispoo lyp ocessed
byendogenous ull-leng hDice .Thisisdue o hega ekeepe  oleo  heN- e minalheli-
case domain, which does no open upon binding long dsRNA.
O no e is ha he human Dice can bind 21-n ssRNAs in i o, independen o hei
sequence and seconda y s uc u e. Dice binds ssRNAs ha ing a 5’-phospha e wi h g ea e
a ini y e sus hosewi ha5’-hyd oxyl.(KiniandWal on,2007).
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Dice -in e ac ing dsRBPs: TARBP2 and PACT
A common Dice in e ac ing pa ne ound ac oss Me azoa is a dsRBP wi h andemly
a ayed dsRBDs. Mammals ha e ou dsRBP wi h andemly a ayed dsRBDs p o eins:
ans-ac i a ion esponsi eRNA-bindingp o ein2(TARBP2),p o einac i a o o PKR
(PACT),S au en1(STAu1),andS au en2(STAu2).Howe e ,onlyTARBP2(alsoknown
asTRBPo TRBP2)andPACTwe eiden i iedasDice bindingpa ne s(Chend imada
e al., 2005; Haase e al., 2005).
TARBP2andPACTa epa alogs,whiche ol ed h oughageneduplica ione en inan
ances alcho da e(DanielsandGa ignol,2012).Thes uc u eo humanTARBP2hasbeen
pa ially esol ed (Benoi and Ple in, 2013). Each p o ein consis s o h ee dsRBDs, whe e
he i s  wodomainscanbinddsRNA(o miRNA)while he hi ddomainhasapa ial
homology o dsRBD and does no bind dsRNA. Ins ead, i media es p o ein-p o ein in e -
ac ions and is a pa o a la ge p o ein-p o ein in e ac ing C- e minal egion e e ed o
asMedipaldomainasi in e ac swi hMe lin,Dice ,andPACT( e iewedinDanielsand
Ga ignol,2012).TARBP2andPACTcanalso o mhomodime sandhe e odime s h ough
he Medipal domain (La aki e al., 2008).
Thebindingsi eo TARBP2andPACTonDice was ecen lyde e minedusingc yo-
EM and c ys allog aphy (Wilson e al., 2015). Homology-based modelling showed ha
Dice -binding esiduesa econse edinTARBP2andPACT, implica ing ha binding o
TARBP2andPACT oDice ismu uallyexclusi e(Wilsone al.,2015).
TARBPhasaposi i ee ec onDice ac i i y.HumanDice ismuch as e a p ocessing
a p e-miRNA subs a e compa ed o a p e-siRNA subs a e unde bo h single and mul iple
u no e condi ions.Maximalclea age a es(Vmax) calcula ed by Michaelis-Men en analy-
sisdi e edbymo e han100- oldunde mul iple u no e condi ions.TARBP2was ound
in i o o s imula e Dice -media ed clea age o bo h, p e-miRNA and p e-siRNA sub-
s a es; his s imula ion equi es he wo N- e minal dsRBDs (Chak a a hy e al., 2010).
Thus,while hes uc u eo  hesubs a ea ec s he a e a which Dice gene a es small
RNAs,TARBP2s imula esdicingbyp esumablyenhancing hes abili yo Dice -subs a e
complexes (Chak a a hy e al., 2010).
Whencompa ed oDice andDice :TARBP2complex,PACTinhibi sDice p ocessing
o p e-siRNAsubs a es(Leee al.,2013).The woN- e minaldsRBDscon ibu e o he
obse ed di e ences in dsRNA subs a e ecogni ion and p ocessing beha iou o Dice :ds-
RNA-bindingp o eincomplexes(Leee al.,2013).Inaddi ion,PACTandTARBP2ha e
non- edundan e ec s on he gene a ion o di e en -sized miRNAs (isomiRs) (Kim e al.,
2014;Leee al.,2013;Wilsone al.,2015).CellslackingTARBP2exhibi al e edclea age
si es in a subse o miRNAs bu no e ec on gene al miRNA abundance o A gonau e load-
ing(Kime al.,2014).Thus,impac o TARBP2andPACTonmiRNAsbiogenesisin i o
seems o be ela i ely mino (Kim e al., 2014; Wilson e al., 2015). Howe e , i should be
poin ed ou ha any change in he 5’ end posi ion o any miRNA will ha e a s ong e ec on
i s a ge  epe oi e.Taken oge he ,TARBP2andPACTa e egula o y ac o s ha con ib-
u e o hesubs a especi ici yandclea age ideli ydu ingmiRNAandsiRNAp oduc ion.
Mo eo e ,TARBP2andPACTha eanaddi ional oleinac oss- alko  hein e e on
(IFN) esponseandsmallRNApa hways( e iewedinDanielsandGa ignol,2012).The
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Y393 phospho yla ion–Ty osine393(Y393)wasimplica edinEGFR-media ed ep es-
sion o miRNA biogenesis du ing hypoxia (Shen e al., 2013). Acco ding o he model,
Y393 nega i ely impac s he in e ac ion be ween AGO2 and Dice and inhibi s ma u a ion
o long-loop p e-miRNAs ca ying umou -supp esso -like miRNAs (Shen e al., 2013).
P olyl 4-hyd oxyla ion
P olyl 4- hyd oxyla ion has been implica ed in AGO s abiliza ion and inc eased RNAi.
Mass spec ome y analysis hyd oxyla ion o he endogenous AGO2 a p oline 700 (P700)
and P700A mu a ion esul ed in des abiliza ion o AGO2 (Qi e al., 2008). P olyl hyd ox-
yla ion was obse ed unde hypoxic condi ions, whe e i lead o inc eased AGO2 s abili y
(Wu e al., 2011). AGO2 hyd oxyla ion co ela ed wi h inc eased miRNA le els as well as
he endonuclease ac i i y o AGO2 (Wu e al., 2011). Con e sely, human cells deple ed
andmouseemb yonic ib oblas cellsdeple edo aspeci icp olyl-4-hyd oxylaseshowed
educed s abili y o AGO2 and impai ed RISC ac i i y (Qi e al., 2008). Hyd oxyla ion o
AGO2 was equi ed o i s associa ion wi h HSP90 (see u he below), which is implica ed
in he RISC loading wi h miRNAs and ansloca ion o s ess g anules (Wu e al., 2011).
SUMOyla ion
Thesmallubiqui in-likemodi ie (SuMO) egula es a iouscellula p ocesses.AGO2was
iden i iedasasubs a e o SuMOE3ligasePIAS3.AGO2wasSuMOyla edinmamma-
lian cells by bo h SUMO1 and SUMO2 p ima ily a lysine 402. Mu a ion o he SUMO
consensus si e educed RNAi ac i i y o AGO2, sugges ing ha SUMOyla ion migh eg-
ula e endonucleoly ic ac i i y o AGO2 (Josa-P ado e al., 2015)
Ubiqui ina ion
Ubiqui in-p o easome appa en ly unes AGO le els o adjus miRNA, AGO and Dice s oi-
chiome y (Smibe e al., 2013). I was ound ha le els o AGO1 a e adjus ed acco ding o
miRNA exp ession in a ubiqui in-p o easome-dependen manne (Smibe e al., 2013). Sim-
ila ly, lowe s abili y o AGO2 in Dice -knockou cells could be escued by p o easome inhi-
bi ion o Dice exp ession (Smibe e al., 2013). AGO and GW182 p o ein le els also depend
onHSP90a ailabili y(Johns one al.,2010).Twos udiesshowexampleso de elopmen-
ally egula ed ubiqui ina ion, which is appa en ly used o supp ess AGO ac i i ies du ing
de elopmen al ansi ions. Fi s , he le -7 a ge Lin-41geneinmiceisas emcellspeci icE3
ubiqui in ligase a ge ing AGO1, AGO2, and AGO4 p o eins (Rybak e al., 2009). Second,
AGOp o einsa edown egula edinap o easome-dependen manne du ingTcelldi e en-
ia ion, p esumably as a pa o gene exp ession ep og amming (B one e sky e al., 2013).
Poly-ADP- ibosyla ion
ThisAGOmodi ica ionsseems obelinked osupp essiono RNAsilencing.Poly(ADP- i-
bose) has been associa ed wi h he assembly o s ess g anules, which accumula e
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RNA-binding p o eins egula ing mRNAs s abili y and ansla ion upon s ess. S ess g an-
ulep o einsmodi iedbypoly(ADP- ibose)includeAGO1—4(Leunge al.,2011).In e -
es ingly, poly-ADP- ibosyla ion o RISC associa ed wi h educed RISC ac i i y has been
obse ed upon i al in ec ion (Seo e al., 2013). Acco ding o he model, poly-ADP- ibo-
syla ion a e i al in ec ion eleases miRNA-media ed ep ession o in e e on-s imula ed
genes, hence boos ing inna e an i i al pa hways (Seo e al., 2013).
O he Dice and AGO in e ac ing p o eins
Apa  om heRISC-loadingcomplexandmiRISCcomponen ssuchasGW182/TNRC6
o DDX6 and o he s men ioned abo e and elsewhe e, a la ge numbe o AGO-in e ac ing
pa ne shasbeeniden i iedin hepas and epo edindi idually(see u he below)o 
comp ehensi ely (Meis e e al., 2005). He e, I p o ide an o e iew o hose in e ac ing
pa ne s.
DDX3 – DEAD-box helicase 3 is one o he helicases sensing i al double-s anded
RNAs. DDX3 was also among he P-body componen s ec ui ed o he Wes Nile i us
eplica ion si es and egula ing i al eplica ion (Chaha e al., 2013). DDX3 was also iden-
i iedbyanRNAisc eenasanessen ial ac o in ol edinRNAipa hway(Kasime al.,
2013). DDX3 is co-localized wi h AGO2 and a dominan nega i e mu an o DDX3 a ec -
ed he RNAi ac i i y (Kasim e al., 2013).
CLIMP-63–Thecy oskele on-linkingendoplasmic e iculum(ER)memb anep o ein
o 63kDa(CLIMP-63)wasiden i iedasano elDice -in e ac ingp o ein h oughayeas 
wo-hyb id sc eening. CLIMP-63 in e ac s wi h Dice o o m a high molecula weigh
complex,whichisca aly icallyac i einp e-miRNAp ocessing(Pepine al.,2012).These
esul s a e consis en wi h analysis o Dice compa men aliza ion, which showed ha load-
ing o small RNAs in o RISC, cogna e mRNA binding, and Ago2-media ed mRNA slicing
in mammalian cells a e nuclea ed a he ough endoplasmic e iculum (S alde e al., 2013).
While he majo RNAi pa hway p o eins a e ound in mos subcellula compa men s,
he miRNA- and siRNA-loaded AGO2 popula ions co-sedimen almos exclusi ely wi h
he oughendoplasmic e iculummemb anes, oge he wi hDice ,TARBP2,andPACT
(S alde e al., 2013).
NUP153–Thenuclea po ecomplexp o einNuP1was ound oassocia ewi hhuman
Dice p o ein.Theassocia ionwasde ec edmainlyin hecy oplasmbu wasalsoappa en 
a he nuclea pe iphe y. Acco dingly, i has been sugges ed ha NUP153 plays a ole in he
nuclea localiza ion o Dice (Ando e al., 2011b)
FMRP – X men al e a da ion p o ein (FMRP) is included in he lis despi e i s ques-
ionable ole in mammalian RNA silencing. In any case, ou li e a u e sea ch e ealed
a numbe o a icles dealing wi h mammalian FMRPs because FMMRP is a highly con-
se ed p o ein and i s D osophila o holog dFXR was implica ed in RNAi (Caudy e al.,
2002; Ishizuka e al., 2002). Acco ding o he a ailable da a, FMRP is associa ed wi h
RNA silencing ac o s. FMRP co-localized wi h AGO2 (Goodie e al., 2007) and immu-
nop ecipi a ion sugges ed ha a po ion o Dice and AGO we e associa ed wi h each o he
and wi h FMRP (Lugli e al., 2005). In i o da a using ecombinan p o eins, sugges ed
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ha human FMRP can ac as a miRNA accep o p o ein o Dice and acili a e he assem-
blyo miRNAsonspeci ic a ge RNAsequences(Plan ee al.,2006).The equi emen o 
FMRP o e icien RNAiwasalsosuppo edin i o by epo e assays suppo ing he ole
o FMRP in he mammalian RISC (Plan e e al., 2006). Howe e , he loss o mammalian
FMRP did no e eal any appa en di ec impac on RISC unc ion (Didio e al., 2009;
Madsen e al., 2009).
Hun ing in – AGO2 was ound as one o he Hun ing in associa ed p o eins by co-im-
munp ecipi a ion. Fu he mo e, Hun ing in and AGO2 co-localized in P-bodies and, impo -
an ly, deple ion o Hun ing in comp omised RNA-media ed gene silencing (Sa as e al.,
2008).Howe e , hemolecula mechanismbywhichHun ing inwouldin luenceRNA
silencing emain unknown.
14–3–3 – Cell cycle egula ing 14–3–3 p o eins we e epo ed o bind he amino e mi-
nus o AGO1 and AGO2 (S oica e al., 2006). O e exp ession o he Ago1 amino e minus
in yeas esul ed in cell cycle delay a he G(2)/M bounda y p omp ing a hypo hesis ha
14–3–3 p o eins con ibu e o A gonau e p o ein unc ions in cell cycle and/o gene-silenc-
ing pa hways (S oica e al., 2006).
UPF1 – mRNA su eillance p o ein appea s o p o ide a nexus be ween h ee di -
e en mechanisms o RNA me abolism: adenosine deamina ion, mRNA su eillance
(non-sense-media ed decay) and RNA silencing. Bo h, human ADAR1 and UPF1 we e
ound associa ed wi hin nuclea RNA-splicing complexes (Ag ana e al., 2008). A he
same ime, UPF1 was connec ed o RNA silencing (Jin e al., 2009). UPF1 in e ac s
wi h human AGO1 and AGO2 and co-localizes wi h hem in o P-bodies. UPF knock-
down yielded up egula ion o miRNA a ge s while i s o e exp ession esul ed in hei
down egula ion(Jine al.,2009).Thiswouldsugges  ha uPFmaycon ibu e oRNA
silencing, maybe a he le el o RISC binding o i s a ge s and accele a ing hei decay
(Jin e al., 2009).
RBM4–TheRNA-bindingmo i p o ein4(RBM4)playsmul iple olesinmRNA
me abolism.RBM4 was ound du ing p o eomic analysis o AGO-con aining miRNPs (i.e.
miRISC) and RBM4 knockdown showed ha i is equi ed o miRNA-guided gene egula-
ion (Hock e al., 2007). I was also ound o co-localize wi h AGO2 du ing muscle cell di -
e en ia ion(LinandTa n,2009).RBM4in e ac sdi ec lywi hAGO2andmayselec i ely
enhancemiRISCassocia ionwi h a ge mRNAs(LinandTa n,2009).RBM4wasalso
implica edi miRNA-media ed ep essioninin lamma ionwhe ein lamma ion-induced
miRNA-146p omo esa eed- o wa dloop ha modi ies h oughphospho yla ion hesub-
cellula localiza ion RBM4 and p omo es i s in e ac ion wi h AGO2 and, subsequen ly,
amesanexcessi eacu ein lamma o y esponse(B udeckie al.,2013)
TRIM32–TRIM-NHL32p o ein egula esp o eindeg ada ionandmiRNAac i i yin
neu al p ogeni o cells o con ol he balance be ween di e en ia ing neu ons and daugh e
cells e aining hep ogeni o  a e.TRIM32wasshown obindAGO1andinc ease he
ac i i yo speci icmiRNAs,suchasLe -7(Schwambo ne al.,2009)
QKI-6 – QKI-6 is one o he p o ein iso o ms encoded by he qkI gene in mice. QKI-6
was ound o in e ac wi h AGO2 and o co-localize wi h AGO2 in o s ess g anules (Wang
e al., 2010). A he same ime QKI-6 deple ion lead o inc eased miR-7 exp ession while
QKI-6 p esence inhibi s p ocessing o p i-miR-7 in o miR-7 in glioblas oma cells (Wang
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e al., 2013). I has been sugges ed ha OKI-6 media es selec i e nuclea e en ion o p i-
miR-7, hence p e en ing i s p ocessing (Wang e al., 2013). Fu he esea ch is needed o
cla i y hese wo seemingly dis an ac i i ies o OKI-6.
RACK – ecep o o ac i a ed p o ein kinase C (RACK1), a cons i uen o he euka -
yo ic 40S subuni , was epo ed o be impo an o miRNA-media ed gene egula ion in
C. elegans and humans, essen ially linking miRISC wi h he ibosome (Janno e al., 2011).
RACK1wasalsoiden i iedasagenenecessa y o  ullmiRNA unc ionasc een o genes
egula ing miRNA unc ion (O suka e al., 2011). RACK1 in e ac s wi h componen s o he
miRISC in nema odes and mammals; he al e a ion o RACK1 exp ession al e s miRNA
unc ion and impai s he associa ion o he miRNA complex wi h he ansla ing ibosomes
(Janno e al., 2011). Ano he s udy ound ha RACK1 binds o KH- ype splicing egula o y
p o ein (KSRP) and is equi ed o he ec ui men o ma u e miRNAs o RISC (O suka
e al., 2011)
PTB–Polypy imidineT ac BindingP o ein(hnRNPI)was ounddu ingasea ch o 
p o einsin ol edinle -7media edgene egula ion.(Engelse al.,2012).PTBin e ac s
wi h miRNAs and human AGO2 h ough RNA and he e is a popula ion o cellula a ge s
ha a eco- egula edbyPTBandAGO2(Engelse al.,2012).
LRRK2 – leucine- ich epea kinase 2 (LRRK2) gain-o - unc ion mu a ions cause
age-dependen degene a ion o dopamine gic neu ons. he analysis o he molecula
mechanism o pa hogenesis in D osophila and humans e ealed ha LRRK2 associa es
wi h D osophila AGO1 o human AGO2 (Geh ke e al., 2010) and ha he gain-o - unc-
ion LRRK2 mu an an agonizes le -7, causing de ep ession o Le -7 a ge s (Geh ke
e al., 2010)
APOBEC3G – he apolipop o ein-B-mRNA-edi ing enzyme ca aly ic polypep ide-like
3G (APOBEC3G o A3G) is cy idine deaminase. APOBEC3G is an an i i al ac o is ound
in P-bodies (Izumi e al., 2013; Wich oski e al., 2006). APOBEC3H also inhibi s miR-
NA-media ed ep ession o ansla ion (Huang e al., 2007) by compe i i ely inhibi ing
bindingo MOV10 oAGO2,causingei he abno malassemblyo abno malma u a iono 
miRISC (Liu e al., 2012a).
AGO loading and RISC o ma ion
Thenex impo an s epis o ma iono RISC, hee ec o complexo miRNAandRNAi
pa hways. I in ol es o ma ion o he RISC Loading Complex (RLC), ans e o a small
RNA on an AGO-p o ein, and RISC ac i a ion.
RISC Loading Complex (RLC)
RISC assembly was so a explo ed mo e in D osophila (Iwasaki e al., 2010; Pham e al.,
2004;Toma ie al.,2004a;Toma ie al.,2004b) haninmammals(Be na de al.,2015;
G ego y e al., 2005; MacRae e al., 2008) pe haps because o he obus in i o sys em
o D osophila emb yo lysa e. Mammals di e om D osophila because hey do no use
di e en Dice and A gonau e p o eins dedica ed o RNAi and miRNA pa hway al hough
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i is assumed ha bo h pa hways use a simila i no he same RLC. Ou knowledge o he
mammalian RLC comes mainly om cells whe e RLC no mally loads miRNAs o om
in i o econs i u iono  heRLCwi hpu i iedp o eins.TheminimalRLCiscomposed
o Dice ,TARBP2andAGO2(G ego ye al.,2005;MacRaee al.,2008).In i o econ-
s i u ed mammalian RLC con ains one copy o each p o ein and has dicing, guide-s and
selec ion, loading, and slicing ac i i ies (Be na d e al., 2015; G ego y e al., 2005; MacRae
e al., 2008; Ma inez e al., 2002).
AGO in e ac s wi h Dice h ough a sub egion o he PIWI domain ( he PIWI-box),
whichbindsdi ec ly o heDice RNaseIIIdomain.(Tahbaze al.,2004).Single-pa icle
EManalysissugges ed ha Dice ’sN- e minalDExH/Ddomainin e ac swi hTARBP2,
whe eas i s C- e minal ca aly ic domains in he main body a e p oximal o AGO2 (Wang
e al., 2009). In e es ingly, binding o AGO o Dice inhibi s dicing ac i i y in i o(Tah-
baz e al., 2004). Analysis o indi idual siRNA posi ions e ealed ha RNA sequences
a posi ions9–12and15–18we eassocia edwi hTARBP2whileposi ions19–21wi h
AGO.AGObindingwasenhancedbyposi ions15–18(Takahashie al.,2014).AGO2
was epo ed o binds p ima ily o he 5’- and al e na i ely, o he 3’-end o p e-miRNAs.
