Introduction to RNAi and miRNA pathways

Introduction to RNAi and miRNA pathways Petr Svoboda KAROLINUM PRESS PRAGUE 2020 Introduction_to_RNAi.indd 1Introduction_to_RNAi.indd 1 09.07.20 8:3409.07.20 8:34 KAROLINUM PRESS Karolinum Press is a publishing department of the Charles University www.karolinum.cz © 2020 by Petr Svoboda First edition ISBN 978-80-246-4372-4 (pdf) https://doi.org/10.14712/9788024643724 Introduction_to_RNAi.indd 2Introduction_to_RNAi.indd 2 09.07.20 8:3409.07.20 8:34 CONTENTS 5 Preface 7 Introduction 29 RNAi and miRNA pathways in mammals I – molecular mechanisms 81 RNAi and miRNA pathways in mammals II – biological roles 119 RNAi and miRNA pathways in birds 133 RNAiandmiRNApathwaysinfish 143 RNAi and miRNA pathways in arthropods 177 RNAi and miRNA pathways in Annelids and Molluscs 191 RNAi and miRNA pathways in nematodes 215 RNAi and miRNA pathways in plants I – molecular mechanisms 255 RNAi and miRNA pathways in plants II – mobility of small RNAs 275 SpecificityoftargetinginRNAiandmiRNApathways 313 Extracellular small RNAs and their transfer between species and kingdoms Introduction_to_RNAi.indd 3Introduction_to_RNAi.indd 3 09.07.20 8:3409.07.20 8:34 Introduction_to_RNAi.indd 4Introduction_to_RNAi.indd 4 09.07.20 8:3409.07.20 8:34 https://doi.org/10.14712/9788024643724.1 5 PREFACE Small RNA pathways or RNA silencing is a group of pathways, which utilize small (20– 30nt)RNAsasguidesforsequence-specificrepression.Thiscollectionoftextsonsmall RNA pathways originates from a report I prepared for the European Food and Safety Authority (EFSA) in 2016 and 2017. EFSA requested literature survey as it was concerned because of several works reporting that small RNAs, which naturally exist in plants, can enterintoamammalianbodyandaffectgeneexpression.ThemainissuewasthatRNA interference (one of a small RNA pathways), represented a promising way for developing sequence-specificpesticides.IfplantsmallRNAscouldenterintoamammalianorganism and regulate genes, RNAi-based pesticides could represent a potential health hazard. With help of my colleagues, who set up a systematic literature search, I wrote an extensive systematic literature review for which I inspected ~10,000 titles and abstracts during oneyearofwriting.TheoriginalEFSAreporthadanumberoftechnicalsectionsdescribing searchmethodologyandwascompilinginformationfrompublishedworksforspecific tasksdefinedbyEFSA.OncethereportwascompletedandpresentedtoEFSA,Ibecame interestedinconvertingthescientificpartofthereportintoamorecoherentoverviewof RNA silencing (particularly of RNAi and microRNA pathways) across different animal groupsandplants.Ithoughtitwouldbebetterifthescientificpartwouldberevised,and provided as a coherent collection of chapters for studying RNAi and related pathways. I subsequently contacted EFSA and discussed possible options for producing a set of chapters based on the report, which could serve as a study material for my lecturing. EFSA representatives agreed that I could produce a set of study materials from the report that would be published by a university publishing house under conditions that EFSA would be credited and the book would not be sold – as a solution, the material is provided as an openaccesscollectionofchapters.TosatisfythesecondEFSArequirement,Iacknowledge hereby that the contents have been produced under a contract with EFSA (OC/EFSA/ GMO/2015/01-CT01)andthattheopinionsexpressedarethoseofthecontractoronlyand donotrepresentEFSA’sofficialposition. Regarding the conversion, the original text was reorganized into twelve chapters, which were reformatted and revised in order to remove copyrighted material from third parties and provide a introductory parts for stand alone chapters. Nine of the chapters focus on smallRNApathwaysinanimals(mammals,birds,fish,arthropods,nematodes,molluscs, andannelids)andplants.Theremainingthreechaptersincludeageneralintroductionand reviews of important phenomena – off-targeting and extracellular small RNAs. I hope that this collection will serve as a useful source for many. Petr Svoboda Introduction_to_RNAi.indd 5Introduction_to_RNAi.indd 5 09.07.20 8:3409.07.20 8:34 Introduction_to_RNAi.indd 6Introduction_to_RNAi.indd 6 09.07.20 8:3409.07.20 8:34 https://doi.org/10.14712/9788024643724.2 7 INTRODUCTION Introduction Keywords:dsRNA,siRNA,miRNA,Dicer,TARBP2,PACT,Argonaute PETRSVOBODA Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska1083,14220Prague4,CzechRepublic Correspondence to: Petr Svoboda, Institute of Molecular Genetics ASCR, Videnska1083,14220Prague4,CzechRepublic,tel.#+420241063147, e-mail: [email protected]. ABSTRACT RNAsilencingdenotesagroupofpathways,whichutilizesmallRNAsassequence-specificguidesforrepressing geneexpression.TworelatedRNAsilencingpathwaysexistinanimalsandplants:RNAinterference(RNAi)and microRNA (miRNA) pathway. While the miRNA pathway regulates endogenous protein-coding gene expression, RNAi serves as a form of innate immunity targeting viruses and mobile elements, although it occasionally also acquiredfunctioninprotein-codinggeneregulation.Theaimofthefollowingtextistoprovideanelementary introductionintoRNAiandmiRNApathwaysforaseriesoftaxon-specificandfeature-specificreviews,which follow.Theideaistobringupcommongeneralprinciplesallowingthereadertobetternavigatethroughcommon andderivedmechanismsandfunctionsofRNAsilencingthatarepresentedintaxon-orientedreviews.Theentire review series was derived from an expert report for the European Food and Safety Agency, which was reorganized tobemoreaccessibleforthescientificcommunity. Introduction to mechanistic principles and roles of RNA silencing ThevolumeoftheRNAsilencinganddouble-strandedRNA(dsRNA)-relatedpublished data is stunning. In 2016, me and my colleagues did literature assessment for the European FoodandSafetyAgency,whichidentifiedover200000publications(Pacesetal.,2017) and which served as a foundation for this article series. While selected taxons are reviewed separately,Ithoughtthecollectionwouldbenefitfromintroducingthecoremolecular mechanisms of RNAi and miRNA pathways (admittedly animal-centric). As mentioned in the abstract, RNA silencing (reviewed in Ketting, 2011) designates repression guided by small RNA molecules (20–30 nucleotides long) and includes diverse silencing mechanisms including RNA degradation, translational repression, induction of repressive chromatin, and even DNA deletions. RNA silencing research evolved from parallelstudiesinseveraldifferentmodelsystems,primarilyfloweringplantmodelsandanimal Introduction_to_RNAi.indd 7Introduction_to_RNAi.indd 7 09.07.20 8:3409.07.20 8:34 INTRODuCTION 8 models including Caenorhabditis elegans, Drosophila melanogaster,zebrafish,mouse,and humans. Some form of RNA silencing exists in almost every eukaryote. Here, the primary focus will be on the RNA interference (RNAi) and microRNA (miRNA) pathways (Fig. 1 and2).ThetermRNAihasbeenoriginallyusedforsequence-specificmRNAdegradation inducedbylongdsRNA(Fireetal.,1998).Thismechanism,whichemployssmallRNAs produced from long dsRNA, is the canonical RNAi. However, the term RNAi is also used as a common name for a broad range of RNA silencing pathways (Ketting, 2011). Here, I will use the term RNAi strictly in its original connotation. miRNAs are genome-encoded short RNAs that regulate gene expression by translational repression and/or degradation of cognate mRNAs. Historically,thefirstdiscoveredRNAsilencingpathwaywasplantco-suppression, whichappearedassequence-specificsilencingofendogenousgenesinducedbytransgeneexpression(Napolietal.,1990).ThemiRNApathwaywasfirstfoundin1993in Figure 1 