(Tane al.,2011).All ou humanAGOp o einsshow ema kablysimila s uc u alp e -
e ences o small-RNA duplexes: cen al misma ches p omo e RISC loading, and seed
o 3’-mid (guide posi ion 12–15) misma ches acili a e unwinding. All hese ea u es o
humanAGOp o einsa ehighly eminiscen o  lyAGO1bu no  lyAGO2.(Yodae al.,
2010).Biochemicalands uc u alanalysissugges s ha TARBP2is lexiblybound o he
Dice DExH/Ddomain(Danielse al.,2009;Wange al.,2009).TARBP2seems ob idge
eleaseo  hesiRNAbyDice andloadingo  heduplexon oAGO2.BindingbyTARBP2
may allow he siRNA in e media e o s ay associa ed wi h he RLC a e elease om Dic-
e andmayalsohelpino ien a iono  hesiRNA o AGO2loading.Jus asin lies,human
RISC assembly is uncoupled om dicing (Yoda e al., 2010).
Analysis o miRNA-ca ying RISC (miRISC) yielded a simila pic u e. Since loading
o miRNA duplexes o AGO p o eins is assis ed by HSP70/ HSP90 chape ones (Mania aki
and Mou ela os, 2005b; Yoda e al., 2010), HSP90 is some imes also included as he com-
ponen o miRLC(Liue al.,2012b).A  hesame ime,AGO2andDice a esu icien  o 
p ocessingandloadingo miRNAsin oRISC(Tane al.,2011).
Combina ion o in i o s udies in Dice -/- cells econs i u ed wi h wild- ype o ca aly -
ically inac i e Dice showed ha he miRNA loading complex (miRLC) is he p ima y
machine ylinkingp e-miRNAp ocessing omiRNAloadingandleadalso ode ini ion
o a miRNA P ecu so Deposi Complex (miPDC) o Dice -independen RISC loading
exempli iedbymiR-451(Liue al.,2012b).miPDCis o medo AGO,p e-miRNA,and
HSP chape one. I unc ions in Dice -independen miRNA biogenesis (e.g. miR-451) and
also p omo es miRNP assembly o ce ain Dice -dependen miRNAs (Liu e al., 2012b).
Ea lie s udiessugges edadi e encebe ween lyandhumansys emsbecausehuman
RISCassemblyusingimmunopu i iedo  econs i u edhumanRLCcon ainingAGO2,Dic-
e andTARBP2didno  equi eATPhyd olysis,(G ego ye al.,2005;MacRaee al.,2008;
Mania akiandMou ela os,2005b).Recen da asugges  ha ATP acili a esalsohuman
RISC loading while i is dispensable o unwinding (Yoda e al., 2010).
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Accesso y RLC ac o s
Apa om he h ee es ablished RLC componen s, se e al p o eins eme ged as RLC co ac-
o s, among which s and ou HSP70/90 chape ones. Hea shock p o ein 90 was ac ually he
i s iden i iedAGO-associa edp o eine enbe o eAGOwasassocia edwi hRNAsilenc-
ing(Tahbaze al.,2001).Inhibi iono HSP90 educesAGOle els(Johns one al.,2010;
Ma inezandG ego y,2013;Tahbaze al.,2001)aswellasGW182p o einle elsand
abolishes P-bodies (Johns on e al., 2010). In addi ion, s able binding be ween AGO and
Dice isdependen on heac i i yo Hsp90(Tahbaze al.,2004)andassocia iono AGO2
wi h HSP90 in ol es p olyl-hyd oxyla ion o AGO2 (Wu e al., 2011). HSP90 ac i i y is
no  equi ed o associa iono AGOwi hin acellula memb ane(Tahbaze al.,2001)bu 
appea s o chape on AGO p o eins be o e binding RNA and may acili a e loading o small
RNAs (Johns on e al., 2010). In e es ingly, miRNA*s (miRNA* is an equi alen o he
passenge s and) wi h as u no e exhibi ed di e en sensi i i y o HSP90 inhibi ion sug-
ges ing di e en ial HSP90 equi emen s o di e en miRNA*s (Guo e al., 2015). HSP90
is also a nega i e egula o o PKR; i is able o bind and inhibi PKR phospho yla ion
andp e en apop osis(Donzee al.,2001).Thus,HSP90p o idesa ac o b idgingRNA
silencing and inna e immuni y.
Fu he mo e, HSP90 co-chape ones FKBP4/5 con ol AGO2 exp ession and acili a e
RISCassembly(Ma ineze al.,2013).FKBP4/5we eiden i iedasAGO2-associa edp o-
eins in mouse emb yonic s em cells. Inhibi ion o FKBP4/5 lead o dec eased Ago2 p o ein
le els while o e exp ession s abilized AGO2 exp ession (Ma inez e al., 2013). Ano he
s udy has ound ha FKBP4 o ms a s able complex wi h human AGO2 be o e small RNA
loadingin hecy oplasmandis equi ed o e icien RNAi(Pa ee al.,2013).
Ano he componen epo ed o unc ion as an RISC-loading ac o is RNA helicase
A (RHA, also known as DHX9) Dice (Robb and Rana, 2007). RHA is a conse ed p o ein
wi h wo dsRBDs (Naga a e al., 2012) wi h mul iple oles in he gene exp ession o cellula
and i al mRNAs. RHA ecognizes highly s uc u ed nucleo ides and ca aly ically ea ang-
es he a ious in e ac ions be ween RNA, DNA, and p o ein molecules o p o ide a pla -
o m o he ibonucleop o ein complex. RHA was shown in human cells o unc ion in
heRNAipa hwayandin e ac wi hsiRNA,AGO2,TARBP2,andDice (RobbandRana,
2007). RHA-deple ed cells, showed educed RNAi, appa en ly as a consequence o lowe
ac i e RISC sugges ing ha RHA unc ions in RISC as an siRNA-loading ac o (Robb and
Rana, 2007). A la e s uc u al analysis o dsRBDs showed ha bo h dsRBDs a e equi ed
o RISC associa ion, and such associa ion is media ed by dsRNA (Fu and Yuan, 2013).
A e mammalian miRNAs so ed?
As men ioned abo e, o he ou AGO p o eins ha can be loaded wi h small RNAs equally
well (Meis e e al., 2004). All ou mouse AGO p o eins seem o be unc ionally edundan
in he miRNA pa hway as shown by escue expe imen s in ESCs lacking all ou A gonau e
genes (Su e al., 2009). Consis en wi h his, all ou AGOs a e unc ionally equi alen when
accommoda ing bulged miRNA duplexes, whe eas AGO1 and AGO2 appea o be mo e
e ec i e a u ilizing pe ec ly ma ched siRNAs (Su e al., 2009). Fu he mo e, AGO2 can
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execu e endonucleoly ic clea age o cogna e RNAs while all ou can media e ansla ional
ep ession.This aisesaques ionwhe he smallRNAsmayunde gosomekindo so ing
ha would esul inp e e en ialloadingon ospeci icAGOhomologs.
S uc u al analysis showed ha all ou human AGO p o eins showed simila s uc u al
p e e ences o small-RNA duplexes, which we e highly eminiscen o D osophila AGO1
bu no o AGO2 (Yoda e al., 2010). Human AGO2 and AGO3 immunop ecipi a ion and
subsequen sequencing o small RNAs e ealed ha bo h AGOs we e associa ed wi h
21–23n RNAs,majo i yo whichwe emiRNAs(Azuma-Mukaie al.,2008).While i een
miRNAsshowedmo e han2- oldsigni ican di e enceinloadingon oAGO2o AGO3,
i is no clea whe he his disc imina ion occu s also in i o (Azuma-Mukai e al., 2008).
A de ailed analysis o small RNAs associa ed wi h all ou human AGO p o eins e ealed
app oxima ely equi alen amoun s o sequence ags de i ed om miRNA loci associa -
edwi hindi idualAGOswi hsomeexcep ions ha couldbecoupled ospeci icAGOs
(Bu oughs e al., 2011). Howe e , u he analysis sugges ed exis ence o some so ing
mechanism a ec ing a subse o dis inc isomiRs ha seemed o be di e en ially associa ed
wi hdis inc AGOp o eins(Bu oughse al.,2011).Thisobse a ioncon as swi hano he 
cloning and deep sequencing expe imen add essing dis ibu ion o endogenous miRNAs
associa edwi hAGO1–3,whichdidno  inde idence o miRNAso inginhumancells.
(Dueck e al., 2012).
I is possible ha so ing o small RNAs on AGO p o eins may no be a gene al phenom-
enon while di e en ial p esence o small RNAs on AGO p o eins can also eme ge om
selec i emechanismsope a inga e loading.Thiscanbeillus a edonselec i ep og es-
si e 3’ sho ening o AGO2-bound miRNAs obse ed in he b ain (Ju una e al., 2012).
Fu he mo e,Duecke alalso epo ed ha AGOiden i yappea s oin luence heleng h
o somemiRNAs,whileo he s emainuna ec ed(Duecke al.,2012).Taken oge he ,i 
seems ha miRNAs a e gene ally no so ed o loading on o AGO p o eins. No able excep-
ions include miRNAs wi h unique biogenesis such as miR-451 whose biogenesis equi es
AGO2 slicing ac i i y (Dueck e al., 2012).
Loading asymme y
While bo h siRNA s ands can guide pos - ansc ip ional silencing in mammals (Wei e al.,
2009), selec ion o he loaded s and exhibi s a clea and long-known he modynamic bias
whe e hes andwhose5′-endisless he modynamicallys ableisp e e en iallyloaded
on o AGO as he guide s and (Kh o o a e al., 2003; Schwa z e al., 2003). Selec ion o
he guide s and in ol es mul iple senso s – his includes AGO2 s and selec ion capabil-
i y (Noland and Doudna, 2013; Suzuki e al., 2015), which is enhanced in complex wi h
Dice andTARBP2o PACT.Inaddi ion,s andselec ion o somemiRNAsisenhanced
incomplexescon ainingPACTbu no TARBP2(NolandandDoudna,2013).No ably,
TARBP2wasp edic ed obeasenso o  he he modynamics abili yo 5’siRNAins and
selec iondu ingRISCloading,simila ly oDCR-2andR2D2(aTARBP2homolog)in
D osophila (Wang e al., 2009). Howe e , he suppo ing e idence is inconclusi e (Haase
e al.,2005)al houghsomea gue ha TARBP2canindeedac sasasenso (G edelle al.,
2010).Fu he mo e,whileTARBP2 unc ionissimila  o ha o R2D2,TARBP2sequence
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is mo e closely ela ed o Loquacious han R2D2 (Mu phy e al., 2008). Finally, quan i a i e
analysis o RISC assembly and a ge silencing ac i i y in he p esence o absence o Dice
sugges ha he mammalian Dice is nonessen ial o asymme ic RISC loading in i o and
in i o.(Be ancu andToma i,2012).
RISC ac i a ion
Thenex s epa e AGOloadingis emo alo  hepassenge s and om heloadedduplex
RNA. In some cases, he passenge s and can be elimina ed by he slice ac i i y whe e he
RISCcomplexuses heguidesiRNA oclea e hepassenge s and.Ino he wo ds he i s 
clea age ac ually a ge s he passenge s and o a loaded siRNA duplex o ee he guiding
s and,soi canbasepai  ocogna emRNAs(Ma angae al.,2005).Theclea age-assis ed
mechanismis ypical o AGO2-loaded lyandhumansiRNAsin heRNAipa hwaywhile
passenge s and clea age is no impo an o loading miRNAs (Ma anga e al., 2005).
Slice -independen mechanism is needed o emo e he passenge s ands om non-slic-
ing AGO p o eins and om miRNA duplexes bound o AGO2 whe e he passenge s and
canno beclea ed.Asslice -de icien hAGO1,hAGO3,andhAGO4a eable oejec  he
passenge s and o siRNA duplexes a 37°C, i is appa en ha AGO1, 3, and 4 can be
eadily p og ammed wi h siRNAs a he physiological empe a u e (Pa k and Shin, 2015).
Thisimplies ha aslice -independen mechanism,which elieson he he maldynamics
o  hePAZdomain(Gue al.,2012a;Pa kandShin,2015),islikelyacommon ea u eo 
human AGOs.
Impo an ly, RISC ac i a ion has been associa ed wi h addi ional ac o s. One o hem
is C3PO, an endonuclease ha ac i a es RISC (Ye e al., 2011). Acco ding o he model
o RISC ac i a ion ha in eg a es he C3PO c ys al s uc u e, Ago2 di ec ly binds duplex
siRNA and nicks he passenge s and, and hen C3PO ac i a es RISC by deg ading he
Ago2-nicked passenge s and (Ye e al., 2011)> Ano he ac o s is La, Sjog en’s synd ome
an igen B (SSB)/au oan igen, which is ac ing as an ac i a o o he RISC-media ed mRNA
clea ageac i i y.(Liue al.,2011).Thus,simila ly oC3PO,Laisa egula o y ac o 
helping o emo e AGO2-clea ed p oduc s in o de o p omo e ac i e RISC o ma ion (Liu
e al., 2011).
Addi ional small RNAs associa ed wi h AGO p o eins
Ou li e a u e sea ch e ealed a he e ogeneous g oup o publica ions desc ibing small
RNAs loaded on AGO p o eins ha we e clea ly dis inc om canonical miRNAs – small
RNAs gene a ed by he mechanism desc ibed abo e. A canonical miRNA is ansc ibed by
polyme ase II, he p ima y ansc ip con ains a ~ 70 n sho hai pin p ecu so p e-miRNA,
which is eleased by he Mic op ocesso complex, anspo ed o he cy oplasm whe e Dice
clea es o he loop and one o he s ands o he miRNA duplex is loaded on o miRISC.
Howe e , nex gene a ion sequencing e ealed exis ence o AGO-loaded small RNAs ha
we e appa en ly gene a ed om di e en subs a es and by molecula mechanisms, which
de ia ed om he canonical pa hway. Below is an o e iew o di e si y o AGO-bound
RNAs, which eme ged om he li e a u e sea ch.
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Non-canonical miRNAs can be di ided ac oss wo axes – (I) acco ding o he RNA
p ecu so and (II) acco ding o he p o eins in ol ed in (o omi ed om) hei biogene-
sis. Non-canonical miRNAs we e disco e ed du ing sys ema ic analyses o small RNAs
in di e en model sys ems, such as disease models (e.g. (Xia e al., 2013) o cul u ed
cells (Babia z e al., 2011; Babia z e al., 2008). A good expe imen al s a egy o iden i y
non-canonical miRNAs is a high h oughpu sequencing analysis o gene ic models lacking
some o he componen s o RNA silencing such as Dice o DGCR8 (Babia z e al., 2011;
Babia z e al., 2008). Pheno ypic di e ence and di e en ial exp ession o dis inc miR-
NA-like sequences can indica e biological oles o non-canonical miRNAs while knock-ou
da a o e an insigh in o he non-canonical biogenesis mechanism
Pe haps he bes known non-canonical miRNA class, which comes om unique, Mic op o-
cesso -independen p ecu so s, a e mi ons, miRNA-like molecules a ising om spliced-ou
in ons, which a e Mic op ocesso -independen (Babia z e al., 2011; Be eziko e al., 2007;
Ladewig e al., 2012; Schambe ge e al., 2012; Sibley e al., 2012; Wes holm e al., 2012).
In e es ingly, some p edic ed mi on-like miRNAs (miR-1225 and miR-1228) a e splic-
ing-independen (sim ons) and hei biogenesis in ol es D osha bu nei he DGCR8 no
Dice (Ha ens e al., 2012). O he non-canonical subs a es can be, o example, 5‘-Capped
RNAs (Xie e al., 2013), SINE epea -de i ed, (Babia z e al., 2008; Cas ellano and S eb-
bing, 2013), small aul RNA (s RNA2–1a) (Minones-Moyano e al., 2013), o RNase III
ansc ip s (Mau in e al., 2012) including anno a ed RNAs such as snoRNAs (Bu oughs
e al., 2011; Ende e al., 2008; Li e al., 2012), 7SL RNA (Ren e al., 2012), RNA agmen s
(Bu oughs e al., 2011; Haussecke e al., 2010; Kuma e al., 2014; Li e al., 2012; Mani-
a akiandMou ela os,2005a;Mau ee al.,2013;Venka eshe al.,2016).Non-canonical
miRNAs can be also p oduced om i al RNAs (Boge d e al., 2010; Kincaid e al., 2014;
Lie al.,2009;Xue al.,2009).Anon-canonicalsmallRNAclasso unclea signi icancea e
semi-mic oRNAs (smiRNAs), which a e ~ 12n sho RNA agmen s appa en ly eme ging
om o he miRNAs, such as le -7 o miR-223 (Plan e e al., 2012).
Non-canonicalmiRNAscanbealsoclassi iedby hei biogenesisasMic op ocesso ,
DGCR8-, o Dice -independen . Fo ins ance, he abo e-men ion mi ons do no equi e
he Mic op ocesso complex while sim ons equi e D osha bu nei he DGCR8 no Dice .
Non-canonicalmiRNAscanbealsop oduced ombona- idemiRNAp ecu so s,which
gi e a ise o a small RNA in a non-canonical way, o example by a dual ole o AGO
p o ein (Diede ichs and Habe , 2007). A classic example is miR-451, a Dice -independen
miRNAbiogenesispa hway ha  equi esAgoca alysis(Chelou ie al.,2010).Aunique
ype o non-canonical miRNAs a e loop-miRs, which a e eleased om he loop egion o
a p e-miRNA (Okamu a e al., 2013; Win e e al., 2013).
Ta ge ecogni ion and modes o silencing
Ta ge ecogni ion
Ta ge  ecogni ionbyRISCismedia edbybasepai ingbe weenRISC-loadedsmallRNAs
and cogna e RNAs. Consis en ly wi h he s uc u al analysis o AGO p o eins, a ge
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ecogni ion by siRNAs exhibi s a dis inc 5’ bias. Analysis o miRNA- a ge ed mRNAs in
D osophilaandmammals e ealed ha miRNAbases2–8 o madis inc „seed“,which
basepai spe ec ly o he a ge  ansc ip (En igh e al.,2003;Lewise al.,2003).Thisis
consis en wi h he ac ha he 5’ hal o a small RNA p o ides mos o he binding ene gy
ha  e he sRISC oa a ge RNA(Doenche al.,2003;HaleyandZamo e,2004).S uc u -
al ea u es o he a ge si e a e only impo an o RISC binding, while sequence ea u es
suchas heA/ucon en o  he3’uTRa eimpo an  o mRNAdeg ada ion.(Hausse e al.,
2009). Acco ding o analyses o RISC kine ics, small RNAs loaded on o AGO p o eins a e
ac uallycomposedo  i edis inc domains(Fig.5): heancho ,seed,cen al,3’supplemen-
a y, and ail (Wee e al., 2012).
Biochemical analysis o a ge ecogni ion by mammalian RISC showed ha he RISC
is appa en ly no sys ema ically scanning ansc ip s. RISC is unable o un old s uc u ed
RNA.Thus,RISC andomly ansien lycon ac ssingle-s andedRNAandp omo essiR-
NA- a ge base pai ing whe e he 5’end o he loaded siRNA c ea es a he modynamic
h eshold o s able associa ion o RISC wi h i s a ge (Ame es e al., 2007).
The ac  ha 5’and3’endso asiRNAa eboundbydis inc bindingpocke sand ha 
bo h ends con ibu e di e en ly o binding o he a ge lead o a „ wo-s a e model o A go-
nau e unc ion p oposed based on he D osophilamodel(Toma ie al.,2004b).In his
model, he3’endisboundin hePAZdomainand he5’end oinapocke a  hein e ace
be ween heMIDand hePIWIdomains.The5’endisp e-o ganized oin e ac wi h he
cogna e mRNA and, upon binding, he 3’ end is dislodged om he binding pocke o allow
o base pai ing o he 3’ end.
Impo an ly, kine ics o silencing is c i ical o unde s anding a ge ecogni ion and
silencing by o small RNAs. A kine ic s udy o D osophila and mouse AGO2 ound ha
mouse AGO2, which mainly media es miRNA-di ec ed ep ession in i o, dissocia es ap-
idly and wi h simila a es o ully pai ed and seed-ma ched a ge s (Wee e al., 2012). An
impo an conclusion om his s udy is ha low-abundan miRNAs a e unlikely o con ib-
u e much biologically meaning ul egula ion because hey a e p esen a a concen a ion
less han hei KD o seed-ma ching a ge s (Wee e al., 2012).
These esul swe esubsequen lyco obo a edbysinglemoleculeanalysis.Single-mol-
ecule luo escenceexpe imen susingaminimalRISC(asmallRNAandAGO2)showed
ha a ge binding s a s a he seed egion o he guide RNA (Chand adoss e al., 2015;
Jo e al., 2015a; Jo e al., 2015b). AGO2 ini ially scans o complemen a i y o nucleo ides
2–4 o he miRNA and his in e ac ion p opaga es in o a s able associa ion when a ge
complemen a i y ex ends ac oss he seed (Chand adoss e al., 2015). S able RISC binding is
huse icien lyes ablishedwi h heseedma chonly,p o idingapo en ialexplana ion o 
he seed-ma ch ule o miRNA a ge selec ion (Chand adoss e al., 2015; Jo e al., 2015a;
Jo e al., 2015b). Rema kably, mouse AGO2 binds igh e o miRNA a ge s han i s RNAi
clea age p oduc , e en hough he clea ed p oduc con ains mo e base pai s (Salomon
e al., 2015). In con as , a ge clea age equi ed ex ensi e sequence complemen a i y and
accele a ed co e-RISC dissocia ion o ecycling (Jo e al., 2015b) and sensi i ely depended
on hesequence(Joe al.,2015a).RISC husu ilizessho RNAsasspeci ici yde e mi-
nan s wi h he modynamic and kine ic p ope ies mo e ypical o RNA-binding p o eins
while a small RNA loaded on AGO no longe ollows ules by which sole oligonucleo ides
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h ps://doi.o g/10.14712/9788024643724.4 81
RNAi AND miRNA PATHWAYS
IN MAMMALS II – BIOLOGICAL ROLES
Mammals II
Keywo ds:dsRNA,siRNA,miRNA,Dice ,TARBP2,PACT,A gonau e
PETRSVOBODA
Ins i u e o Molecula Gene ics, Academy o Sciences o he Czech Republic,
Videnska1083,14220P ague4,CzechRepublic
Co espondence o: Pe S oboda, Ins i u e o Molecula Gene ics ASCR,
Videnska1083,14220P ague4,CzechRepublic, el.#+420241063147,
e-mail: [email p o ec ed].