RNAi pathway overview Canonical RNAi is triggered by some form of long dsRNA. dsRNA can originate from various sources including viruses and their replication intermediates or base pairing if RNAs transcribed in the genome (either as an intramolecular duplex (hairpin dsRNA), or by base pairing RNAs transcribed in cis (convergent transcription) or in trans (from interspersed elements, pseudogenes etc.). The core mechanism of RNAi has three steps: dicing – cleavage of long dsRNA into siRNA duplexes by RNase III Dicer, loading – where one strand of siRNA duplex is selected and loaded onto an Argonaute protein from AGO subfamily forming the RNA-induced silencing complex (RISC), and slicing – where siRNA guides RISC to cognate RNAs. Upon making a perfect duplex with a cognate RNA, AGO proteins performs endonucleolytic cleavage of the cognate RNA in the middle of the base-paired sequence. In some species, RNAi also involves an RNA-dependent RNA polymerase (RdRp), which may generate initial substrates or participate in amplification of the response by converting cognate RNAs into dsRNA. Introduction_to_RNAi.indd 8Introduction_to_RNAi.indd 8 09.07.20 8:3409.07.20 8:34 INTRODuCTION 9 the nematode Caenorhabditis elegans(Leeetal.,1993).TheideaofaconservedmiRNA pathway emerged upon discovery of Let-7 miRNA in 2000, which was is conserved from Caenorhabditis elegans to mammals (Pasquinelli et al., 2000). In the meantime, RNAi was found in Caenorhabditis as well (Fire et al., 1998). Around the year 2000, it became apparent that earlier observations, which included the aforementioned plant co-suppression, quelling in fungi, and animal RNAi and miRNA pathways (Lee et al., 1993; Napoli et al., 1990; Romano and Macino, 1992; van der Krol et al., 1990), belong to one group of related molecular mechanisms commonly called RNA silencing. ThecoreprincipleofRNAsilencing(repressionmediatedbyaribonucleoproteincomplex guided by a small RNA) was deciphered during 1998–2004 using a combination of genetic and biochemical approaches. Key steps in understanding how RNA silencing works were biochemical studies in Drosophilaembryolysates(Tuschletal.,1999;Zamoreetal., 2000) and genetic studies in Caenorhabditis elegans and plants (e.g. (Bohmert et al., 1998; Dalmay et al., 2000; Fagard et al., 2000; Grishok et al., 2000; Lynn et al., 1999; Mourrain etal.,2000;Smardonetal.,2000;Tabaraetal.,1999)).Thelastdiscovery,whicharguably closed the era of deciphering the key principles of RNA silencing, was the structural Figure 2 Canonical animal miRNA pathway overview miRNAs are genome-encoded. Their synthesis starts with Pol II-mediated transcription of long primary miRNA transcripts (pri-miRNAs), which carry one or more local short hairpins, which are released as precursor miRNAs (pre-miRNAs) by the activity of the nuclear “Microprocessor complex”. Pre-miRNAs are transported into the cytoplasm via Exportin 5. In the cytoplasm, Dicer cleaves a pre-miRNA and one strand of the duplex is loaded onto an AGO protein, which forms the core of the effector complex (RISC or miRISC). The effector complex contains additional proteins, which mediate translational repression and RNA degradation. The key bridge between AGO and proteins mediating deadenylation and decapping is GW182 protein. Targeted mRNAs usually localize to P-bodies, which are cytoplasmic foci associated with RNA metabolism Introduction_to_RNAi.indd 9Introduction_to_RNAi.indd 9 09.07.20 8:3409.07.20 8:34 INTRODuCTION 16 Table 2 Overview of Argonaute proteins and associated RNAs in key model organisms The table was compiled form the following literature (Batista et al., 2008; Buckley et al., 2012; Das et al., 2008; Duran-Figueroa and Vielle-Calzada, 2010; Fischer et al., 2011; Forstemann et al., 2007; Iwasaki et al., 2015; Liu et al., 