ABSTRACT
RNAsilencingdeno essequence-speci ic ep essionmedia edbysmallRNAs.Inmammals, he ea e woclose-
ly ela ed pa hways, which sha e se e al p o ein ac o s: RNA in e e ence (RNAi) and mic oRNA (miRNA)
pa hway.ThemiRNApa hway egula esendogenousp o ein-codinggeneexp ession.I hasbeenimplica ed
in many biological p ocesses and majo i y o mammalian genes appea o be di ec ly o indi ec ly exposed o
miRNA-media ed egula ions. RNAi gene ally se es as a o m o inna e immuni y a ge ing i uses and mobile
elemen s,al houghi occasionallyalsoacqui ed unc ioninp o ein-codinggene egula ion.The unc iono 
RNAi in mammals is s ill poo ly unde s ood bu i is clea ha p o eins suppo ing RNAi a e also in ol ed in
miRNA biogenesis and unc ion. Because o he la ge olume o he exis ing li e a u e, he e iew o mammalian
miRNAandRNAipa hwayswasdi idedin o wopa s,whe e i s one e iewedcomponen so  hepa hways
and hesecondone,p esen edhe e, e iews olesandsigni icanceo  hepa hways.
In oduc ion
In he i s pa o  he e iewo mammalianRNAiandmiRNApa hways,I ocusedon
mechanis ic desc ip ion o he pa hways. He e, I will p o ide an o e iew o biological
oles and biological phenomena associa ed wi h mammalian RNAi and miRNA pa hways
(Fig. 1).
miRNA-media ed con ol o gene exp ession – impo an unc ional aspec s
Thecu en miRBase(Kozoma aandG i i hs-Jones,2014)edi ion22.1anno a es1917hum-
anmiRNAloci ha gi e ise o2654anno a edmiRNAs.The ea e1234p ecu so sand
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1978 ma u e miRNAs anno a ed in mouse. A simple connec ion o hese coun s wi h he
ac  ha onlynucleo ides2–8o amiRNAa esu icien  o  a ge  ecogni ionandsup-
p ession implies ha miRNA-media ed ep ession is a widesp ead and ex emely e ol -
able egula o y sys em o gene exp ession. A he same ime, one should no o ge he
abo e-men ioned s oichiome y be ween miRNAs and hei a ge si es ha is needed o
e icien silencing.
E olu ion o miRNAs is as – he e a e only a ew miRNAs conse ed be ween D osoph-
ila and mammals. Gi en he di e si y o canonical and non-canonical miRNAs, i is con-
cei able ha miRNAs o eme ge om andom o ma ion o D osha/Dice subs a es. New-
ly e ol ing miRNAs likely o m a conside able po ion o anno a ed miRNAs, especially
inspecieswhe emiRNAsa edeeplysequencedandlow-abundan miRNAsa eiden i ied.
Acco ding o he e olu iona y heo y, new miRNAs would ei he acqui e unc ion and
become ixeddu inge olu iono  heywouldbelos .Inaddi ion, he a ge  epe oi eo 
exis ing miRNAs can also apidly e ol e since a single poin mu a ion can weaken an
exis ing egula iono c ea eanewone.Thisideaisconsis en wi h heda ashowing ha 
mammalianmRNAsa eunde selec i ep essu e omain ainand/o a oidspeci ic7-nucle-
o ideseeding egions(Fa he al.,2005).I canbenicelyexempli iedon heTexelsheep
pheno ype whe e a single mu a ion c ea ing a no el miRNA a ge si e in myos a in causes
he excep ional mea iness o his b eed (Clop e al., 2006).
Figu e 1 Mechanis ical me ging o miRNA and RNAi pa hways in mammals
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These o miRNAsineachcell ype o msacombina o ialpos - ansc ip ional eg-
ula ion sys em s abilizing gene exp ession pa e n. miRNAs ha e widesp ead impac
onexp essionande olu iono p o ein-codinggenes(Fa he al.,2005).Thenumbe o 
mRNAs ha ha e unc ionally impo an in e ac ion wi h miRNAs (i.e. supp ession o his
in e ac ion yields a pheno ype) in a s udied model sys em is p esumably small and ce ain-
lydi icul  odisce namong hepossiblein e ac ions.Thus,e e ysea ch o  unc ionally
impo an in e ac ions be ween miRNAs and hei a ge s has o ace he ac ha miRNAs
ep esen a dynamically e ol ing sys em wi h coun less andom in e ac ions, which a e no
biologically ele an .
Ex acellula mic oRNAs
Anin e es ing esea ch ieldde elopeda ound he eleaseo miRNAs omcells,de ec ion
o ex acellula miRNAs, and ans e be ween cells. Impo an ly, he as majo i y o he
e e ences p o ided desc ip i e and co ela i e da a documen ing p esence o ci cula ing
miRNAs unde di e en condi ions (e.g. (A oyo e al., 2011; Bellingham e al., 2012;
Huange al.,2013b;Luoe al.,2009;No ellinoe al.,2012;Tu chino iche al.,2011).
I will no e iew he bulk o he ci cula ing RNA li e a u e, which p o ides da a conce ning
bioma ke po en ial o ci cula ing miRNAs, undoub edly o ex eme clinical ele ance bu
o minimal ele ance o his e iew. Below, I summa ize esul s, which admi edly aise
mo e ques ions han p o ide sa is ac o y answe s.
Small RNAs can be ansmi ed om one cell o ano he unde physiological condi ions,
as e idenced, o example, by sys emic RNAi in a h opods o plan s. Small RNAs can
u ilize dedica ed anspo e s, common communica ion channels, o sec e o y ehicles. I
was also epo ed ha Gap junc ions can se e o miRNA ans e om mic o ascula
endo helialcells ocoloncance cells(Thu inge e al.,2016).Ci cula ingmammalian
miRNAs we e epo ed 2008 when hey we e ound in se um o lymphoma pa ien s; hey
we e immedia ely ecognized as po en ial non-in asi e bioma ke s o cance diagnos ics
and ea men (Law iee al.,2008).Thesameyea ,placen almiRNAswe e oundci cula -
ing in ma e nal plasma (Chim e al., 2008), which was one o he disco e ies leading o he
no ion ha miRNAs could be a mobile egula ing molecule (Iguchi e al., 2010) and ha
could e en media e ansgene a ional epigene ic he i ance (Sha ma, 2015) o be ansmi ed
ac ossspecies(Bucke al.,2014;Zhange al.,2012).Since hen,ex acellula miRNAs
we eiden i iedinab oad angeo biological luids,includingplasma,aqueoushumou ,
ce eb ospinal luid,nasalmucus,o milk(Baglioe al.,2015;Dismukee al.,2015;Huang
e al., 2013b; Izumi e al., 2015; K opp e al., 2014; Peg el e al., 2011; Wu e al., 2015a).
miRNAswe eiden i iedin heca goo exosomes,memb anous esicles40 o100nmin
diame e , which a e cons i u i ely eleased by almos all cell ype and a e ound essen ially
ine e ybiological luid( e iewed, o example,inRak,2013;Yoone al.,2014)
Howe e , ex acellula miRNAs do no need o be necessa ily encapsula ed in ex acel-
lula esicles, as wo s udies showed ha 95–99% o ex acellula miRNA a e no in ex a-
cellula esicles bu associa ed wi h AGO p o eins in se um and cell cul u e media (A oyo
e al.,2011;Tu chino iche al.,2011).Fu he mo e,mos indi idualexosomesins anda d
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p epa a ionsdono seem ocon ainbiologicallysigni ican numbe so miRNAs(Che ille 
e al.,2014).Themolecula mechanismo miRNA elease,ei he asaca goina esicleo 
ee, is poo ly unde s ood and he cu en knowledge does no allow o building a cohe -
en model as he li e a u e is sca ce. Non- empla ed nucleo ide addi ions we e ound o
dis inguish be ween cellula miRNAs, which we e 3’ end adenyla ed in cells whe eas 3’
end u idyla ed iso o ms appea ed o e ep esen ed in exosomes sugges ing a possible ole
o 3’ e minalmodi ica ionsinso ingmiRNAsin oex acellula  esicles(Koppe s-Lalic
e al., 2014). Recen ly, ALIX, an accesso y p o ein o he endosomal so ing complex, i
has been implica ed in so ing miRNAs in o ex acellula esicles based on i s in e ac ion
wi h AGO2 and educed miRNAs le els in ex acellula esicles upon Alix knock-down
(Ia ello e al., 2016).
Impo an ly, any model whe e miRNAs would be ca ied o e o egula e gene exp ession
by he canonical miRNA ac i i y mus ace he kine ic da a men ioned abo e (Wee e al.,
2012). While one canno exclude a non-canonical signalling unc ion o ci cula ing miRNAs
(which has no been conclusi ely demons a ed ye ), he li e a u e on ci cula ing RNAs may
include misleading s a emen s, which a e unsuppo ed by expe imen al e idence.
Taken oge he ,whileexis enceo ci cula ingmiRNAshasbeendemons a edbeyond
a doub , expe imen al e idence o hei unc ion (i any) is no conclusi e. Exosomal
esicles can ca y miRNAs and siRNAs – in he la e case, exosomes we e adap ed o
a deli e y ool o siRNAs, which has a good po en ial o u he de elopmen o siRNA
he apy (El-Andaloussi e al., 2012; Kuma e al., 2015; Lasse , 2012; Lee e al., 2012;
Nguyen and Szoka, 2012; Sh am e al., 2013; Wahlg en e al., 2012; Wahlg en e al., 2016).
RNAi pa hway in mammals– impo an unc ional aspec s
I should ei e a ed ha  he,“so-called”RNAiknock-downwi hsiRNAsinmammali-
an cells is essen ially using he miRNA pa hway wi h e ained he abili y o clea e pe -
ec lycomplemen a y a ge sbyAGO2.ThemammaliancanonicalRNAi(i.e.longdsR-
NA-d i en) is a do man pa hway, a bes . By ha is mean ha he p o ein ac o s p esen
ine e ymammaliancells(Dice ,TARBP2,andAGO2)a ecompe en  osuppo RNAi
bu longdsRNAdoesno e icien lyinduceRNAiinmos mammaliancells(Nejepinska
e al.,2012).Thisno ionissuppo edby he econs i u iono humanRNAin e e encein
buddingyeas demons a es ha Dice ,TARBP2,andAGO2a esu icien  o unc ionally
econs i u eRNAi(Suke al.,2011).Thisdemons a es ha  hese h eep o einscons i-
u e he essen ial co e o RNAi mechanism al hough RNAi is no p ope ly econs i u ed
whenbona ideRNAip ecu so swe eco-exp essed(Wange al.,2013).Thep oblemis
appa en ly a he le el o Dice p ocessing as he human slice AGO2 RNAi ole is so con-
se ed ha i could unc ion in RNAi in he ea ly di e gen p o ozoan T ypanosoma b ucei,
demons a ing conse a ion o basic ea u es o he RNAi mechanism (Shi e al., 2006).
In an analogous expe imen , human AGO2 could no eplace A abidopsis haliana AGO1
in he miRNA pa hway (De eson e al., 2013). In a sense, hese di e en esul s a e no
ha su p ising conside ing he minimal equi emen s o RNAi and he complexi y o he
miRNA pa hway, which p o ides a la ge space o e olu ion o incompa ible adap a ions.
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Howe e , he e a e some cases indica ing ha RNAi is s ill ac i e in mammals and, unde
unique ci cums ances, may be e en an essen ial pa hway.
Themainbo leneck o canonicalRNAiinmammalsise icien p oduc iono siRNAs
om long dsRNA, which is poo in mos mammalian cells (Flem e al., 2013; Nejepinska
e al., 2012). Howe e , se e al epo s showed ha induc ion o RNAi wi h in acellu-
la exp ession o long dsRNA can be achie ed in ans o med and p ima y soma ic cells
(Dialloe al.,2003;Elbashi e al.,2001;Gane al.,2002;ShinagawaandIshii,2003;T an
e al.,2004;Yie al.,2003).Theseda aimply ha RNAicanoccu i  he eisasu icien 
amoun o long dsRNA, which is di ec ed p e e en ially o RNAi bu no in o o he dsRNA
pa hways. Unde hese ci cums ances, he limi ing ac o is jus Dice ’s abili y o p oduce
siRNA (Flem e al., 2013).
Endogenous RNAi in he ge mline
Re o ansposon ep ession in mouse oocy es
RNAi-media ed mobile elemen silencing has also been documen ed in he mouse ge mline
(Tame al.,2008;Wa anabee al.,2006;Wa anabee al.,2008).Mu a ionsin hepiRNA
pa hway componen s a e de imen al o spe m de elopmen , sugges ing ha piRNAs a e
he dominan class o small RNAs con olling mobile elemen ac i i y in he male ge mline
( e iewedinTo he al.,2016).Incon as , emalemicelacking unc ionalpiRNApa hway
a e e ile wi h no ob ious de ec s in oocy es (Ca mell e al., 2007). Endo-siRNAs supp ess
TEssilencinginmammalianoocy esasdocumen edbyde ep essiono some e o anspos-
ons in oocy es deple ed o Dice o AGO2 (Mu chison e al., 2007; Wa anabe e al., 2008).
As al eady p oposed o in e eb a es, he piRNA and endo-siRNA pa hways likely coop-
e a e in c ea ing a complex silencing ne wo k agains mobile elemen s in he mammalian
ge mline.Long e minal epea MTelemen sandSINEelemen sa es onglyup egula ed
in Dice -/- oocy es, while he le els o IAP ansposon a e ele a ed in he absence o MILI
p o ein bu no in Dice -/- oocy es (Mu chison e al., 2007; Wa anabe e al., 2008). S ill many
locicomposedo o he  ypeso TEs,e.g.LINE e o ansposons,gi e ise obo hpiRNAs
and endo-siRNAs, again sugges ing ha he biogenesis o hese small RNAs is in e de-
penden .The oleo endogenousRNAiinTEsilencingex ends omge mcells op eim-
plan a ion emb yo s ages. Apa om ma e nally de i ed piRNAs and endo-siRNAs, which
pe sis in he emb yos o a la ge pa o p eimplan a ion de elopmen , zygo ic endo-siR-
NAs a e gene a ed de no omainly ocon ol heac i i yo zygo icallyac i a edMuERV-L
e o ansposon (Ohnishi e al., 2010; S oboda e al., 2004). SINE-de i ed endo-siRNAs
also inc ease in abundance in ea ly emb yo s ages, which is consis en wi h he obse a ion
ha B1/Alu SINE endo-siRNAs accoun o a as majo i y o endo-siRNAs sequenced
om mouse ES cells (mESCs) (Babia z e al., 2008). Whe he hese SINE endo-siRNAs
playanac i e oleinTEsilencinginmESCssimila ly oo he TE-de i edendo-siRNAs
in oocy es emains o be de e mined. RNAi-dependen silencing o LINE ansposons has
also been desc ibed in cul u ed HeLa cells, whe e endo-siRNAs de i ed om bidi ec ional
ansc ip s o sense and an isense L1 p omo e we e p oposed o con ol L1 ac i i y (Yang
and Kazazian, 2006). Al hough some e idence o e o ansposon-de i ed endo-siRNAs
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om mammalian soma ic cells was ob ained om deep sequencing da a (Kawaji e al.,
2008),acon incingsuppo  o  he unc iono endo-siRNAsinTEsilencinginmammalian
soma ic issues, has ye o be p o ided.
Con ol o endogenous genes in mouse oocy es
In mice, pe u ba ion o he endo-siRNA pa hway in oocy es is esponsible o se e e mei-
o icde ec sand esul ing emalein e ili y.Ta ge edoocy e-speci icknockou o bo hDice
and Ago2 lead o simila pheno ypes including ch omosome misalignmen and de ec i e
spindle(Kanedae al.,2009;Mu chisone al.,2007;Tange al.,2007).Thesee ec s
we e o iginally a ibu ed o he loss o ma e nal miRNAs. Howe e , miRNA pa hway is
supp essed in mouse oocy es and oocy es lacking Dgc 8, which is equi ed o canonical
miRNAbiogenesis,canbe e ilizedanddono showanysigni ican dis u banceo  he
ansc ip ome(Mae al.,2010;Suhe al.,2010).Thismeans ha  hecanonicalmiRNA
pa hway is non-essen ial and la gely inac i e in mouse oocy es despi e in ac biogene-
sis o miRNAs (Fig. 2). In ac , he spindle pheno ype is caused by he loss o a highly
ac i e RNAi pa hway in mouse oocy es. High- h oughpu analysis o small RNAs in mouse
oocy es e ealedauniqueclasso endo-siRNAsde i ed omp ocessedpseudogenes(Tam
e al.,2008;Wa anabee al.,2008).T ansc ip omeso oocy eslackingDice and Ago2
(including oocy es exp essing ca aly ically-dead AGO2) a e simila ly a ec ed (Kaneda
e al., 2009; S ein e al., 2015). A he same ime, genes ma ching pseudogene-de i ed
endo-siRNAs a e en iched in he g oup o up egula ed genes in bo h knockou s (Kaneda
e al.,2009;S eine al.,2015;Tame al.,2008;Wa anabee al.,2008).
In addi ion, pu a i e endo-siRNA a ge s a e en iched in cell cycle egula o s and genes
in ol edinmic o ubuleo ganiza ionanddynamics(Tame al.,2008).These indingssug-
ges ha egula ion o p o ein-coding genes by endo-siRNAs con ols he equilib ium o
p o ein ac o s equi ed o p ope spindle o ma ion, ch omosome seg ega ion and meiosis
p og ession in mouse oocy es. As pseudogenes a e apidly e ol ing sou ce o dsRNA o
endo-siRNA p oduc ion, i will be in e es ing o in es iga e whe he he ole o RNAi in
spindle o ma ion du ing meio ic ma u a ion o oocy es is conse ed in mammals.
The eason o highle elso endo-siRNAsand hehighRNAiac i i yinmouseoocy es
is he a o emen ioned unca ed Dice iso o m ha lacks he N- e minal helicase domain
(Flem e al.,2013)(Fig.2).I e icien lygene a essiRNAs omlongdsRNAs,andissu -
icien  o enhancingRNAiincul u edcellswhilei slossinmouseoocy esyields hesame
pheno ype as condi ional knock-ou s o Dice o Ago2 (Flem e al., 2013).
Endo-siRNAs ha e also been p oposed o con ibu e o he sel - enewal and p oli e a ion
o mouse emb yonic s em cells (mESCs), since he p oli e a ion and di e en ia ion de ec s
obse ed in Dice -/- mESCs a e mo e d ama ic han in Dgc 8-/- mESCs (Kanellopoulou
e al., 2005; Mu chison e al., 2005; Wang e al., 2007). A popula ion o endo-siRNAs
de i edmos ly omhai pin o mingB1/Alusubclasso SINEelemen swasiden i ied
in mESCs (Babia z e al., 2008). F agmen s o SINE elemen s a e commonly p esen in
un ansla ed egions o p o ein-coding ansc ip s and i is he e o e possible ha SINE-de-
i ed endo-siRNAs pa icipa e in pos ansc ip ional gene silencing in mESCs. Howe e ,
his hypo hesis has no been es ed expe imen ally.