2009; Tijsterman et al., 2002a; Tijsterman et al., 2002b; Vasale et al., 2010; Vourekas et al., 2012; Wang and Reinke, 2008; Yigit et al., 2006; Zhang et al., 2016; Zheng et al., 2007). Slicer activity “+” indicates that a given Argonaute protein has potential to act as a slicer, not that slicing is its primary mode of action. In some case, slicing potential has been inferred from the sequence, i.e. it is not supported with experimental evidence. Introduction_to_RNAi.indd 16Introduction_to_RNAi.indd 16 09.07.20 8:3409.07.20 8:34 INTRODuCTION 17 step, in which RdRPs generate secondary siRNAs and (2) systemic RNAi where an RNAi response can spread across cellular boundaries. Because dsRNA often originates from viruses, the role of RNAi has been viewed as a form of native immunity. While this role is experimentally supported in some models, RNAi may also have other roles in maintaining genome integrity, and control of gene expression. RNA viruses generate dsRNA during their replication cycle in host cells. DNA viruses often produce complementary sense and antisense transcripts, which can form dsRNA upon annealing.Thus,dsRNAisacommonmarkerofviralinfectionanditisrecognizedby differentmechanismsmediatinganinnateimmuneresponse.TheideathatRNAsilencing may function as a form of innate immunity is supported by several lines of evidence, which werefirstfoundinplantsandlateralsoininvertebrates(reviewedinMarquesandCarthew, 2007; Xie and Guo, 2006): 1) siRNAs derived from viral sequences were found in infected organism (Hamilton and Baulcombe, 1999), 2) inhibition of RNA silencing resulted in increased viral replication (Mourrain et al., 2000), and 3) some viruses produce suppressors ofRNAsilencing(Voinnetetal.,1999). TheroleofRNAivariesamongdifferentorganisms.Vertebratesreplacedtheantiviral defense system provided by RNAi by an array of innate immune sensors of dsRNA molecules,whoseactivationconvergesonasequence-independentinterferonresponse.Thus, the canonical RNAi is generally not a ubiquitous primary mechanism in response to dsRNA invertebratesalthoughitisobservedinspecificcases.Inspecies,whichstilluseRNAias the primary antiviral immunity pathway (such as plants, nematodes, arthropods), it is frequently observed that viruses overcome the RNAi response with various protein inhibitors. Whether the more complex interferon system in vertebrates provides a stronger defense barrier is unclear as the interferon pathway is just a part of a highly complex immune system. One interesting aspect of RNAi and interferon response evolution is the rewiring of the RIG-I helicase family, which is associated with RNAi in Caenorhabditis elegans and interferon response in mammals. Understanding the role of the RNAi module in immunity of molluscs and annelids requires further research. Molluscs are a particularly interesting case, because their genome carries homologs of the genes involved in the interferon response, and studying them might provide an insight into how the interferon response has replaced RNAi, as the main antiviral response. Systemic and environmental RNAi RNAi can either act in a cell autonomous manner, i.e. affecting only cells directly exposed todsRNA,orcanpropagateacrosscellboundaries.Twomodesofnon-cellautonomous RNAi are recognized: (1) environmental RNAi involves processes where dsRNA is taken up by a cell from the environment. (2) systemic RNAi includes processes where a silencing signal spreads from a cell across cellular boundaries into other cells. Both modes can be combinedandsystemicRNAicanfollowenvironmentalRNAi.TwopathwaysfordsRNA uptakeweredescribed:(1)aspecifictransmembranechannel-mediateduptakeand(2)an alternative endocytosis-mediated uptake (reviewed in Huvenne and Smagghe, 2010; Whangbo and Hunter, 2008). Introduction_to_RNAi.indd 17Introduction_to_RNAi.indd 17 09.07.20 8:3409.07.20 8:34 INTRODuCTION 18 Thenon-cellautonomousRNAiwasobservedalreadyduringthefirstRNAiexperiments in Caenorhabditis elegans (Fire et al., 1998). When animals were microinjected with dsRNA into head, tail, intestine or gonad arm, or even just soaked in dsRNA solution or fed bybacteriaexpressingdsRNA,thesetreatmentsinducedaspecificnullphenotypeinthe whole animal and even in its progeny, demonstrating a surprising ability of dsRNA to cross cellularboundaries(Fireetal.,1998;Tabaraetal.,1998;TimmonsandFire,1998).Noncell autonomous RNAi has been discovered also in parasitic nematodes (Geldhof et al., 2007), hydra (Chera et al., 2006), planaria (Newmark et al., 2003; Orii et al., 2003), insects (Tomoyasuetal.,2008;XuandHan,2008),orplants(Himberetal.,2003). miRNA pathway unlikesiRNAs,miRNAsaregenome-encodedshortRNAswithdefinedsequencesthatregulate gene expression by mediating translational repression and/or degradation of cognate mRNAs. miRNAs play important roles in many processes and are one of the most common small RNAs found in animal and plant cells. miRNAs have been implicated in countless cellular and developmental processes; in some cases are changes in their expression linked to pathological conditions. Bioinformatics estimates suggest that miRNAs might directly target over 60% of mammalian genes (Friedman et al., 2009); miRNA-dependent regulation in invertebrates and plants are less extensive. ThousandsofmiRNAshavebeenannotated.ThecentralmiRNAdatabasemiRBase (http://www.mirbase.org,(KozomaraandGriffiths-Jones,2014)includes2654human, 1978 murine, 469 Drosophila melanogaster , 437 Caenorhabditis elegans, and 428 Arabidopsis thaliana mature miRNAs (release 22.1). Remarkably, there are only a few miRNAs conserved between Drosophila and mammals and it is not clear if there are any conserved miRNA genes between plants and animals. Animal miRNAs seem to emerge from random formation of Drosha/Dicer substrates (discussed in detail in (Svoboda and Cara, 2006). Newly evolving miRNAs likely form a considerable portion of annotated miRNAs, especially in species where miRNAs were intensely studied by next generation sequencing (NGS),whichcanidentifylow-abundancemiRNAs.ThenewlyemergingmiRNAseither acquiresignificantrepressivefunctionsandbecomeretainedduringevolutionorthey become lost. Furthermore, target repertoire of individual miRNAs can evolve fast since a single point mutation can weaken an existing regulation or create a new one. Animal miRNAs biogenesis starts with long primary transcripts (pri-miRNAs), which areprocessedbythenuclear“Microprocessor”complex,intoshorthairpinintermediates (pre-miRNAs). Pre-miRNAs are transported to the cytoplasm where they are further processed by Dicer into a small RNA duplex, from which is one RNA strand loaded onto an Argonaute protein where it guides recognition and repression of cognate mRNAs (Fig. 2). TheAGO-containingeffectorcomplexhasbeengivendifferentnames;hereitwillbe referredtoasmiRNA-InducedSilencingComplex(miRISC).Themechanismofactionof an AGO-containing effector complex varies and may include either translational repression and/or RNAi-like endonucleolytic cleavage. Functional base pairing of animal miRNAs with theirmRNAtargetsappearstoinvolvelittlebeyondthe“seed”regioncomprisingnucleotides 2 to 8 of the miRNA (Brennecke et al., 2005; Sontheimer, 2005). Pairing between Introduction_to_RNAi.indd 18Introduction_to_RNAi.indd 18 09.07.20 8:3409.07.20 8:34 INTRODuCTION 19 miRNAs and mRNAs in plants is typically much more extensive and results in direct endonucleolytic cleavage. Imperfect miRNA:mRNA base pairing in animals generally results in translational repression(Doenchetal.,2003;HutvagnerandZamore,2002),whichiscoupledwith mRNAdegradation(Baggaetal.,2005;Limetal.,2005).Themolecularmechanismof