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Endogenous RNAi in he soma
Li le e idence is a ailable o po en ial ole o endo-siRNAs in he egula ion o p o-
ein-codingmRNAsinmammaliansoma ic issues.Thena u alan isense ansc ip ionin
soma ic cells, which has a po en ial o gene a e dsRNA, yields low le els o endo-siR-
NAs, whose biological ele ance is ques ionable. A he same ime, endo-siRNAs de i ed
om na u al an isense ansc ip s o Slc34agenewe eiden i iedinmousekidney,whe e
Na/phospha e co anspo e exe s i s physiological unc ion (Ca lile e al., 2009). How-
e e , changes in exp ession le els o Slc34a upon supp ession o he endo-siRNA pa h-
way ha e no been add essed. In mouse hippocampus, deep sequencing e ealed a se o
po en ial endo-siRNAs gene a ed om o e lapping sense/an isense ansc ip s and om
hai pins uc u eswi hinin onso p o ein-codinggenes(Smalheise e al.,2011).The
mos abundan endo-siRNAs om SynGAP1 gene locus we e also ound in complexes
wi h AGO p o eins and FMRP in i o. In e es ingly, a la ge pa o po en ial hippocampal
endo-siRNA a ge s encode o p o eins in ol ed in he con ol o synap ic plas ici y and
henumbe o endo-siRNAsde i ed om hesegenelociinc easedsigni ican lydu ing
ol ac o y disc imina ion aining (Smalheise e al., 2011). Gi en he ac ha as majo i y
o iden i iedendo-siRNAsequencesmapped oin onic egions, heendo-siRNAscould
Figu e 2 miRNA & RNA a angemen in mouse oocy es
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ac co- ansc ip ionally on nuclea p e-mRNAs, pe haps simila ly o he mechanism o
RNAi-media ed inhibi ion o RNA Pol II elonga ion desc ibed in C. elegans (Guang e al.,
2010). Al e na i ely, endo-siRNAs could con ol co ec dis ibu ion o a ge mRNAs as
unspliced p e-mRNA can be expo ed om he neu onal nucleus and anspo ed o den-
d i es o p ocessing(Glanze e al.,2005).Inanycase, hese indingsopenana ac i e
hypo hesis ha endo-siRNAs pa icipa e in synap ic plas ici y du ing lea ning p ocess and
he neu onal endo-siRNA pa hway migh be also linked o a ious neu odegene a i e dis-
o de s (Smalheise e al., 2011).
An i i al RNAi
In con as o nema odes and insec s, da a suppo ing in ol emen o mammalian RNAi
in an i i al de ense is weak ( e iewed in de ail in Cullen, 2006; Cullen e al., 2013). I is
unlikely ha RNAi subs an ially ac s as an an i i al mechanism in mammals whe e long
dsRNA induces a complex sequence-independen an i i al esponse, commonly known as
he in e e on esponse ( e iewed in Gan ie and Williams, 2007). Consis en wi h his,
no siRNAs o i al o igin ha e been ound in human cells in ec ed wi h a wide ange o
i uses (P e e e al., 2005). Occasional obse a ions, such as de ec ion o a single siRNA
inHIV-1in ec edcells(Bennasse e al.,2005)doesno p o ideanyconclusi ee idence
ha RNAi is p ocessing i al dsRNA and supp esses i uses unde physiological condi-
ions in i o.
I mus be s essed ha ci cums an ial e idence sugges ing he ole o RNAi in i al sup-
p ession mus be c i ically examined and in e p e ed. One has o keep in mind, o example,
ha da a,whichappea ase idence o  i alsupp essionbyRNAi,could e lec miR-
NA-media ed e ec s. Since i uses co-e ol e wi h di e en hos s and explo e all possible
s a egies omain ainandinc ease hei  i ness,i isno su p ising ha  i al ep oduc i e
s a egies come in o con ac wi h mammalian RNA silencing pa hways, pa icula ly he
miRNA pa hway, which sha es componen s wi h he RNAi pa hway. Fo example, Eps ein-
Ba  i us(EBV)andse e alo he  i usesencode hei ownmiRNAs(Pa ameswa an
e al., 2010; P e e e al., 2005; P e e e al., 2004; Sulli an e al., 2005) o ake ad an age
o hos cell miRNAs o enhance hei eplica ion (Jopling e al., 2005).
Ano he e idence o an in e ac ion be ween i uses and RNA silencing is he p esence
o pu a i e supp esso s o RNA silencing (SRS) in a ious i uses. As i al genomes ap-
idly e ol e, SRS should be unc ionally ele an . Fo example, B2 p o ein in Noda i uses
(e.g.FHV)isessen ial o  eplica ion,inhibi sDice  unc ion,andB2-de icien FHVcanbe
escuedbya i icialinhibi iono RNAi esponse(Lie al.,2002).B2p o einalsoenhances
he accumula ion o Noda i al RNA in in ec ed mammalian cells (Fenne e al., 2006; John-
sone al.,2004).O he po en ialSRSmoleculesha ebeeniden i iedin i usesin ec ing
e eb a es,suchasAdeno i usVA1noncodingRNA(LuandCullen,2004),In luenza
NS1p o ein(Lie al.,2004),Vaccinia i usE3Lp o ein(Lie al.,2004),Ebola i usVP35
p o ein(Haasnoo e al.,2007),TASp o eininp ima e oamy i us(Lecellie e al.,2005),
o HIV-1TATp o ein(Bennasse e al.,2005).
Theexis enceo SRSin i usesin ec ingmammalsdoesno p o e ha  hese i usesa e
a ge ed by mammalian RNAi. Fi s , i uses may ha e a b oade ange o hos s (o ec o s),
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including,e.g.bloodsuckinginsec s.Thus,a i uscanbe a ge edbyRNAiinonehos 
and by ano he de ense mechanism in ano he one. Fo example, he Dengue i us, whose
li e cycle akes place in humans and mosqui oes, is a ge ed by RNAi in mosqui oes and i
likelye ol edanadap a ion oci cum en RNAi(Sanchez-Va gase al.,2009).Second,
i al SRS in mammalian cells may ha e o he pu pose han coun e ac ing i al supp ession
by RNAi. Since biogenesis and mechanism o ac ion o mammalian miRNAs o e laps
wi h RNAi, i is possible ha he ole o such SRS is o modi y cellula gene exp ession by
supp essing heac i i yo miRNAs.Thi d, hemaine ec o SRSmaybeaimeda o he 
de ense mechanisms ecognizing and esponding o dsRNA and, as a consequence, SRS
e ec s on RNAi a e obse ed.
Sys emic RNAi in mammals
Non-cell au onomous RNA wi h an ex en simila o ha o C. elegans o in some insec s
is highly unlikely o unc ion in e eb a es. Howe e , a limi ed en i onmen al o sys emic
RNAi may exis he e as he homologs o sid-1 ha e been ound in all sequenced e eb a e
genomes(JoseandHun e ,2007).Twosid-1homologs(SidT1andSidT2)a ep esen in
miceandhumanswi hadocumen ed ole o SidT1indsRNAup akeinhumans(Duxbu y
e al., 2005; Wol um e al., 2007). Fu he mo e, expe imen al o e exp ession o human
SidT1signi ican ly acili a edcellula up akeo siRNAsand esul edininc easedRNAi
e icacy(Duxbu ye al.,2005).Asi willbediscussedla e , hemammalianimmunesys-
em employs a numbe o p o eins esponding o dsRNA independen ly o RNAi (Gan ie
and Williams, 2007), while RNAi does no seem o pa icipa e in he inna e immuni y
(Cullen,2006;Cullene al.,2013).Thus, hep ima y oleo adsRNAup akemechanism
in mammals is likely no in ol ing RNAi e en hough i could ha e se ed such a ole in
an ances al o ganism.
Nuclea unc ion o small RNAs
Theli e a u esea chyieldedala gehe e ogeneousg oupo publica ionsconce ningnucle-
a localiza ion o Dice and AGO p o eins as well as nuclea e ec s, including ansc ip-
ional gene silencing. Some o hese obse a ions migh come om physiologically ele an
nuclea silencing mechanisms. Howe e , when c i ically e alua ing published s udies, no
enough e idence was ound, o es ablish a model o ansc ip ional silencing in mammals;
excep o he PIWI-induced ansc ip ional silencing in he ge mline (REF). He e, I will
p o ide an o e iew o nuclea aspec s o RNA silencing and highligh hose obse a ions
which migh be ela ed o he miRNA pa hway o long dsRNA esponse.
Homology-dependen phenomenaandobse a ions ha may e lec nuclea mechanisms
in ol ing small RNAs can be so ed in o se e al a eas, which will be discussed u he
below:
Indi ec e ec s o miRNAs on ch oma in
Nuclea RNAi (nuclea pos - ansc ip ional silencing)
T ansc ip ional egula ions (s imula ion/ ep ession) by exogenous small RNAs
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2012; Wei e al., 2012). Wha is somewha con using in DNA- epai associa ed small
RNAsis he oleo miRNA-speci ic ac o sD osha(F anciae al.,2012)o DGCR8
(Swaha i e al., 2016).
Despi e he he e ogenei y o he nuclea e ec s and many unknowns, some common
hemes eme ged, allowing o o mula ing es able hypo heses ha could be c i ically e al-
ua ed. Fi s , nuclea e ec s can be media ed by small RNAs p o ided in ans. Second,
smallRNAs ec ui AGOp o einsinasequence-speci icmanne ,mos likely ecognizing
alocal ansc ip (pe hapsanncRNA).Thi d, hee ec in ol esachangein hech oma in
s uc u e.Thus,byca e ullyexaminingessen ialexogenoussiRNAp ope iesinp e iously
epo ed nuclea e ec s, one should be able o demons a e ha he silencing phenomenon
uly in ol es an AGO-loaded small RNA engaging ano he nuclea RNA and whe he
hee ec  equi es he“slice ”ac i i y.De ailedexamina iono  heseedsequencewould
alsodisce nbe weenspeci icnuclea e ec sando - a ge ing.Fu he mo e,should he
e ec in ol e small RNA loaded AGO p o ein, he kine ics o he phenomenon should be
in ag eemen wi h known RISC kine ics discussed abo e. Finally, i he a o emen ioned
phenomena ely on localized ec ui men o AGO-loaded small RNAs, one should be able
omimic hosee ec sby e he ingAGOp o eins h oughsequence-speci icDNAbinding
modulessuchas hoseemployedbyTALENo CRISPRnucleases.These esea chdi ec-
ions should be combined wi h alida ed an ibodies o ch oma in immunop ecipi a ion
andimmuno luo escence(o epi opeknock-inin ocandida egenes),mo eex ensi euseo 
mu an s de ec i e in RNA silencing, de ailed quan i a i e analysis o cellula ac iona ion
andiden i ica ionso in e ac ingpa ne ,s udieso pu a i enuclea impo andexpo sig-
nals o Dice and AGO p o eins, and ad anced imaging echniques.
O he dsRNA–associa ed mechanisms I – dsRNA sensing
in he in e e on pa hway
Long dsRNA is no a usual RNA molecule in euka yo ic cells while RNA i uses p oduce
dsRNA du ing eplica ion. A common mechanism ep essing i uses in non- e eb a e
species is RNA silencing (Wang e al., 2006; Wilkins e al., 2005). Howe e , esponse o
o eign long dsRNA in mammals is much mo e complex and in ol es a se o sequence-in-
dependen senso s igge ingexp essiono ade inedse o genesknownasin e e on-s im-
ula edgenes(ISGs).Thein e e onpa hwayis hemos ubiqui oussequence-independen 
pa hwayinducedbydsRNAinmammaliancells( e iewedinde ailindeVee e al.,2005).
Among he ele an senso s ecognizing cy oplasmic dsRNA a e p o ein kinase R (PKR),
hehelicaseRIG-I,MDA5,2’,5’-oligoadenyla esyn he ase(2’,5’-OAS),o Toll-like ecep-
o s(TLR3,7,8)( e iewedinGan ie andWilliams,2007;Sadle andWilliams,2007).
No ably, he e a e also dsRNA-independen mechanisms ha can ac i a e in e e ons in
mammalian cells. Al oge he , di e en s imuli a e being sensed and con e ge on ac i a ion
o o e lappingbu dis inc se so ISGs(Geisse al.,2001).Thesi ua ionise enmo e
con olu ed by cellula di e si y as some cell ypes, pa icula ly immune cells, can elici
hein e e on esponsebyaddi ional,cell- ype-speci icpa hways( e iewedinSchleeand
Ha mann, 2010).
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PKR
PKR is he oldes known mammalian dsRNA sensing p o ein. A pionee ing wo k by Hun e
e al. showed ha di e en ypes o dsRNA can block ansla ion in e iculocy e lysa es
(Hun e e al.,1975).Analysiso  hephenomenoniden i iedPKR ha isac i a edupon
binding o dsRNA and blocks ansla ion by phospho yla ing he alpha subuni o euka -
yo icini ia ion ac o 2(eIF-2α)(Meu se al.,1990).Ac i a iono PKRalsoincludes
ac i a iono  heNFκB ansc ip ion ac o andala genumbe o in e e on-s imula ed
genes (ISGs) (Geiss e al., 2001). PKR esponse o i al dsRNA can be coo dina ed wi h
o he dsRNA senso s, such as RIG-I and MDA5 (Sen e al., 2011). PKR can also espond
o endogenous RNAs in unique physiological egula ions (Be ilacqua e al., 1998; Bomme
e al., 2002). Howe e , endogenously exp essed long dsRNA does no necessa ily induce
canonical PKR esponse wi h in e e on ac i a ion, al hough PKR binding o dsRNA and
es ic ed ansla ional ep ession can be obse ed (Nejepinska e al., 2012; Nejepinska
e al., 2014). I was belie ed ha dsRNA <30-bp in leng h does no induce PKR. Howe e ,
Ma ques e al. epo ed ha , siRNAs can bind and ac i a e PKR in i o ega dless o siR-
NA e mini (Ma ques e al., 2006) a guing agains he long-es ablished 30-bp leng h as he
minimal size-limi o PKR ac i a ion. The ea ealsoo he da aindica ingsensi i i yo 
PKR o dsRNA mo i s sho e han 30-bp (Pu hen ee il e al., 2006; Reynolds e al., 2006;
ZhengandBe ilacqua,2004).
RIG-I-like ecep o s (RIG-I, MDA5, LGP2)
Mammalian soma ic cells can espond o dsRNA in a sequence-independen manne .. In
addi ion o PKR, se e al o he p o eins ecognizing dsRNA a e in eg a ed o he in e -
e on esponse, including helicases RIG-I ( e inoic-acid-inducible gene-I, also known as
DDX58), MDA5 (IFIH1), and LGP2 (DHX58), which sense cy oplasmic dsRNA and ac i-
a e in e e on exp ession.
RIG-I is a cy oplasmic senso di e en ia ing be ween endogenous and o eign RNAs
s uc u es. In pa icula , RIG-I is ac i a ed by blun -ended dsRNAs wi h o wi hou
a 5’- iphospha e, by single-s anded RNA ma ked by a 5’- iphospha e, and by polyu-
idine sequences. RIG-I domains o ganize in o a ing a ound dsRNA, capping one end,
while con ac ing bo h s ands; he s uc u e is consis en wi h dsRNA ansloca ion wi hou
unwinding and coope a i e binding o RNA (Jiang e al., 2011a; Jiang e al., 2011b). Like
RIG-I and LGP2, MDA5 p e e en ially binds dsRNA wi h blun ends (Li e al., 2009a).
RIG-I,MDA5,andLGP2exhibi di e encesin ecognizingspeci icRNAs uc u esand
di e en ypes o i uses p o iding a b oade ange o coo dina ed sensi i i y do di e en
po en ial h ea s (Ka o e al., 2006; Li e al., 2009b; McCa ney e al., 2008; Sen e al.,
2011; Sla e e al., 2010; Wu e al., 2015b). In e es ingly, RIG-I can become ac i a ed also
wi hsiRNAslacking2-n 3’o e hangs(Ma quese al.,2006).Theseda aimply ha 2-n 
3’ o e hangs gene a ed by Dice a e he s uc u al basis o disc imina ing be ween Dice
p oduc s and o he sho dsRNA. Roles o MDA5 and LGP2 in siRNA-media ed in e e -
on esponse emains o be add essed. Fu he mo e, ecogni ion 5’ iphospha e RNA ends
RIG-I (Ho nung e al., 2006; Pichlmai e al., 2006) highligh s impo ance o app op ia e
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p ocessing o 5’ e mini o RNAs p oduced by phage polyme ases when such RNAs a e
used in mammalian cells.
I is no clea how PKR and RIG-I pa hways a e in eg a ed. RIG-I binds siRNAs (wi h o
wi hou 2-n 3’ o e hangs) in i o and i shows g ea e unwinding o blun -ended siRNAs.
Unwinding is hen ansla ed in o he in e e on ac i a ion media ed ia IRF-3.
Toll-like Recep o 3 (TLR3)
TLR3isamembe o  heToll-like ecep o (TLR) amilyand unc ionsasasenso o 
ex acellula , in acellula and i al dsRNAs (Ama an e e al., 2011; Seo e al., 2013; Wang
e al.,2015b;Wue al.,2015b;Yange al.,2006b).TLR3hasdis inc o complemen a y
oles o RIG-I and ela ed helicases in sensing o eign molecules and ac i a ing downs eam
esponses (Li engood e al., 2007; McCa ney e al., 2009; Sla e e al., 2010; Wu e al.,
2015b).
Oligoadenyla e Syn he ase (OAS)
In e e on and dsRNA also ac i a e 2’,5’-oligoadenyla e syn he ase (2’,5’-OAS) ha p o-
duces 2’,5’ oligoadenyla es wi h 5’- e minal iphospha e esidues ha subsequen ly induce
ac i a iono RNAseL;ap o ein esponsible o gene alRNAdeg ada ion(deVee e al.,
2005).
TARBP2 and PACT
In e ac ionsbe weenRNAi,miRNA,andin e e on esponsea epoo lyunde s ood.The e
a e woclea mechanis icconnec ionsbe ween hese wopa hways.Fi s ,TARBP2and
PACT, wodsRNAbindingp o eins,whichwe emen ionedea lie asDice -in e ac ing
p o eins,in e ac alsowi hPKR.No ably,whileTARBP2inhibi sPKR(Cosen inoe al.,
1995;Pa ke al.,1994),PACThas heopposi e ole(Pa elandSen,1998).Whilecy o-
plasmic long dsRNA in soma ic cells appa en ly igge s he in e e on esponse, i is no
clea i he same dsRNA is also ou ed in o he RNAi pa hways. Expe imen s in oocy es
and undi e en ia ed emb yonic s em cells (S ein e al., 2005; Yang e al., 2001) sugges
ha RNAi domina es esponse o cy oplasmic long dsRNA in he absence o a s ong
in e e on esponse and ha he in e e on pa hway domina es when i s ele an compo-
nen s a e p esen . On he o he hand, his iew may be oo simplis ic as i does no explain
he lack o bo h, RNAi and in e e on esponse, in soma ic cells exp essing long dsRNA
(Nejepinska e al., 2012; Nejepinska e al., 2014). In any case, unde s anding he ole o
TARBP2andPACTiso o msin ou inglongdsRNAin oRNAiandin e e onpa hways
equi es u he s udies.
The eisaclea e olu iona yconnec ionbe weenRNAiandin e e on esponse.The
abo e-men ioned mammalian RNA helicases RIG-I, LGP2 and MDA5 a e he closes
homologs o helicases in ol ed in p ocessing o long dsRNA du ing RNAi in C. elegans.
No ably, RIG-I is an es ablished componen o he in e e on esponse o long dsRNA
(Yoneyamae al.,2004).Thissugges s ha  hein e e on esponse,whichhasacommon
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igge and e ol ed a e he RNAi pa hway, adop ed se e al componen s om he la e
pa hway. I emains o be de e mined whe he hese and o he componen s o RNAi los
hei unc ion in RNAi en i ely o media e some o m o a c oss- alk be ween RNAi and
in e e on esponse.
Finally, he e is also a complex ela ionship be ween miRNA and in e e on pa hways
(Ingle e al., 2015; Os e mann e al., 2012; Shapi o e al., 2014; Xu e al., 2011). One con-
nec ionisexempli iedby i almiRNAs,which i usesuse o egula e hehos  esponse,in
pa icula ac o s o he in e e on pa hway (Os e mann e al., 2012) o o he cellula sig-
nalling (Xu e al., 2011). Howe e endogenous cellula miRNAs may also ac o supp ess
he in e e on esponse ac o s, such as he case mi -485, which has a dual ole in a ge ing
RIG-Iaswellas hein luenza i us H5N1 (Ingle e al., 2015).
O he dsRNA-associa ed mechanisms II – Adenosine deamina ion
A- o-Iedi ingisaco alen RNAmodi ica ionsys emo b oadsigni icance( e iewed
in Nishiku a, 2016). I is media ed by adenosine deaminases ac ing on RNA (ADARs),
enzymes ha ca y wo o h ee dsRBD and ecognize bo h in e - and in amolecula dsR-
NAs longe han 20–30 bp (Nishiku a e al., 1991). ADARs con e adenosines o inosines,
which base pai wi h cy osines, which a e in e p e ed as guanosines du ing ansla ion.
Thus,RNAedi inga ec scodingpo en ial, ideli yo RNA eplica ion e e se ansc ip-
ion, o o ma ion/s abili y o RNA seconda y s uc u es whe e a change o a single base in
a sequence may esul ei he in dsRNA des abiliza ion (inosine-u idine pai ) o s abiliza ion
(inosine-cy idine pai ) (Nishiku a, 2010). Such ansi ion in he local and global s abili y
o dsRNAs uc u ecanin luence u he p ocessingo dsRNA,suchas heselec iono  he
e ec i emiRNAs and(Ba el,2004;Meis e andTuschl,2004).
Mammals (and e eb a es in gene al) ha e h ee ADAR genes ( e iewed in Nishiku-
a,2016)(Fig.3).Twoencodep o einsca yingdeaminaseac i i y:ADAR1,whichis
in e e on-inducible, and ADAR2, which is cons i u i ely exp essed. ADAR3 is mos ly
exp essedin heb ainbu i sedi ingac i i yhasno beenshownye .Thespeci ici yo 
he ADAR1 and ADAR2 deaminases anges om highly si e-selec i e o non-selec i e,
dependen on he duplex s uc u e o he subs a e RNA.
Thecomple eADARs uc u ehasno beensol edye bu s uc u eo se e aldomains
isknown– heZalphadomaino  hehumanedi ingenzymeADAR1(Schwa ze al.,
1999)anddsRBDso ADAR2(S e le al.,2010).Theanalysiso dsRBDsp o idedan
insigh in oedi ingo aspeci icsubs a eand e ealed ha dsRBDso ADARno only
ecognize heshapebu also hesequenceo  hedsRNA(S e le al.,2010).Theunex-
pec ed di ec eadou o he RNA p ima y sequence by dsRBDs is achie ed ia he mino
g oo eo  hedsRNAand his ecogni ionisc i ical o bo hedi ingandbindinga ini y
o edi edRNA(S e le al.,2010).I wasalsoshown ha ADAR2 o msdime sin i o
and ha dsRBDsa enecessa yandsu icien  o dime iza iono  heenzyme(Poulsen
e al., 2006).
ADARs exhibi complex egula ion o localiza ion. Fo example, i was shown ha
mouse ADAR1 iso o ms a e di e en ially localized in cellula compa men s and ha hei
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localiza ion is con olled by se e al independen signals, which include a nuclea locali-
za ion signal (NLS), he nucleola localiza ion signal (NoLS), he nuclea expo e signal
(NES)nea  heN e minus(Niee al.,2004).ADAR1in e ac swi hTuDOR-SNnuclease
(Nishiku a, 2010; Scadden, 2005; Weissbach and Scadden, 2012; Yang e al., 2006a) and
localizes o s ess g anules upon s ess induc ion (Weissbach and Scadden, 2012) while
udo -SN deg ades hype edi ed dsRNA (Scadden, 2005).
RNA edi ing conce ns a b oad ange o RNAs including i al and cellula RNAs. Many
long pe ec dsRNAs (>100 bp) unde go ex ensi e edi ing wi h a con e sion o app ox-
ima ely 50 % o adenosines o inosines (Nishiku a e al., 1991; Polson and Bass, 1994).
Ex ensi e edi ing (hype edi ing) is linked wi h nuclea e en ion ( e iewed in DeCe bo
and Ca michael, 2005). On he o he hand, sho RNAs (~20–30 bp) o impe ec long
dsRNAsa eedi edselec i ely;usuallyonlya ewadeninesa speci icsi esa edeamina ed
(Lehmann and Bass, 1999). High h oughpu analyses e ealed he ex en o RNA edi ing
o mammalian RNAs in e ms o subs a e di e si y and equency o edi ing in he an-
sc ip ome (Ca mi e al., 2011; Peng e al., 2012). Edi ed endogenous RNAs (Dawson e al.,
2004; Hundley e al., 2008; Mo se e al., 2002; Salameh e al., 2015) include mRNAs, epe -
i i e sequences (mainly Alu (A hanasiadis e al., 2004)), and miRNAs. I was p edic ed ha
mo e han 85% o p e-mRNAs may be edi ed, p edominan ly in he non-coding egions
(A hanasiadis e al., 2004).
Se e al p i-miRNAs (e.g. miR-142) a e known o unde go edi ing, which inhibi s D o-
shaclea ageo e encausesdeg ada iono p i-miRNAbyTudo SN(Nishiku a,2010;
Scadden,2005;Yange al.,2006a).Ino he cases,p i-miRNAedi ingdoesno in luence
D osha ac i i y bu inhibi s p ocessing o p e-miRNA by Dice (e.g. miR-151) (Kawaha a
e al., 2007a). Las bu no leas , RNA edi ing migh also inhibi expo o miRNAs om
he nucleus (Nishiku a, 2010). A sys ema ic analysis o edi ed miRNAs in he human b ain
showed ha edi ing o miRNAs a ec s se e al miRNAs bu i is no widesp ead (Alon
e al., 2012). A simila pic u e was ob ained om analysis o emb yonic miRNAs (Ga -
cia-Lopeze al.,2013;Veselye al.,2012)
One o he oles o ADARs in immuni y is o p e en inna e immune sensing o sel -RNA
(He aud-Fa low and Walkley, 2016). ADARs also a ec i al RNAs in a ious ways –
ADARs a e bo h an i i al and p o i al; he e ec on i us g ow h and pe sis ence depends
Figu e 3 Domain composi ion o mammalian ADAR p o eins
NES, nuclea expo signal, NLS, nuclea localiza ion signal; dsRBD, dsRNA binding domain.
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upon hespeci ic i us.(Samuel,2011).Vi uses a ge edbyADARsinmammalsinclude
HIV(Cle ziuse al.,2009),he pes i us(Gandye al.,2007),HRSV(Ma inezandMele-
o,2002),HCMV(Nachmanie al.,2014),VSV(Niee al.,2007),andHDV(Wongand
Lazinski, 2002).
C oss alk be ween RNA edi ing and o he dsRNA pa hways.
ADARs a ec o he dsRNA pa hways in se e al ways. In RNA silencing, ADARs can
compe ewi hRNAi o dsRNAsubs a es(includingsiRNAs).TheADAR1iso o m
(ADAR1p150) s ongly binds siRNA and educes hus he a ailabili y o dsRNA o RNAi,
esul inginlesse icien RNAiinno malcellscompa ed oAda 1-/- cells (Yang e al.,
2005). In e es ingly, injec ion o high doses o siRNAs enhances ADAR1 exp ession, sug-
ges ing a ole o ADAR1 in a cellula eedback mechanism in esponse o siRNA (Hong
e al., 2005).
Edi ing a ec s base pai ing quali y o dsRNA subs a es as well as a ge ecogni ion
since a single nucleo ide misma ch be ween siRNA and a ge mRNA can educe RNAi
e icacy(ScaddenandSmi h,2001)o modi y a ge speci ici y,especiallywhenoccu ing
in he seed sequence (Kawaha a e al., 2007b). MiRNAs would be a ec ed in a simila
way. A mode a e deamina ion (one I-U pai pe siRNA) does no p e en Dice p ocessing
osiRNAs(Zamo ee al.,2000)bu ,hype edi ing(~50%o deamina edadenosines)can
make dsRNA esis an o Dice p ocessing (Scadden and Smi h, 2001).
Thus,ADARsa e ac o scon e ing o o ma iono RNAi esis ance(Honge al.,2005),
which may be one o he i al s a egies o a oid being a ge ed h ough a dsRNA- espond-
ingpa hway(Zhenge al.,2005).ADARsin luence heinna eimmuni yei he indi ec lyby
p e en ing sensing o sel -RNA (He aud-Fa low and Walkley, 2016) o by in e ac ing wi h
inna eimmuni y ac o s,suchasPKR(Cle ziuse al.,2009).Theimmunosupp essi e ole
o ADAR1couldexplain hepheno ypeo  heAica di-Gou ie essynd ome(AGS,OMIM#
225750), an au oimmune diso de caused by ADAR1 mu a ions (Rice e al., 2012). I has
been p oposed ha in he absence o ADAR1, accumula ion o cy oplasmic dsRNA may
p o oke in e e on signalling and cause up egula ion o in e e on-s imula ed genes, which
is obse ed in AGS (Rice e al., 2012).
Summa y
In mammals (Fig. 4), he miRNA pa hway seems o be he dominan small RNA pa hway
in he soma while he exis ence and unc ionali y o endogenous RNAi emains unclea .
Theonlycell ypewi hwelldocumen ed obus andmechanis icallyexplainedendo-RNAi
is he mouse oocy e. Soma ic cells ypically espond o long dsRNA wi h a sequence-inde-
penden in e e on esponse, which is employing mul iple dsRNA senso s, which igge
a complex in e e on esponse.
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Acknowledgemen
Iwouldlike o hankmycolleaguesJanPaces,Milosla Nic,andTomasNo o ny o help
wi hcollec ingli e a u e o  he e iew.The e iewcon en wasp oducedunde acon-
ac OC/EFSA/GMO/2015/01-CT01wi hEu opeanFoodSa e yAu ho i y(EFSA); he
opinionsexp esseda e hoseo  hecon ac o onlyanddono  ep esen EFSA’so icial
posi ion. Publica ion o he e iew was unded by LO1220 and LM2015063 by he Minis y
o Educa ion, You h and Spo s.
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h ps://doi.o g/10.14712/9788024643724.5 119
RNAi AND miRNA PATHWAYS IN BIRDS
Bi ds
Keywo ds:dsRNA,siRNA,miRNA,Dice ,TARBP2,PACT,A gonau e
PETRSVOBODA
Ins i u e o Molecula Gene ics, Academy o Sciences o he Czech Republic,
Videnska1083,14220P ague4,CzechRepublic
Co espondence o: Pe S oboda, Ins i u e o Molecula Gene ics ASCR,
Videnska1083,14220P ague4,CzechRepublic, el.#+420241063147,
e-mail: [email p o ec ed].
ABSTRACT
RNAsilencingdeno essequence-speci ic ep essionmedia edbysmallRNAs.In e eb a es, he ea e woclosely
ela ed pa hways, which sha e se e al p o ein ac o s: RNA in e e ence (RNAi) and mic oRNA (miRNA) pa h-
way.ThemiRNApa hway egula esendogenousp o ein-codinggeneexp essionandhasbeenimplica edinmany
biological p ocesses. RNAi gene ally se es as a o m o inna e immuni y a ge ing i uses and mobile elemen s.
This ex  e iewsmiRNAandRNAipa hwaysinbi ds.Al hough hea ailableli e a u eonRNAsilencingin
bi ds is e y limi ed, many ea u es can be deduced om he genomic da a in he public domain. miRNA, RNAi
ando he dsRNA- espondingpa hwaysinbi dsappea  e ymuchlike hoseinmammals,impo an bi d-speci ic
ea u eso RNAsilencingpa hwaysa eye  obeiden i ied.ThemiRNApa hwayislikely hedominan small
RNA pa hway while he exis ence and unc ionali y o endogenous RNAi emains unclea . Some a ia ions may
be p esen in he main bi d an i i al in e e on sys em.
In oduc ion
Bi ds (A es)belong oge he wi hmammalsand ishes o heg oupC ania a wi hin cho -
da es. Some o he bi ds a e o high economic impo ance ( ood indus y) o medical el-
e ance ( i al ec o s causing zoonoses). Bi d ances o s b anched o mammalian ances o s
o e 300 MYA when he synapsid lineage leading o mammals b anched o he sau opsid
lineageleading odinosau sandbi ds.The ea e~9000ex an bi dspecies(Ma gulisand
Schwa z, 1998). Du ing hei e olu ion, bi ds e ol ed nume ous physiological adap a ions
in which hey di e om mammals, including ea he s, shelled eggs wi h ex e nal de el-
opmen , o di e en sex ch omosome sys em, o name a ew. A he same ime, hey a e
he closes mammal- ela ed g oup co e ed in his se ies, in e ms o syn eny and sequence
simila i y.Thisisuse ul o assessing ea u eso dsRNAandmiRNApa hwaysbecause
he a ailable li e a u e on RNA silencing in bi ds is e y limi ed. Howe e , many ea u es
can be deduced om he genomic da a in he public domain. miRNA, RNAi and o he
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dsRNA- esponding pa hways in bi ds a e e y much like hose in mammals and he li e a u e
doesno  epo animpo an bi d-speci ic ea u einRNAsilencingpa hways.Sincemech-
anis icalp incipleso  e eb a emiRNAandRNAipa hwayswe ein oducedin he i s 
wo e iews o his se ies (S oboda, 2019a, b) and in u he de ail elsewhe e (Ba el, 2018;
S oboda, 2014), I will ocus he e di ec ly on ea u es o hese pa hways desc ibed o bi ds.
Dice
Acco ding o hecomple egenomesequenceso chickenandZeb aFinch,bi dsha eone
Dice p o ein. Chicken Dice has been assigned o he ch omosome 5 acco ding o he adi-
a ionhyb idmapping(Tiane al.,2007)whichisinag eemen wi h hecu en chicken
genomemap.The eisnode ailedanalysiso a ianDice speci ici yandac i i y,whichha e
o be in e ed indi ec ly om o he esul s. Chicken Dice can p ocess bo h, long dsRNA and
miRNA p ecu so s, as e idenced by induc ion o RNAi wi h long dsRNA (Mau i e al., 2008;
Peka ik e al., 2003) and hund eds o a ian miRNAs in he miRBase.
ThecommonDice p oduc sizeseems obe21–23n wi ha ypicalsizeo 22n .This
in o ma ioncanbein e ed oma ailablemiRBaseda a(Fig.1).Thus, hea ianDice 
p oduces small RNAs wi h he same sizes as he mammalian Dice (Fig. 1). Ano he pos-
siblesubs a eo Dice inbi dsmigh besnoRNAs,al hough hebiologicalsigni icanceo 
hisobse a ion emainsunclea (Ta e al.,2009).
I is unclea i he e a e unc ionally di e en a ian Dice iso o ms as is he case in
mu ineoocy esandsoma iccells(Flem e al.,2013).The eisone epo o di e en Dice 
splice a ian in goose (Anse cygnoides) whe e one a ian lacks a linke be ween DEAD
boxandhelicaseCdomainsa  heN- e minus(gDice -b)(Hue al.,2014).Thesho e iso-
o mgDice -bisp esen inmul iple issues,howe e i s unc ionalsigni icanceisunclea .
The unca ionis oundin heN- e minus,whichisassocia edwi hsubs a eselec i i yand
e icien p ocessing.The e o e,onemigh specula eabou some unc ionaldi e gencein
subs a e p ocessing be ween he wo iso o ms. Howe e , he e is no expe imen al e idence
a  hemomen .Theonlya ailableda a,so a ,conce ncloningo  hesho iso o mand
exp essionanalysiso se e al issuesand ollicula s agesbyRT-PCR(Hue al.,2014).
dsRBPs
dsRBP binding pa ne s o Dice ha e no been s udied, so a . In e es ingly, he chick-
engenomecon ainsadsRBP,whichis ela ed oTARBP2andPACT,sugges ingamo e
ances al e eb a e s a e and a educed c oss alk be ween RNAi and he in e e on pa hway.
A gonau e p o eins
A gonau e amilyp o einsa ee ec o so RNAsilencingmechanisms.Theya edi ided
in o wo sub amilies: AGO p o eins, which accommoda e miRNAs and siRNAs, and PIWI
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p o eins, which accommoda e piRNAs. A ian AGO p o eins ha e no been cha ac e ized
in a published epo bu public chicken genome da a show ha he se up is he same as
in mammals: S udies in chicken e ealed ou AGO p o eins, whe e AGO1, 3, and 4 a e
encoded wi hin one locus on ch omosome 23 and AGO2 is encoded sepa a ely on ch o-
mosome2.Thisa angemen appea s obesha edwi hinmammalsandbi ds(Zhoue al.,
2010). Addi ional in o ma ion abou a ian AGOs can be in e ed indi ec ly om he exis -
ence o unc ional RNAi and miRNA pa hways (discussed below), which implies ha a
leas oneAGOp o einisa“slice ”(p esumablyAGO2,gi eni sconse ed oleasaslice 
om D osophila o mammals). A ian AGO p o eins can also media e pos - ansc ip ional
silencing guided by impe ec ly base pai ed miRNAs.
In addi ion, he e we e wo publica ions ound, which men ion a ian PIWI p o eins,
which p ima ily con ol genome in eg i y in he ge mline and a e no wi hin he scope o
his epo (Kim e al., 2012; Lim e al., 2013).
O he ac o s
Bi ds ha e addi ional p o eins in ol ed in o he dsRNA esponses, which a e ei he asso-
cia ed wi h adenosine deamina ion (He be e al., 1995) o in e e on esponse. In e e on
esponse ac o s, which ecognize some o m o dsRNA and a e also ound in mammals,
include MDA5 (Hayashi e al., 2014; Lee e al., 2012, 2014), RIG-I (Chen e al., 2015; Li
e al., 2014a; Xu e al., 2015), and PKR (Gonzalez-Lopez e al., 2003; Los ale-Seijo e al.,
2016;Zhange al.,2014).In e es ingly,chickenlack heRHA/DHX9homolog(Sa oe al.,
2015).Thean i i al esponse odsRNAwillbediscussed u he below.
miRNA pa hway
Acco ding omiRBase(Kozoma aandG i i hs-Jones,2014),bi dgenomesencodehun-
d edso miRNAs(Table1)Du ing hesys ema icli e a u e e iew,miRNA- ela edpub-
lica ions lacking a mechanis ic molecula insigh in o he miRNA pa hway we e he mos
commonclasso anno a edpublica ions o bi ds(~50%o allselec edpublica ions).These
publica ions all in o ou basic ca ego ies:
a) anno a ions o no el miRNAs, including high- h oughpu exp ession analyses ( o exam-
ple(Godnice al.,2013;Luoe al.,2012;Ta e al.,2009)andmanyo he s).This
ca ego yalsoincludes heo iginalchickenandZeb aFinchgenomeanno a ionpape s
(In e na ional Chicken Genome Sequencing, 2004; Wa en e al., 2010).
b) s udies o miRNAs in di e en biological con ex s, including ep oduc ion (Lee e al.,
2015; Lee e al., 2011), skele omuscula appa a us (Chen e al., 2009a), bi d song phys-
iology (Guna a ne e al., 2011), g ow h/weigh gain (Li e al., 2013), and many o he s;
hei comp ehensi e lis ing would be beyond he scope o his epo .
c) s udies o ela ionship be ween miRNAs and he immune sys em, especially an i i-
al – hese will be discussed u he below in he sec ion 3.1.2.7. O he dsRNA esponse
pa hways
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h ps://doi.o g/10.14712/9788024643724.6 133
RNAi AND miRNA PATHWAYS IN FISH
Fish
Keywo ds: dsRNA,siRNA,miRNA,Dice ,TARBP2,PACT,A gonau e
PETRSVOBODA
Ins i u e o Molecula Gene ics, Academy o Sciences o he Czech Republic,
Videnska1083,14220P ague4,CzechRepublic
Co espondence o: Pe S oboda, Ins i u e o Molecula Gene ics ASCR,
Videnska1083,14220P ague4,CzechRepublic, el.#+420241063147,
e-mail: [email p o ec ed].
ABSTRACT
RNAsilencingdeno essequence-speci ic ep essionmedia edbysmallRNAs.In e eb a es, he ea e woclosely
ela ed pa hways, which sha e se e al p o ein ac o s: RNA in e e ence (RNAi) and mic oRNA (miRNA) pa h-
way.ThemiRNApa hway egula esendogenousp o ein-codinggeneexp essionandhasbeenimplica edinmany
biological p ocesses. RNAi gene ally se es as a o m o inna e immuni y a ge ing i uses and mobile elemen s.
This ex  e iewsmiRNAandRNAipa hwaysin ish.RNAiando he dsRNA- espondingpa hwaysin isha e
e ymuchlike hoseinmammals,whichis ema kableconside ing hemammalianand ishlineagessepa a edin
Paleozoicumsome400millionyea sago.ThemiRNApa hwayislikely hedominan smallRNApa hwaywhile,
simila ly o o he e eb a es, he exis ence and unc ionali y o endogenous RNAi emains unclea .
In oduc ion
Fish a e an aqua ic he e ogeneous pa aphyle ic g oup wi h he majo i y o he species
belonging o hebony ishclass(Os eich hyes) g oup, which has ~25 000 species, he
highes species di e si y han any o he e eb a e g oup (Ma gulis and Schwa z, 1998).
O e all, heo ganiza iono smallRNApa hwaysin ishis e ysimila  o ha o mammals
(Fig.1),whichisno ableconside ing hecommonances o o  ishandmammalsexis ed
3602–4500millionyea sago(Vol ,2005).
Since mechanis ical p inciples o e eb a e miRNA and RNAi pa hways we e in o-
ducedin he i s  wo e iewso  hisse ies(S oboda,2019a,b)andin u he de ailelse-
whe e (Ba el, 2018; S oboda, 2014), I will ocus he e di ec ly on ea u es o hese pa h-
ways epo ed om ishmodels.Nex gene a ionsequencinganalysiso zeb a ishsmall
RNAsiden i iedmiRNAsandge mlinepiRNAsascommonsmallRNAs.Thedominan 
smallRNApa hwayin ishis hemiRNApa hway.S udieso  oleso miRNAsaccoun 
o  he as majo i yo  heli e a u eonsmallRNAin ish.A  hesame ime, hemolecula 
In oduc ion_ o_RNAi.indd 133In oduc ion_ o_RNAi.indd 133 09.07.20 8:3409.07.20 8:34

FISH
134
mechanismo miRNAandRNAipa hwayswasseldomd ec lys udiedin he ishmodel.
Thesecondmos s udieds udiedsmallRNApa hwayin ishis hepiRNApa hway,which
p o ec s he ge mline om mobile elemen s (Houwing e al., 2007; Huang e al., 2011;
Kamminga e al., 2010) and di e s om miRNA and RNAi pa hways in small RNA bio-
genesis, which does no equi e Dice .
Dice
Fish genomes ca y a single gene o Dice , which is an o holog o he mammalian Dice
and Dice -1 in D osophila(Mu phye al.,2008).Thisno ionissuppo edbyanno a ed ish
genome da a in he UCSC genome b owse (h ps://genome.ucsc.edu/), and blas n sea ch
o  ishsequencesa NCBI(e.g. Salmo sala , Danio e io, Taki ugu ub ipes, Gas e os eus
aculea us (s ickleback), O yzias la ipes (medaka)). Exis ence o a single Dice gene in
ishgenomesis ema kablein eleos  ishspecies,whichunde wen genomeduplica ion
(Howee al.,2013;Meye andScha l,1999).Thissugges sselec i ep essu ecouldexis 
agains Dice geneduplica ion.Zeb a ishDice isessen ial o de elopmen andi sp i-
ma y oleseems obemiRNAbiogenesis(Wienholdse al.,2003).The oleo Dice in
endogenousRNAiin ishhasno beenadd essedinmuchde ail.As udyo Dice ing ass
ca p C enopha yngodon idella e ealed a CDS encoding Dice p o ein ca ying all known
unc ional domains ound ypically in o he Dice s (Shen e al., 2013). C enopha yngodon
idella Dice is abundan ly exp essed in b ain, gill, head kidney, li e , spleen, hea , muscle
and in es ine. A posi i e co ela ion was ound be ween C enopha yngodon idella Dice
mRNAexp essionandin ec ionwi hg assca p eo i us(GCRV)in ec ionincul u ed
kidney cells and in he li e (8.46- old, P < 0.01, 12 h pos -in ec ion) and spleen in i o
(Shene al.,2013).Thissugges s ha C enopha yngodon idella Dice is an inducible gene
esponding o i al in ec ion al hough e idence o i us-de i ed endogenous siRNAs has
no been p o ided.
dsRBPs
Fishgenomes ypicallycon aino hologso TRBP2andPACT,whicha emammalian
Dice -in e ac ing dsRBPs (Mu phy e al., 2008). Howe e , hei unc ion in small RNA
silencingwasno s udiedin he ishmodel,so a .
A gonau e p o eins
Fish AGO p o eins a e o hologs o AGO p o eins in o he e eb a es (Mu phy e al.,
2008).Howe e , eleos  ishcladecon ainsanaddi ionalAGOpa alog,whicheme ged
oma ish-speci icgenomeduplica ione en  ha occu edapp oxima ely350million
yea sago(McFa lanee al.,2011).All i eAgogenomiclociin eleos scon ainspeci -
ic, conse ed sequence elemen s in non-coding egions indica ing ha he eleos AGO
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pa alogs a e di e en ially egula ed, which is consis en wi h exp ession analysis in he
zeb a ishmodel.Mul iplesequencealignmen sshow ha  eleos homologspossessc i ical
aminoacid esidues o AGO unc ionaswellaso holog-speci ic ea u es e ained h ough-
ou he e eb a e lineage (McFa lane e al., 2011).
miRNA pa hway
In e mso smallRNA esea ch, ishmodelsa e ypicallyused o explo ingmiRNApop-
ula ions and s udying biological oles o di e en miRNAs. A icles anno a ing miRNAs
andanalyzing hei exp essionand unc ion ep esen  hebulko  he ish- ela ed e e ences.
We ha e ound 348 a icles, which anno a ed and/o analyzed exp ession and unc ion o
miRNAsin ish.Howe e ,almos noneo  hesea iclesb ough anyspeci icmechanis ic
insigh in o hemolecula mechanismo miRNAin ish.
Acco ding o hemiRBase( elease22.1),miRNApopula ionin ishappea slesscom-
plex haninmammals– ishmodelsha elessanno a edmiRNAs(Table1)al hough he e
a e dozens o epo s on nex gene a ion sequencing analysis and miRNA anno a ion. How-
e e ,zeb a ishisanexpe imen allyeasilyaccessiblemodel o explo ingconse ed oleso 
miRNAsindi e en  issues,whichisalso e lec edin henumbe o  e e ences.
Table 1 Numbe s o anno a ed miRNAs in selec ed ishes in miRBase 22.1
species miRNA p ecu so s ma u e miRNA
Cyp inus ca pio 134 146
Danio e io 355 373
Fugu ub ipes 131 108
Ic alu us punc a us 281 205
O yzias la ipes 168 146
Salmo sala 371 498
Te aodon nig o i idis 132 109
In e mso  hemolecula mechanismo RNAsilencing,s udiesin hezeb a ishando he 
ishmodelsb ough se e alin e es ingdisco e iesconce ningspeci icmiRNA unc ions
anduniqueadap a ionsin ish.Se e alexamplesha e ele ance o  he esea cho  he
molecula mechanism o miRNA pa hway:
The i s exampleis hebiologyo  hemiR-430 amilyo miRNAs.WhilemiRNAs
in mice a e essen ially i ele an o he oocy e- o-emb yo ansi ion (Suh e al., 2010),
hezeb a ishoocy e- o-emb yo ansi ioninco po a eszygo ically-exp essedmiR-430
amily in ma e nal mRNA deg ada ion (Gi aldez e al., 2006; Mishima e al., 2006). Fu -
he mo e, heonse o miR-430ac i i yin hezeb a ishzygo eallowed o add essing
he ela ionship be ween miRNA-induced ansla ional ep ession and mRNA deg ada ion
(Bazzinie al.,2012;Mishimae al.,2012).using ibosomep o ilingo zygo ics ages,i 
was showed ha miR-430 educes ansla ion be o e causing mRNA decay (Bazzini e al.,
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2012).Asigni ican  echnologicalou comeo  heses udieswasde elopmen o  a ge p o-
ec o s,mo pholinooligonucleo idesspeci icallydis up ingmiRNA-media ed ep ession
ia hyb idizing o and masking miRNA-binding si es (Choi e al., 2007).
Ano he con ibu ion osmallRNAbiologycoming om ishmodelwasdisco e yo one
o he non-canonical miRNA biogenesis mechanisms, namely Dice -independen miRNA
biogenesis o miR-451, which uses AGO2 slicing ac i i y ollowed by u idyla ion and im-
ming(Ci uen ese al.,2010).Thep ocessalsoemploys ansla ionini ia ion ac o eIF1A,
which di ec ly in e ac s wi h AGO2 and p omo es miR-451 biogenesis (Yi e al., 2015).
Da a omzeb a ishalsocon ibu ed ounde s andingo he miRNA egula ions.Two
ela ed e minalu idyl ans e ases(TuTases),Zcchc6(TuT7)andZcchc11(TuT4),selec-
i ely3‘monou idyla easubse o miRNAs(Tho n one al.,2014).TuTaseinhibi ionin
zeb a ishemb yoscausesde elopmen alde ec sandabe an Hoxgeneexp ession(Tho n-
on e al., 2014).
Ano he miRNA egula o disco e edin hezeb a ishisdeadend1(DND1),whichis
nega i ely egula ing miRNA a ge ing. DND1 is an e olu iona y conse ed RNA-binding
p o ein(RBP) ha coun e ac s he unc iono se e almiRNAsinzeb a ishp imo dialge m
cells as well as in human cells. DND1 binds mRNAs and p ohibi s miRNAs om binding
cogna e mRNAs. DND1 e ec s in ol e u idine- ich egions p esen in he miRNA- a ge ed
mRNAs (Kedde e al., 2007)
Taken oge he s udieso molecula mechanismo miRNA-media ed ep essionin ish
did no e eal any no able de ia ion om wha has been obse ed in mammals. As he
p o ein machine y appea s o be well-conse ed, miRNA pa hways among e eb a e ax-
ons p ima ily di e in se s o miRNAs and hei a ge s, which dynamically e ol e o e
ime.Thiswas o exampledemons a ed o zeb a ishmiR-430andmu inemiR-290–295
miRNA clus e s, which sha e common ances y, bo h a e associa ed wi h ea ly de elopmen
bu do no egula e he same genes al hough some a ge s seem o be conse ed (S oboda
and Flem , 2010).
RNAi
Thep esenceo RNAi esponsewasexaminedinzeb a isha  hesameasino he animal
modelsdu ing he u no  hecen u y.Howe e ,unlikespeci icRNAiobse edinmouse
oocy esandea lyemb yos(S obodae al.,2000;WiannyandZe nicka-Goe z,2000),long
dsRNAinjec ionin ozeb a ishhadbeenyieldinginconsis en  esul s(Lie al.,2000;Man-
gose al.,2001;Oa ese al.,2000;Zhaoe al.,2001).Whilesome epo edspeci icknock-
downe ec s(Lie al.,2000;Mangose al.,2001),o he sobse ednon-speci ice ec s
(Oa ese al.,2000;Zhaoe al.,2001).Non-speci ice ec s emaineda ecu ing heme
alsoinla e s udies(Wange al.,2010;Zhaoe al.,2008)al houghsomeau ho swe eable
oachie especi icRNAie ec s(DeRienzoe al.,2012;Donge al.,2013;Yinge al.,
2010).Non-speci ice ec sinzeb a ishemb yoswe eno  emediedby heuseo siRNAs
andi wasla e shown ha  hebasiso  henon-speci ice ec sisin e e encewi hmiRNA
unc ion(Zhaoe al.,2008).Injec iono zeb a ishzygo eswi hsiRNAcausedasigni ican 
educ ioninmiR-430le elsleading ounspeci icde elopmen alde ec s(Zhaoe al.,2008).
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In e es ingly, li e a u e su ey e ealed ha expe imen s wi h he pa asi ic sea lamp ey
(Pe omyzon ma inus),ajawless ish ela i e,showed ha up akeo  eesiRNAa 5μg/ml
did no igge an RNAi esponse (Hea h e al., 2014). In any case, RNAi did no become
a a ou i eknock-downs a egy os udygenesdu ingzeb a ishde elopmen ;mic oinjec-
ion o mo pholino oligonucleo ides (Blum e al., 2015; Eisen and Smi h, 2008) became he
p e e ed app oach ins ead.
Taken oge he ,success ulRNAiexpe imen swi hlongdsRNAdemons a e ha zeb a -
ishholds hemolecula machine y o execu ingRNAi:Dice ,TARBP2,andAGO2.How-
e e ,i scapaci y o media ingspeci icknock-downe ec sislimi edbecause hesame
machine y is being simul aneously u ilized by he miRNA pa hway. Impo an ly, he a ail-
abili y o he machine y abo e he minimum capaci y sus aining he miRNA pa hway unc-
ionali y likely di e s du ing de elopmen and among di e en cell ypes.
Whilezeb a ishholds hemolecula machine y o execu ingRNAi, heques ion emains
whe he  heendogenousRNAihasanysigni ican  olein ish.A ailableda adono p o ide
unequi ocale idence o signi ican endogenousRNAiin ish.Nex gene a ionsequencing
o small RNAs con ains ac ions o non-miRNA small RNAs o endo-siRNA size, ye i
is no clea i hese agmen s uly ep esen bona ide endo-siRNAs. Some o he da a
indi ec lypoin  oapossiblean i i al ole,namelyGCRV-induced ansien up egula ion
o Ago2 in a e minnow (Gobiocyp is a us) and Dice up egula ion in g ass ca p (C en-
opha yngodon idella)(Guoe al.,2012;Shene al.,2013).In e ac iono GCRVwi h he
small RNA machine y ( he miRNA pa hway should no be excluded) has been sugges ed
basedon heobse a ioning assca pkidneycells ha GCRVdsRNAcouldbep ocessed
in osiRNAsbu GCRVin ec iondidno yieldGCRV-de i edsiRNAswhileDice up egu-
la ionoccu ed(Go esmane al.,2014).I hasbeen husp oposed ha anuniden i iedRNAi
supp esso migh con ibu e o hesu i alo  he i algenomeande icien  i al eplica-
ion(Go esmane al.,2014).Thep esenceo a i us-de i edinhibi o o RNAsilencing
ina ishRNA i uswouldbeindica i eo anexis ingan i i al oleo smallRNAs,which
is being supp essed. Howe e , an al e na i e scena io ha should be conside ed as well is
ha dsRNA o med du ing i al eplica ion is no accessible o Dice -media ed clea age.
The e o e, u he  esea chisneeded oadd ess hisissue.
O he no able silencing phenomena
The ea e wophenomena,whichclea lyo e lapwi hRNAsilencingbu  hei unde lying
molecula mechanism emains unclea and will equi e u he in es iga ion.
And ews e al ound ha in oduc ion o ansgenes con aining con e gen ansc ip ion
uni sinzeb a ishemb yosinduceds able ansc ip ionalgenesilencingincis and ans.
Thesilencingwassupp esseduponDice knockdown,indica ingp ocessingo double
s anded RNA. ChIP e ealed ha silencing was accompanied by en ichmen o he cons i-
u i e he e och oma in ma k H3K9me3 (And ews e al., 2014). While small RNA-induced
ansc ip ional silencing is well es ablished in ungi and plan s (and seems o be a p oduc
o con e gen e olu ion), he molecula mechanism unde lying seemingly ela ed obse a-
ions in e eb a es is unclea .
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a e wi h, some excep ions (e.g. a madillo bug an ela i es known as woodlice), aqua ic
andha edi e en ia edsegmen edbodyandbi amousappendages.Theyincludesh imp,
c ay ish,lobs e s,c abs,ba nacles,p awnsando he s.Hexapoda comp ise insec s and
insec -like animals wi h six ho acic legs.
Thekeymodelo ganism o a h opodsisD osophila, which has been a wo kho se o
biology o o e hund ed yea s. miRNA, RNAi and o he dsRNA pa hways in D osoph-
ila a e well unde s ood and will se e as benchma ks o he en i e phylum. D osophi-
la e ol ed an ex ensi e gene ic sepa a ion o miRNA and RNAi pa hways whe e each
pa hway has a dedica ed Dice , dsRBP, and A gonau e p o ein. Gi en he complexi y o
he phylum and e olu iona y ime, one could ques ion how ep esen a i e o a h opods
is he D osophila model. Howe e , analysis o Dice and AGO indica es ha D osophila
isamo e-o -lessaccep ablemodel o mos a h opodsas he“ woDice sys em”can
be ecognized wi hin phylogeny o Dice and AGOs also in Chelice a a (whose common
ances o s wi h D osophila b anched in he mos dis an pas ), My iapoda, and C us acea
(Palme and Jiggins, 2015). Howe e , i should be kep in mind ha some a iabili y could
eme ge du ing hal a billion yea s o a h opod e olu ion.
Since mechanis ical p inciples o e eb a e miRNA and RNAi pa hways we e in o-
ducedin he i s  e iewo  hisse ies(S oboda,2019)andin u he de ailelsewhe e(Ba -
el, 2018), I will ocus he e di ec ly on ea u es o hese pa hways disco e ed in A h opods.
The o mals uc u eo  he epo willbeasino he animal axons–uponmiRNAdna
RNAi molecula ea u es o key indi idual componen s o e iewed mechanisms, I will
discuss he silencing mechanisms and hei biological oles. Impo an ly, o p o ide an
o e iew o miRNA and dsRNA mechanisms in a h opods, I will ocus on desc ip ion o
molecula mechanismsiden i iedinD osophilaandwillhighligh anddiscusssigni ican 
de ia ions obse ed elsewhe e in a h opods, especially in mo e s udied o ganisms, such as
mosqui os, lowe bee le,silkmo h,andsh imps.
The Mic op ocesso complex
D osophila u ilizes he same Mic op ocesso complex as he ea lie discussed Me azoa,
i.e. a complex o D osha and DGCR8 homologs, he la e being named Pasha (pa ne o
Figu e 1 Simpli i ed di ision o A h opoda used in he ex
The scheme efl ec s he Mandibula a model o a h opod phylogene ics desc ibed in (Regie e al., 2010)
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D osha)(Denlie al.,2004;Filippo e al.,2000;Land hale e al.,2004).Thecomplex
clea es he p i-miRNA in o p e-miRNA in he nucleus. Supp ession o Pasha in D osoph-
ila in e e es wi h p i-miRNA p ocessing, leading o an accumula ion o p i-miRNAs and
a educ ion in ma u e miRNAs (Denli e al., 2004; Land hale e al., 2004). Like in o he
animals, Pasha is essen ial o p ocessing o canonical miRNAs bu is dispensable o mi -
ons (Flyn e al., 2010; Ma in e al., 2009; Smibe e al., 2011). D osophila Pasha is pos-
sibly phospho yla ed by ERK/MAPK, as sugges ed by phospho yla ion o human DGCR8
in insec cells; he phospho yla ion appea s o inc ease p o ein s abili y wi hou al e ing
miRNA p ocessing ac i i y (He be e al., 2013). miRNA biogenesis in D osophila also
in ol es SmD1, a componen o he D osophila small nuclea ibonucleop o ein pa icle
(snRNP), which in e ac s wi h bo h he mic op ocesso componen Pasha and p i-miRNAs,
and is indispensable o op imal miRNA biogenesis (Xiong e al., 2015).
Analysiso  ansc ip omechangesuponD oshaknock-downinS2cellsiden i ied137
D osha- egula ed RNAs, including 11 ela i ely long (>10 kb) p i-miRNAs (Kadene e al.,
2009). In e es ingly, >100 RNAs no anno a ed as miRNAs could be di ec a ge s o D os-
ha ac ion (Kadene e al., 2009), which is consis en wi h o he model sys ems whe e D o-
sha is ha ing oles beyond miRNA biogenesis. D osha- egula ed RNAs con ain conse ed
hai pins simila o hose ecognized by he D osha-Pasha/DGCR8 complex in p i-miRNAs,
one o such hai pins is ound also in Pasha sugges ing a nega i e eedback loop egula ing
miRNA-biogenesis (Kadene e al., 2009). miRNA-independen oles o he Mic op o-
cesso complexcomponen sseem obe e lec edinpheno ypeso someo  hei mu an s
(Luhu e al., 2014).
In e ms o e olu iona y di e si y o he Mic op ocesso complex in a h opods, he
miRNA pa hway seemed o expand in he pea aphid (insec , Hemip e a), whose genome
ca ies ou exp essed copies o Pasha (Jaube -Possamai e al., 2010). A he same ime,
he b own plan hoppe (insec , Hemip e a), he all a mywo m (insec , Lepidop e a) o he
dese locus (insec , O hop e a) all ha e a single Pasha (Ghosh e al., 2014; Wynan e al.,
2015; Xu e al., 2013), which appea s he common case among a h opods when b owsing
a ailable genome da abases. Analysis o Pasha in Li openaeus annamei (sh imp) e ealed
high sequence conse a ion and nuclea localiza ion, sugges ing a well-conse ed ole in
miRNA biogenesis (Chen e al., 2012). Conse a ion o miRNA pa hway in sh imps is u -
he suppo ed by equi emen o D osha, Dice 1 and Ago1 o p oduc ion o i al RNAs
inin ec edsh imps(HeandZhang,2012;Huange al.,2012).
Dice
D osophila u ilizes wo Dice p o eins (Fig. 2), Dice -1 (DCR-1) and Dice -2 (DCR-2),
whicha ededica ed omiRNAandRNAipa hways, espec i ely(Leee al.,2004).This
makes D osophila (and a h opods in gene al) unique among he e iewed me azoan model
sys ems (Fig. 3), which employ a single Dice p o ein p oducing mul iple classes o small
RNAs (miRNAs, endo-siRNAs, exo-siRNAs). Sepa a ion o miRNA and RNAi a Dice
le el could ha e an ad an age in e ms o uncoupling an agonis ic e olu iona y o ces ac -
ing on Dice , i.e. (i) selec i e p essu e on conse a ion o he miRNA pa hway machine y
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and (ii) hos -pa hogen a ms ace whe e Dice e ol es o a oid i al p o eins in e e ing
wi h i s unc ion.
Thedomaino ganiza iono D osophila Dice p o eins is gene ally he same as in o he
me azoan Dice p o eins – hey a e composed o domains o de ed om he N- o he C- e -
minus as ollows: N- e minal helicase domains, a domain o unknown unc ion DUF283,
PAZdomain,RNaseIIIaandRNaseIIIbdomains,and heC- e minaldsRBD(Fig.2).As
o o he me azoan Dice s, D osophila Dice p o eins ha e no been c ys allized ye bu
hei s uc u e can be in e ed om biochemical s udies o ecombinan Dice and indi id-
ualdomains(Tsu sumie al.,2011;Yee al.,2007), hec ys als uc u eo Gia dia in es-
inalis Dice (MacRae e al., 2007; MacRae e al., 2006), domain modelling o c yo-EM
s udies (Lau e al., 2012).
Dice -1
Dice -1waso iginallyiden i iedasoneo  wohomologsinD osophila, which was able
o p oduce siRNAs in i o and pa icipa ed in RNAi (Be ns ein e al., 2001). Subsequen
analysis o Dice mu an s showed ha mu a ion in dice -1 blocked p ocessing o miRNA
p ecu so s while dice -2 mu an s we e de ec i e o p ocessing siRNA p ecu so s (Lee
e al., 2004). Howe e , consis en wi h he ini ial s udy, Dice -1 was also implica ed in
RNAi (Lee e al., 2004). Biochemical analysis o Dice -1 showed ha i s unc ional co e
consis so aDuF283domain,aPAZdomain,and woRIIIdomains(Yee al.,2007).
Wi h espec o he size o clea age p oduc s, Dice -1 appa en ly does no di e om o he
me azoan Dice s, as he ypical p oduc size is 22 n long (Fig. 4). DCR-1 also unc ions in
biogenesis o mi on class o miRNAs (Okamu a e al., 2007).
Dice -1di e s omDice -2insubs a especi ici iesandATP equi emen s(Jiange al.,
2005).LikehumanDice ,Dice -1gene a essmallRNAsinanATP-independen manne 
(Jiange al.,2005),whe easDice -2o Dice -2/R2D2 equi edATPhyd olysis o e icien 
siRNA p oduc ion (Liu e al., 2003). Dice -1 shows a p e e ence o p e-miRNAs (Jiang
e al.,2005;Tsu sumie al.,2011).I  ecognizes hesingle-s anded e minalloops uc u e
o p e-miRNAs h ough i s N- e minal helicase domain, checks he loop size and measu es
hedis ancebe ween he3′o e hangand he e minalloop– hisallowsDice -1 oinspec 
heau hen ici yo p e-miRNAs uc u es(Tsu sumie al.,2011).
Figu e 2 Compa ison o C. elegans, human and D osophila Dice p o eins
Domain composi ion was adop ed om (Jaskiewicz and Filipowicz, 2008).
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In e ms o e olu iona y di e si y o Dice -1 in a h opods, as men ioned abo e, he
miRNA pa hway seemed o expand in pea aphid (insec , Hemip e a) which u ilizes wo
ac i e copies o Dice 1 (Jaube -Possamai e al., 2010; O iz-Ri as e al., 2012). Howe e ,
hisduplica ionisa ela i ely ecen e en whilesingleDice -1wasalsoiden i iedelse-
whe e among a h opods (Jaube -Possamai e al., 2010; O iz-Ri as e al., 2012), including
sh imp (Su e al., 2008), mosqui o (Be nha d e al., 2012), cock oach (Gomez-O e and
Belles, 2009) o locus (Wynan e al., 2015) species.
Dice -2
Dice -2 in D osophila is mainly p oducing siRNAs om long dsRNA and unc ions in
RNAi and an i i al de ense (Galiana-A noux e al., 2006; Kim e al., 2006). Dice -2 has
ac ually a dual ole in an i i al de ense – apa om RNAi, i has an RNAi-independen
oleinp omo ingTollsignalling(Wange al.,2015b),bu biologicalaspec so Dice -2 ole
will be co e ed la e in he ex .
unlikemammalianDice o Dice -1pa alog,Dice -2 equi esATP o p ocessi eclea -
age o dsRNA (Liu e al., 2003; Nykanen e al., 2001; P o os e al., 2002; Welke e al.,
2011;Zhange al.,2002).Rema kably,analysiso shapeso amammalianDice andDic-
e -2 by c yo-EM yielded an L-shaped econs uc ion wi h dimensions s ikingly simila o
hoseo  hehumanenzyme(Laue al.,2012).The e o e,despi es iking unc ionaldi e -
encesinATP equi emen andsubs a ep e e ence, heo e all h ee-dimensionala chi ec-
u e o Dice is well conse ed (Lau e al., 2012).
Dice -2con ainsanN- e minalhelicasemo i andhyd olyzesATP;ATPhyd olysisis
equi ed o Dice -2 o p ocess long dsRNA, bu no p e-miRNA (Cenik e al., 2011). Dic-
e -2wo ksasadsRNA-s imula edATPase ha hyd olyzesATP oADP;andi wassugges -
ed ha Dice -2helicasedomainusesATP ogene a emanysiRNAs omasinglemolecule
o dsRNA be o e dissocia ing om i s subs a e. (Cenik e al., 2011).
Thehelicasedomaino Dice -2alsogo e nssubs a e ecogni ionandclea agee i-
ciency h ough disc imina ing among dsRNA ends. Fi s , i was shown ha he helicase
domain is essen ial o clea ing dsRNA wi h blun o 5’-o e hanging e mini, bu no hose
wi h 3’ o e hangs, as in p e-miRNAs (Welke e al., 2011). Subsequen ly, i was ound ha
he disc imina ion o e mini akes place du ing ini ial binding (Sinha e al., 2015). In he
absenceo ATP,Dice -2binds3′o e hanging(p e-miRNA-like),bu no blun  e mini.
in hep esenceo ATP,Dice -2bindsbo h ypeso  e mini,wi hhighes -a ini ybinding
obse ed wi h blun dsRNA (Sinha e al., 2015).
An impo an ac o in subs a e disc imina ion and p ocessing is ino ganic phospha e,
which inhibi s Dice -2 clea age o p e-miRNAs, bu no long dsRNAs (Cenik e al., 2011).
I wasp oposed ha  heino ganicphospha eoccupiesaPAZdomain5’phospha ebinding
pocke equi ed o bind he 5’ e minal phospha e o sho subs a es, blocking hei use and
es ic ingp e-miRNAp ocessingin lies oDice -1(Fukunagae al.,2014).Bindingo 
longdsRNAisno inhibi edwhen heino ganicphospha eoccupies hePAZdomainbind-
ing pocke because i also in ol es he helicase domain and/o he cen al dsRNA-binding
domain, which migh be combined wi h displacemen o he ino ganic phospha e om i s
binding pocke (Fukunaga e al., 2014)
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In e ms o e olu iona y di e si y o Dice -2 in a h opods, mos species seem o use only
one Dice -2 bu some unde wen duplica ion, such as Daphnia (C us acea, wo Dice -2
pa alogs) o Me aseiulus (Chelice a a, i eDice -5pa alogs)(Palme andJiggins,2015).
Among he expe imen ally app oached species, one Dice -2 was epo ed in expe imen al
esul s om silk mo h (Kolliopoulou and Swe e s, 2013), mosqui o (Lege e al., 2013),
cock oach(Lozanoe al.,2012),Hessian ly(KolliopoulouandSwe e s,2013),plan hop-
pe (Zhange al.,2013),eme aldashbo e (Zhaoe al.,2015),mi e(Hoye al.,2016),
bumble bee (Niu e al., 2016), o sh imp (Niu e al., 2016).
dsRBPs in a h opods – R2D2 and LOQS homologs
D osophila also u ilizes Dice pa ne dsRBPs wi h andemly a anged dsRBDs – Loqua-
cious(LOQS)andR2D2.The i s Dice pa ne dsRBPinD osophila is Loquacious, which
was ound o associa e wi h Dice -1, sugges ing ha he miRNA pa hway in D osophila
employs a dis inc dsRBP in subs a e ou ing (Fo s emann e al., 2005; Sai o e al., 2005).
Howe e , i was also ound ha Dice -2-gene a ed siRNAs in he endogenous RNAi pa h-
way depend p e e en ially on Loquacious and no on R2D2, he canonical Dice -2 pa ne
Figu e 3 Me azoan Dice phylogeny
The un oo ed ee shows phylogene ic ela ionships o Dice p o eins in Me azoa. The blue ame de-
pic s Dice 2 homologs in a h opods. As he leng h o each b anch indica es e olu iona y dis ance (o
sequence di e gence), i is appa en ha a h opod’s Dice 2 p o eins ac ing in RNAi a e e ol ing a
much as e pace han Dice 1 p o ein, which unc ion in he miRNA pa hway. This is consis en wi h he
abo e-men ioned no ion o an agonis ic e olu iona y o ces ac ing on Dice whe e he miRNA pa hway
unc ionali y is being conse ed while he RNAi unc ionali y is e ol ing du ing he hos -pa hogen a ms
ace whe e Dice e ol es o a oid i al p o eins in e e ing wi h i s unc ion.
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(Czech e al., 2008). i u ned ou ha Loquacious gene ac ually p oduces h ee p o ein
iso o ms , which associa e wi h Dice -1 and miRNA pa hway (LOQS-PA and LOQS-PB
iso o m) and Dice -2 and RNAi (LOQS-PD iso o m) (Fukunaga e al., 2012; Ha ig e al.,
2009;Miyoshie al.,2010a;Zhoue al.,2009).
LOQS-PB uses he second dsRNA-binding domain o bind p e-miRNA and he hi d
dsRNA-binding domain o in e ac wi h Dc -1. Bo h domains o LOQS-PB a e equi ed
o e icien miRNAp oduc ionbyenhancing hea ini yo Dc -1 o p e-miRNA(Ye
e al., 2007).
LOQS-PD and R2D2 unc ion sequen ially and non- edundan ly in he endogenous
RNAi pa hway. LOQS-PD s imula es DCR-2-media ed p ocessing o dsRNA whe eas
R2D2 ac s downs eam du ing RISC loading (Ha ig and Fo s emann, 2011; Ma ques e al.,
2010;Miyoshie al.,2010a).Taken oge he ,LOQSandR2D2con ibu e o hep o ound
mechanis ic sepa a ion o miRNA and RNAi pa hways, which e ol ed in D osophila (and
p esumably in a h opods in gene al).
R2D2associa eswi hDice -2andac sinRNAi;i wasco-pu i iedwi hDice -2du ing
pu i ying siRNA-gene a ing ac i i y om D osophila S2 cell lysa es (Liu e al., 2003).
Al hough R2D2 bea s 33% simila i y o RDE-4 (see he sec ion Nema oda) i s ole is di -
e en .R2D2doesno in luenceDCR-2enzyma icac i i y(Liue al.,2003)bu  es ic s
Dice -2 unc ion o p ocessing o long dsRNAs (Cenik e al., 2011; Fukunaga e al., 2014). I
also acili a es passing he clea age p oduc o AGO2 excluding miRNA-like duplexes wi h
impe ec basepai ing(Toma ie al.,2004a).R2D2has wo oles–i issensingsiRNA he -
modynamic asymme y o s and selec ion and i is a licensing ac o o en y o au hen ic
siRNAsin o heRNAipa hway(Nishidae al.,2013;Toma ie al.,2004b).
Unlike he Mic op ocesso complex, Dice o A gonau es, dsRBPs seem unde go a ious
unc ionaladap a ionsbe weendi e en  axons(compa e, o exampleTARBP2,RDE-4,
R2D2o LOQS).Thispossiblyalsohappensamonga h opods.Anexampleis helacko 
he RNAi-associa ed LOQS-PD iso o m ou side D osophila (Haac e al., 2015). Analysis o
dsRBPs in he mosqui o Aedes aegyp i e ealed absence o LOQS-PD iso o m, conse ed
oles o R2D2 and LOQS-PB, and LOQS-PA ole in biogenesis o bo h, miRNAs and
endo-siRNAs (Haac e al., 2015).
A gonau e p o eins
AGO p o eins o a h opods a e conse ed, i.e. hei domain composi ion is he same as ha
o mammalian p o eins, which was discussed in de ail (Pe e s and Meis e , 2007). A h o-
pods ha e a ying numbe o A gonau e p o eins o he AGO and PIWI clade bu i seems
ha hei a che ypal s a e is wo AGO p o eins, each being associa ed wi h one Dice and
one ype o small RNA (Palme and Jiggins, 2015).
AGO1 and i s loading wi h miRNAs
AGO1 RISC loading is simila o ha o human RISC assembly desc ibed ea lie (Yoda
e al., 2010). Dice -1/LOQS-PB he e odime unc ions in assembling AGO1 RISC, which
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is p e e en ially loaded wi h miRNA/miRNA* duplexes while siRNAs a e being excluded
om heassembly(Toma ie al.,2007).
AGO1 equi esATP o miRISCloading,p esumably o igge  hedynamiccon o ma-
ional opening o AGO p o eins so ha hey can accep small-RNA duplexes (Kawama a
e al., 2009). Unwinding o miRNA-miRNA* duplexes is a passi e p ocess ha does no
equi eATPo slice ac i i yo Ago1(Kawama ae al.,2009).
Twodis inc AGOcomplexeswe eiden i ied(Miyoshie al.,2009):(i)AGO1-Dice -1
complex wi h p e-miRNA p ocessing ac i i y whe e he esul an ma u e RNA was loaded
on o AGO1 wi hin he complex – his complex co esponds o miRLC (miRISC loading
complex) (ii) he AGO1-GW182 complex wi h excluded DCR-1, con aining ma u e miR-
NA no p e-miRNA p ocessing ac i i y – his complex co esponds o miRISC. AGO1 load-
ing also in ol es R3D1-L, a dsRBP ha unc ions as a co ac o in e ac ing wi h Dice -1 and
AGO1 (Jiang e al., 2005).
AGO1 migh also ha e miRNA-independen ole in ansla ional ep ession whe e AGO1
is ec ui ed o mRNA ia an RNA-binding p o ein SMAUG and no h ough miRNA:mRNA
in e ac ion (Pinde and Smibe , 2013).
AGO2 and i s loading wi h siRNAs
Themodelo RNAiRISCloadinginD osophila sugges s ha RISC assembly occu s in
se e als eps,whichin ol ease e alcomplexes(Toma iandZamo e,2005).The i s 
complex is o med by siRNA, R2D2 and DCR-2, also known as R1 o R2/D2/DCR-2 ini-
ia o (RDI) complex (Kim e al., 2007; Pham e al., 2004), which de elops in o a ma u e
o mo  heRISCloadingcomplexRLC(Toma iandZamo e,2005).TheRLCde e mines
s and selec ion and ec ui s AGO2 (and o he p o eins) o o m p e-RISC (Kim e al.,
2007), which con ains duplex siRNA. Finally, he elease o he passenge s and om he
duplex p oduces holo-RISC, which can base pai wi h complemen a y mRNA subs a es.
Theloadingisassis edbyHsc70/Hsp90chape ones(Iwasakie al.,2015;Miyoshie al.,
2010b).
Thecouplingo dsRNAclea ageandRISCassemblyisama e o deba e.I wassug-
ges ed ha , a e clea age, small-RNA duplexes need o dissocia e om Dice and hen
ebind o a senso o he he modynamic asymme y o he duplex, because he guide s and
o ansiRNAwillbea  andomo ien a ion(Toma ie al.,2004b).
AGO2 equi esATP o RISCloading(Kawama ae al.,2009;Phame al.,2004;Toma i
e al.,2004a).ATPisp esumablyused o igge  hedynamiccon o ma ionalopeningo 
AGO p o eins so ha hey can accep small-RNA duplexes (Kawama a e al., 2009).
S andselec ionin lyRLCiscon olledbyR2D2.Analysiso  hein e ac iono DCR-
2/R2D2 complex wi h siRNA duplexes showed ha R2D2 o ien s he complex acco ding
o he modynamic s abili ies o siRNA s ands and binds he 5’ phospha e o he passen-
ge s anda  he he modynamicallymo es ableend(Toma ie al.,2004b).Thus,R2D2
unc ions as a licensing ac o o ou ing siRNAs in o he RNAi pa hway. In e es ingly,
a ho ough analysis o AGO2 complexes e ealed ha , unlike ma u e miRNAs, which a e
loadedonAGO1,complemen a ys andso ma u emiRNAs(miRNA*)a ee icien ly
loaded on AGO2 in DCR2/R2D2-dependen manne (Ghildiyal e al., 2010; Okamu a e al.,
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2011).Thus, he oleo R2D2inso ingsmallRNAsiswide andex endsin o hemiRNA
pa hway.
The inals epinassemblyo anac i eRISCis he eleaseo  hepassenge s and om
he siRNA duplex. D osophila is A mi age helicase is a candida e o a mechanism sepa a -
ing he wosiRNAs andswhile heguide emainsbound oAGO2(Toma ie al.,2004a).
Howe e , expe imen al da a suppo a simple solu ion whe e passenge s and clea age by
AGO2 slice ac i i y libe a es he single-s anded guide siRNA s and om he p e-RISC
complex (Kim e al., 2007; Ma anga e al., 2005; Miyoshi e al., 2005). Remo al o siR-
NA passenge s and clea age p oduc s is assis ed by C3PO endo ibonuclease, which was
iden i iedasaRISC-enhancing ac o  ha p omo esRISCac i a ion(Liue al.,2009).
Theclea age-assis edmechanismis ypical o AGO2-loaded lyandhumansiRNAsin
he RNAi pa hway while passenge s and clea age is no impo an o loading miRNAs
(Ma anga e al., 2005).
Small RNA so ing and mRNA a ge ing by AGO1 and AGO2
D osophila so s Dice -p oduced small RNAs on o unc ionally dis inc AGO p o eins
whe e AGO1 is dedica ed o he miRNA pa hway while AGO2 se ed o RNAi. Small
RNA so ing is ini ia ed by subs a e ecogni ion and con inues h ough so ed loading on o
he AGO p o eins. Small-RNA duplexes a e ac i ely so ed in o AGO-con aining com-
plexesacco ding o hei in insics uc u es(Fo s emanne al.,2007;Toma ie al.,2007).
Impo an ly, sepa a ion o miRNA and RNAi a he le el o small RNA so ing on o AGO1
andAGO2isno comple elyp e-de e minedbysmallRNAo igins(Toma ie al.,2007).I 
was ound ha miRNA*s a e o en loaded as unc ional species in o AGO2 (Czech e al.,
2009; Ghildiyal e al., 2010; Okamu a e al., 2009). Fu he mo e, miRNAs p oduced by
Dice -1 and LOQS can be loaded by Dice -2 and R2D2 in o an AGO2 RISC (Fo s emann
e al., 2007). Finally, siRNAs de i ed om long hai pin RNA genes (hpRNA) also show
a hyb id biogenesis combining RNAi ac o s DIce -2 and AGO2 and Loquacious iso o m
(Okamu a e al., 2008c).
Subsequen ly, AGO2-RISC media es RNAi while only AGO1 is able o ep ess mRNAs
wi h cen al misma ches in miRNA-binding si es (Fo s emann e al., 2007). A he same
ime,AGO1canno media eRNAi,becausei isanine icien nucleasewi haca aly ic
a e limi ed by he dissocia ion o eac ion p oduc s (Fo s emann e al., 2007). AGO1 and
AGO2 RISCs also di e in mechanisms o ansla ional ep ession – AGO1-RISC ep ess-
es ansla ionp ima ilybyATP-dependen deadenyla ionwhileAgo2-RISCcompe i i ely
blocks he in e ac ion o eIF4E wi h eIF4G and inhibi s he cap unc ion (Fukaya e al.,
2014; Iwasaki e al., 2009). AGO1-media ed ansla ional ep ession in ol es GW182 in
he same manne as in mammals (GW182 is sepa a ely desc ibed u he below). miR-
NA-media edsilencingin ol es ec ui men o PABP,CCR4-NOTdeadenylaseand
decapping complex o RISC (Behm-Ansman e al., 2006; Chekulae a e al., 2011; Eulalio
e al.,2008;FukayaandToma i,2011;Hun zinge e al.,2010;Hun zinge e al.,2013;
Mo e i e al., 2012; Rehwinkel e al., 2005). miRNA-media ed ep ession occu s on ibo-
some complexes bu is independen o ibosomal scanning(An ic e al., 2015; Kuzuo-
glu-Oz u k e al., 2016).
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E olu iona y pe spec i e
As men ioned abo e, he a che ypal s a e a h opod s a e is wo AGO p o eins, AGO1
and AGO2. Apa om D osophila, such a si ua ion is ound in Daphnia (C us acea)
and Me aseiulus (mi e, Chelice a a) (Palme and Jiggins, 2015). Howe e , upon de ailed
inspec ion,one equen ly inds a iabili yin henumbe o AGOp o einsac oss he
phylumo e enac osssmalle  axonomicuni s.ThemiRNApa hwayseemed oexpand
in pea aphid (insec , Hemip e a), whose genome wo exp essed copies o ago1, one o
which (ago1b) shows signs o posi i e selec ion (Jaube -Possamai e al., 2010). A he
same ime, a single AGO1 bu duplica ions o AGO2 we e ound Ixodes ( ick, Cheli-
ce a a, h ee AGO2 pa alogs), S igamia (cen ipede, My iapoda, wo AGO2 pa alogs),
Mesobu hus (sco pion, Chelice a a, six AGO2 pa alogs) o Te anychus (spide mi e,
Chelice a a, six AGO2 pa alogs) (Palme and Jiggins, 2015). Penaeus monodon (black
ige sh imp)has ou  unc ionallydi e si iedAGOpa alogs(Dechkla e al.,2008;Lee-
bonoi e al., 2015; Phe ungnapha e al., 2013; Yang e al., 2014b). Analysis o A gonau e
genes ac oss 86 Dip e an species showed ha a ia ion in copy numbe can occu apidly,
and ha  he eiscons an  luxinsomeRNAimechanisms; hissugges s ha A gonau es
unde go equen e olu iona y expansions ha acili a e unc ional di e gence (Lewis
e al., 2016).
Addi ional miRNA and RNAi ac o s
The eisala genumbe o accesso y ac o sbeyond hosedesc ibedabo e.Fo example,
a sys ema ic sc een o 40% o he genome o genes ac ing in he miRNA pa hway yielded
45 mu a ions in 24 genes and an es ima e o ~100 genes a e equi ed o execu e he miRNA
p og am (P essman e al., 2012). He e, we will desc ibe se e al addi ional ac o s, which
ha e been associa ed wi h miRNA o RNAi pa hways.
Nibble – Nibble is a 3’-5; exo ibonuclease in ol ed in imming 3’ ends o miRNAs
and piRNAs (Fel zin e al., 2015; Han e al., 2011; Liu e al., 2011; Wang e al., 2016; Yang
e al., 2014a). In he miRNA pa hway, Nibble sho ens dis inc longe miRNAs du ing
RISC assembly, yielding miRNA iso o ms ha a e compa ible wi h he p e e ed leng h o
AGO1-bound small RNAs (Han e al., 2011; Liu e al., 2011). I has been es ima ed ha
abou a qua e o miRNAs unde goes such a imming (Han e al., 2011).
HEN1 – HEN1 (Pime , Dmhen1)is an enzyme ca alyzing addi ion o a 2’-O-me hyl
g oup a he 3’ end o small RNAs (Ho wich e al., 2007; Sai o e al., 2007). While his
modi ica ionisp edominan ly oundonpiRNAsinD osophila, i was also ound on siR-
NAs and miRNAs (Abe e al., 2014; Ho wich e al., 2007; Yang e al., 2014a). Func ionally,
2’-O-me hyla ion o siRNAs loaded on AGO2 p e en s ailing and imming o siRNAs
(Ame es e al., 2010). Gene ally HEN1 and Nibble hus ha e an agonis ic ac i i ies a he
3’ end o small RNAs whe e Nibble p omo es small RNA imming while Hen1 p e en s i
(Ame es e al., 2010; Yang e al., 2014a). 2’-O-me hyla ion is also ound on selec miRNA
iso o ms and appea ed o inc ease wi h age while i s educ ion was associa ed wi h neu o-
degene a ion and sho e li e span (Abe e al., 2014).
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nucleo idyl ans e ases – ailing o sho RNAs is media ed by e minal nucleo idyl-
ans e ases, which p oduce 3’ u idyla ion o adenyla ion. PAPD4hasbeeniden i iedas
a p ima y miRNA adenyla ing enzyme in D osophila, adenyla ion did no appea o a ec
miRNA s abili y on a genome-wide scale (Bu oughs e al., 2010). Ano he non-canonical
adenylase is Wispy, which is esponsible o adenyla ion o miRNAs and biologically i
may acili a e clea ance o ma e nal miRNAs in he emb yo (Lee e al., 2014). U idyla ion
is media ed by Tailo , which is a u idylyl ans e ase ha is equi ed o he majo i y o 3’
endmodi ica ionso mic oRNAsinD osophila and p edominan ly a ge s mi on hai pins
(Reimao-Pin o e al., 2015; Wes holm e al., 2012).
GW182 – GW182 is he key co- ac o o AGO1 in miRISC. I s ole has been desc ibed
inde ailin hemammaliansec ion,hewewillb ie lyno ei skey ea u eswi h espec 
o a h opods. GW182 and i s in e ac ion wi h AGO1 we e ound o be equi ed o miR-
NA-media ed ep ession in D osophila cells (Behm-Ansman e al., 2006; Eulalio e al.,
2008; Rehwinkel e al., 2005). miRNA-media ed ep ession also equi ed he decapping
complexDCP1:DCP2andCCR4-NOTdeadenylase(Behm-Ansman e al.,2006;Reh-
winkel e al., 2005). Mul iple domains o GW182 con ibu e o miRNA-media ed ep es-
sion (Chekulae a e al., 2009; Chekulae a e al., 2011; Chekulae a e al., 2010; Eulalio
e al., 2009). Simila ly o mammals, D osophila GW182 di ec ly in e ac s wi h PABP and
CCR4-NOT(Chekulae ae al.,2011;FukayaandToma i,2011;Hun zinge e al.,2010;
Hun zinge e al., 2013; Mo e i e al., 2012).
A mi age–RNAhelicase,whichwasiden i iedasama e nale ec gene equi ed o 
RNAi(Toma ie al.,2004a).A mi ageisp obablyno  equi ed o RISCac i i y.Ins ead,
i wasp oposed o acili a e emo alo  hepassenge s anddu ingRISC o ma ion(Tom-
a i e al., 2004a). A mi age was also implica ed in piRNA biogenesis (Huang e al., 2014;
Mu o a e al., 2014; Nagao e al., 2010; Qi e al., 2011; Sai o e al., 2010).
dFMR1 – D osophila o holog o human agile X men al e a da ion p o ein (FMRP)
wasiden i iedasaRISCcomponen (Caudye al.,2002;Ishizukae al.,2002;Phame al.,
2004). dFMR1 is associa ed wi h ibosomes h ough in e ac ion wi h ibosomal p o eins
L5 and L1 and wi h complexes con aining miRNAs (Ishizuka e al., 2002). dFMR1 is no
a conse ed RISC componen in ol ed in RNAi as deple ion o dFMR1 educes RNAi
e iciencyinD osophila S2 cells bu no in mammals (Caudy e al., 2002). dFMR has been
also implica ed in he piRNA pa hway (Bozze i e al., 2015; Jiang e al., 2016).
VIG–VasaIn onicGene(Caudye al.,2002;Phame al.,2004).VIGisaconse ed
p o ein, which encodes a pu a i e RNA binding p o ein, whose deple ion educes RNAi
e iciency(Caudye al.,2002).Vig mu an s a e mo e suscep ible o i al in ec ions in
D osophila(Zambone al.,2006).Whe he  his oleo VIGiscoupledwi hi sp esencein
heRISCcomplexisno known.The eisnoe idence ha SERBP1, hecloses mammalian
VIGhomolog,wouldbeassocia edwi hRISC.VIGwasalsoimplica edinhe e och oma in
o ma ion (G ache a e al., 2009).
Tudo -SN –Tudo S aphylococcalNucleaseisap o eincon aining i es aphylococ-
cal/mic ococcalnucleasedomainsandaTudo domain.I isacomponen o  heRISCin
C. elegans, D osophilaandmammals(Caudye al.,2003;Phame al.,2004).The ole
o Tudo -SNinRISCRNAi emainsenigma ic.TSNisno  he„slice “(Schwa ze al.,
2004)andi sknock-downinsilkmo hcellshadno e ec onRNAie iciency(Zhue al.,
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e al.,2015).Thecomplexi yo smallRNAbiologywas e iewedin he i s plan RNA
silencing e iew (S oboda, 2019). He e, I will ocus on mobili y o small RNAs in plan s.
Plan ana omy and ea u es ele an o mo emen o molecules
Plan s a e unique in se e al aspec s when compa ed o cells o o he euka yo ic o ganisms.
Plan cell ea u es impo an o in e cellula exchange o molecules include a polysaccha-
idecellwallandplasmodesma a.Thela e a emic oscopicchannels a e singcellwalls
o plan cells allowing o mo emen (symplas ic mo emen o symplas ) o molecules
be ween adjacen cells ( e iewed in Maule, 2008; Maule e al., 2011). An al e na i e o he
symplas ic mo emen , which in ol es cell cy oplasm, is apoplas ic mo emen (apoplas )
whe e molecules mo e h ough cell walls and in e cellula space.
A lowe ingplan (Fig.2)consis so  h eeana omicalsys ems– oo ,shoo ,andin lo-
escence. I is ancho ed by a oo , abso bs wa e and mine als om he g ounds and ans-
po s hem h ough he xylem ascula u e h ough he plan and in o lea es, which a e he
main pho osyn he ic o gans. Suga s p oduced in lea es a e anspo ed a ound h ough he
phloem ascula u e.
Figu e 1 Complexi y o small RNAs in plan s. The scheme was adap ed om (Bo ges and Ma ienssen,
2015)
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S uc u ally, plan issues a e di ided in o me is ems (con aining ac i ely di iding cells,
husbeing hep ima yplaceo plan g ow h)andpe manen  issuesclassi iedacco ding o
hei shapes and in e cellula space.
Long dis ance mo emen o molecules occu s h ough he a o emen ioned ascula sys-
em, which anspo s wa e , mine al, signaling molecules, nu ien s, and o he molecules.
In pa icula , phloem is he main a enue o anspo ing o ganic molecules (discussed in
(A kinse al.,2011;DeScheppe e al.,2013;Tu geonandWol ,2009)).The eisala ge
numbe o mobile mac omolecules in plan s. In an e o o p o ide a sys ema ic ca alogue
o mobile mac omolecules, a da abase PlaMoM was compiled, which p o ides con enien
and in e ac i e sea ch ools allowing use s o e ie e, o analyze and also o p edic mobile
RNAs/p o eins(Guane al.,2017).Thecu en  e sioncompilesa o alo 17,991mobile
mac omolecules om 14 plan species/eco ypes and is a ailable a : h p://www.sys embi-
oin o.o g/plamom/.
Dis ibu ion o RNA silencing pa hways ac oss plan issues
Plan s exp essed mul iple AGOs, DCLs and RDRs, which suppo di e en ypes o RNA
silencing pa hways employing di e en ypes o small RNAs (Fig. 1). Plan small RNAs a e
ypically me hyla ed a he 3’ end, which p o ec s hem om deg ada ion (Li e al., 2005).
Plan RNA silencing pa hways can be di ided in o miRNA and siRNA pa hways, which use
dis inc small RNA subs a es. F om he pe spec i e o small RNA popula ions o igina ing
om di e en subs a es, wo dis inc ypes o small RNAs can be ecognized when consid-
e ing hei sequencep edic ion(Fig.3):The i s  ype,exempli iedbymiRNAs,comp ises
small RNAs, which occu in cells in many iden ical copies (i.e. wi h he same sequence).
Thesecond ype,exempli iedbysiRNAsde i ed omdsRNA,ischa ac e izedbyexis -
ence o popula ions o small RNAs wi h a iable sequences, which o igina e om a longe
sequence. Al hough hei sequences could be de e mined, hei indi idual anno a ion is
poin less. One kilobase o dsRNA heo e ically p oduces nea ly housand small RNAs di -
e ing a hei 5’ ends whe e each one o hem could egula e a di e en se o genes ( he e
is4096possibleo ahexame sequence).ThesesmallRNAsusually unc ionasade ense
sys em ope a ing on he basis (nea ly) pe ec complemen a i y.
miRNA (Fig. 4) and ela ed pa hways unc ion pos - ansc ip ionally and include he
canonical miRNA pa hway wi h 21n miRNAs (u ilizing DCL1 and AGO1) and i s al e na-
i es employing o he DCL and AGO p o eins and longe hai pin subs a es.
siRNA pa hways use ei he exogenous subs a es (an i i al de ense and ansgene silenc-
ing,Fig.5)o  a iousdsRNAsde i ed omgenomicsequences.Va iousbiogenesispa h-
ways p oduce p ima y and seconda y siRNAs o a ious ypes and leng hs (21, 22, o
24 n ), which media e pos - ansc ip ional o ansc ip ional silencing. siRNA leng hs a e
de e mined by he p ocessing Dice pa alogue. 21n siRNAs can be p oduced by DCL4 o
DCL1, 22n siRNAs by DCL2, and 24 n by DCL3.
The eisala ge olumeo li e a u econce ningNGSanalysiso smallRNApopula-
ions om di e en issues in di e en plan species. A la ge olume o NGS da a can be
accessed h ough Plan MPSS (massi ely pa allel signa u e sequencing) da abases websi e
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(h ps://mpss.dan o hcen e .o g/), which was in oduced in 2005 (Nakano e al., 2006) and
has been con inuously upda ed and expanded. I cu en ly con ains small RNA da a om 19
plan species. Mos da ase s in he da abase (six) come om analysis o small RNAs in ice.
These iceda ase scombine heexp essiona laso  icemRNAsandsmallRNAs(Nobu a
e al., 2007), wi h analysis o small RNAs du ing de elopmen whe e di e en pools o
phasiRNAswe eiden i ied(Feie al.,2016),acomp ehensi eanalysiso smallRNAsin
di e en issues unde no mal condi ions and s ess (Jeong e al., 2011) and unpublished
NGS da a om di e en ice issues and AGO immunop ecipi a es.
Theseando he da a om ice(e.g.(Heisele al.,2008)) e ealcommonand issue-spe-
ci icpopula ionso smallRNAs.Theexis enceo  issue-speci icsmallRNAsshows ha 
sys emicRNAsilencingco-exis swi hRNAsilencingmechanisms es ic ed ospeci ic is-
sues.Se e al ac o smayunde lie issuespeci ici yo pa icula smallRNAmechanisms.
Theseinclude(i) issue- es ic edexp essiono p o einsin ol edinbiogenesisandac i i y
o pa icula small RNA class, (ii) issue- es ic ed p esence o subs a es o biogenesis o
small RNAs, and (iii) es ic ion o mobili y/sp eading o small RNAs, which could di e
be weendi e en cell ypes.Nex ,Iwillb ie lydiscussselec edspeci icexamples,which
p o ide an insigh in o he complexi y o he issue:
Figu e 2 A schema ic o e iew o plan body o ganiza ion.
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Tissue- es ic ed exp ession o p o ein ac o s in ol ed
in small RNA biogenesis and ac i i y
While many genes encoding ac o s in ol ed in small RNA biogenesis (e.g. Dice s, RDRs)
and unc ion (e.g. A gonau es) o small RNAs a e ubiqui ously exp essed, some exhib-
i p e e en ial o issue es ic ed exp ession. Fo example, a sys ema ic analysis o gene
exp ession was done in A abidopsis and ice (Kapoo e al., 2008), whe e mic oa ay p o-
ilingiden i iedse e aldi e en iallyexp essed ac o sdu ingde elopmen andindi e en 
issues.Simila da acouldalsobeex ac ed oma ailableNGSp o ilingo mRNAsa 
he a o emen ioned websi e (h ps://mpss.dan o hcen e .o g/). In gene al, hese exp ession
da aha ep edic i e aluemainly o speci icA gonau epa alogs,whichhos uniquesmall
RNA pools and ha e highly es ic ed exp ession pa e ns.
Tissue- es ic ed p esence o subs a es o biogenesis o small RNAs
This ac o in ol eslocaliza iono exogenoussmallRNAsubs a es odi e en  issues
(i.e. i al in ec ions and ansgene exp ession) o exp ession o endogenous small RNA
subs a es.Thisallows, o example odi e en ia eexp essiono subpopula ionso small
RNAsin imeandspace.Aclassicexampleis issue-speci icexp essionmiRNAs,whose
p ecu so s a e ansc ibed by polII polyme ase and hus can exhibi issue- es ic ed exp es-
sion as mRNAs. As he e is a la ge olume o he li e a u e on miRNA exp ession in plan s,
which is beyond he scope o his epo , I only selec illus a i e examples o analyses o
issue-speci icexp essiono miRNAsin ice.(Mi ale al.,2013;Zhue al.,2008),Nico-
iana(Valoczie al.,2006),andA abidopsis(G an -Down one al.,2009;Valoczie al.,
Figu e 3 Dis inc ypes o small RNAs di e ing in occu ence o RNAs wi h iden ical sequences
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2006).Tissue-speci icexp essiono smallRNAscanbeobse ed o  asiRNAs,which
o igina e omspeci icloci(Ma ine al.,2010;Zabalae al.,2012)o phasiRNA,whe e
aspeci icmiRNAini ia esp oduc iono phasiRNAs(Feie al.,2016).
Res ic ion o mobili y/sp eading o small RNAs.
Res ic iono mobili yassu es ha  issue/cell-speci icexp essionwill emaincon ained.
The ea ese e alcell ypes, omwhichsmallRNAsdono seem obesp eadinga ound.
Theseincludegua dcellso s oma a(Voinne e al.,1998),endospe m(ene gys o ageo 
heseed)(Hou na de al.,2007)o  heseedcoa (Tu ejae al.,2009).
Mo emen o small RNAs be ween cells and issues – miRNA mobili y
miRNAs can also be anspo ed o e long dis ances bu his is no a uni e sal ule o
all miRNAs (Buh z e al., 2008; de Felippes e al., 2011; Knaue e al., 2013; Lin e al.,
2008). Analysis o he phloem sap o oilseed ape B assica napusiden i ied32anno a ed
mic oRNAs (miRNAs) om 18 di e en amilies (Buh z e al., 2008). In addi ion, he
le els o h ee ma u e miRNAs known o espond o nu ien dep i a ion in non- ascula
issue, MIR395 (sulpha e), MIR398 (coppe ) and MIR399 (phospha e), we e inc eased in
phloem sap du ing he g ow h o plan s unde he espec i e nu ien dep i a ion (Buh z
e al., 2008). O he known mobile miRNAs in A abidopsis a e MIR394, which is p oduced
by he su ace cell laye and con ibu es o shoo me is em o ma ion (Knaue e al., 2013),
and MIR165, which egula es di e en ia ion (Miyashima e al., 2011).
MIR395 and MIR399 mobili y h ough phloem was demons a ed in g a ing expe i-
men s while MIR171 was no anspo ed (Buh z e al., 2010; Pan e al., 2008). Consis en
wi h phloem mo emen , ano he analysis o miR399 mo emen using ecip ocal g a ing in
A abidopsis sugges ed ha i mo es om shoo s o oo s (Lin e al., 2008).
Thebasiso selec i i yunde lyingmiRNAmobili yinplan s emainsunclea .I was
shown22n a i icialmiRNAsde i ed omasymme icduplexesmedia ewidesp ead
silencingo  hei cogna egenemo ee icien ly han21n siRNAs omsymme icduplex-
es (McHale e al., 2013).
Mo emen o small RNAs be ween cells
and issues – sys emic RNAi in plan s
RNAi can ei he ac in a cell au onomous manne , i.e. a ec ing only cells di ec ly exposed
o dsRNA, o can p opaga e ac oss cell bounda ies. Ea ly obse a ions o co-supp ession
in pe unia (Jo gensen, 1995; Napoli e al., 1990) and an i i al esis ance in Nico iana ben-
hamiana (Ra cli e al., 1997) sugges ed ha RNA silencing in plan s includes a mobile
silencingsignal.Se e als udiessubsequen lycon i med ha  ansgene-inducedsilenc-
ingismobile(Palauquie al.,1997;Voinne andBaulcombe,1997;Voinne e al.,1998,
2016).Theseexpe imen susedg a ingandag oin il a ions a egies oshow ha silencing
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sp eads om he lowe silenced lea es o he uppe non-silenced lea es (Palauqui e al.,
1997;Voinne andBaulcombe,1997).Basedon hedis anceo silencingsp ead,sho  ange
and long ange/sys emic sp ead can be dis inguished (Fig. 6).
Sho ange silencing
Sho  angesilencingmani es sasRNAsilencinginade ineda ea.I wasshown ha
a sho -dis ance sp eading o RNA silencing, once ini ia ed om a small g oup o cells,
can sp ead o e 10–15 cells independen ly o he p esence o cogna e ansc ip s (Himbe
e al., 2003). Sho ange silencing was obse ed o bo h ansgenes and endogenous genes
(Dunoye e al., 2007; Kalan idis e al., 2006; Ryabo e al., 2004; Schwach e al., 2005;
Smi h e al., 2007).
Rega ding he na u e o he sho - ange mobile signal, i is s ill a ma e o deba e. Ini-
ially, i was p oposed ha sho ange sp eading in ol es DCL3-dependen 24n siRNAs
(Hamil on e al., 2002; Molna e al., 2010). Subsequen ly, DCL4-dependen 21 n siRNA
we e also implica ed in sho - ange sp eading (Dunoye e al., 2007; Dunoye e al., 2005;
Himbe e al.,2003;Smi he al.,2007).I wasshown ha  heya esu icien  o  helimi ed
mo ing o he silencing and ha he sho ange silencing is independen o an RdRP SDE1
and helicase SDE3 (Himbe e al., 2003). Fu he mo e, i was sugges ed ha he mobile
signal is a e siRNAs hemsel es an no hei longe p ecu so s and AGO-bound single
s and molecules bu his e idence came in one o he ecen ly e ac ed pape s (discussed
in Addendum a he end o he e iew).
Besides 21 siRNAs, se e al o he ypes o plan small RNAs can sp ead cell- o-cell.
TheseincludemiRNAs(discussedsepa a ely u he below), asiRNAso  heTAS3locus
(Chi wood e al., 2009; de Felippes e al., 2011; Ma in e al., 2010; Schwab e al., 2009).
In e ms o he mechanism o cell- o-cell silencing mo emen , ea ly s udies implied
ha pos - ansc ip ional silencing sp eads h ough plasmodesma a (Palauqui e al., 1997;
Voinne e al.,1998).Howe e , heamoun o e idence o cell- o-cellsilencingmo emen 
h ough plasmodesma a is a he mode a e. Suppo o he symplas ic mo emen h ough
plasmodesma a comes om an obse a ion ha symplas ically isola ed gua d cells o s o-
ma aescapesho - angesp eading(Himbe e al.,2003;Kalan idise al.,2006;Voinne 
e al., 1998). Fu he mo e, analysis o sho - ange sp eading in A abidopsis emb yos, sug-
ges ed ha he sp ead is a ec ed by and posi i ely co ela es wi h plasmodesma ape u e
(KobayashiandZamb yski,2007).While hese esul sa einag eemen wi h heassump-
ion ha sho - ange sp eading o silencing occu s h ough di usion and plasmodesma a,
al e na i e ou es, such as sec e o y esicles should s ill no be excluded.
In addi ion, gene ic analysis e ealed se e al ac o s in ol ed o cell- o-cell silencing
mo emen (summa izedinTable1).Someo  hese ac o swe eal eadymen ionedabo e.
In e es ingly, among he ac o s whose mu a ions educe cell- o-cell silencing mo emen s
we e also RDR2 CLSY1, and NRPD1a, which a e equi ed o 24n siRNA-media ed
ansc ip ional silencing (Dunoye e al., 2007; Smi h e al., 2007). I was sugges ed
ha hese ac o s migh unc ion in ecep ion and/o downs eam unc ional in eg a ion
o mobilesiRNAsin ecipien cells(B osnanandVoinne ,2011).Ano he nuclea  ac-
o whose mu a ion a ec s cell- o-cell silencing mo emen is JMJ14, a H3K4 his one
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