mRNA degradation induced by imperfect base pairing differs from the RNA-like cleavage described above (Schmitter et al., 2006) and involves mRNA deadenylation and decapping activities (Chen et al., 2014; Djuranovic et al., 2012; Nishihara et al., 2013; Rouya et al., 2014). RNA degradation might actually be the dominant component of cognate gene repression (Eichhorn et al., 2014). Repressed mRNAs, miRNAs, and AGO proteins localize to cytoplasmic foci known as P-bodies (Liu et al., 2005; Pillai et al., 2005), which contain mRNA degrading enzymes such as the decapping complex, deadenylases, and the exonuclease XRN1 (reviewed in Decker and Parker, 2012). ThereareonlyminordifferencesinmiRNApathwaysacrossanimals.Themainoneis genetic separation between miRNA and RNAi pathways in arthropods, which utilize miRNA-dedicated Dicer, dsRBP, and AGO while other animals use one Dicer to produce miRNAsandsiRNAs.Thereisacleardifferencebetweenanimalsandplants.Plantsemploy a single RNase III, one of their Dicer paralogs, to process pri-miRNA into pre-miRNA and thenintomiRNAduplexinthenucleus.ThesemiRNAsare2’-O-methylatedattheir3’ termini.ThismodificationisabsentinanimalmiRNAs(butfoundinpiRNAsmallRNAs in the germline). In addition, animals employ two distinct RNase III enzymes – Drosha in the Microprocessor complex in the nucleus, which releases pre-miRNA from pri-miRNA, and Dicer, which produces miRNA duplex in the cytoplasm. Other relevant pathways in Metazoa Adenosine deamination A-to-I editing is mediated by Adenosine Deaminases Acting on RNA (ADAR) enzymes, which contain dsRBD domains and recognize both interand intramolecular dsRNAs longer than 20–30 bp (Nishikura et al., 1991). ADARs convert adenosines to inosines, which translation and reverse transcription interpret as guanosines. ADARs were found in animals (including earliest branching groups) but not plants, yeasts or protozoa (Grice and Degnan, 2015; Nishikura, 2010). It was predicted that more than 85% of pre-mRNAs could be edited, predominantly in the non-coding regions (Athanasiadis et al., 2004). RNAeditingcannegativelyinfluenceRNAiinseveralways.First,ADARscancompete with RNAi for dsRNA substrates including siRNAs. A change of a single base in a sequence may result either in destabilization of dsRNA structure (inosine-uridine pair) or in its stabilization(inosine-cytidinepair)(Nishikura,2010).ThistransitioninthelocalandglobalstabilityofdsRNAstructurecaninfluencefurtherprocessingofdsRNA,suchastheselection oftheeffectivemiRNAstrand(Bartel,2004;DuandZamore,2005;MeisterandTuschl, 2004). While moderate deamination (one I-U pair per siRNA) does not prevent Dicer processingtosiRNAs(Zamoreetal.,2000),hyperediting(~50%ofdeaminatedadenosines) Introduction_to_RNAi.indd 19Introduction_to_RNAi.indd 19 09.07.20 8:3409.07.20 8:34 INTRODuCTION 20 can make dsRNA resistant to Dicer processing (Scadden and Smith, 2001). Hyperedited dsRNAisalsodegradedbyTudor-SN(TSN)nuclease(Scadden,2005).ADARmutantsin Caenorhabditis elegans exhibit defective chemotaxis while the phenotype can be rescued byRNAi-deficiency(TonkinandBass,2003).Inmammaliancells,ADAR1limitssiRNA efficiency(Yangetal.,2005).Editingcanaffecttargetrecognition;amismatchbetween siRNAandtargetmRNAcanreduceRNAiefficacy(ScaddenandSmith,2001)ormodify targetspecificity(Kawaharaetal.,2007b).Severalpri-miRNAs(e.g.miR-142)undergo editing, which inhibits miRNA biogenesis or causes even degradation of pri-miRNA by TSN(Kawaharaetal.,2007a;Nishikura,2010;Scadden,2005;Yangetal.,2006). Interferon pathway Mammalian somatic cells can respond to dsRNA in a sequence-independent manner. A pioneering work by Hunter et al. showed that different types of dsRNA can block translation inreticulocytelysates(Hunteretal.,1975).Analysisofthephenomenonidentifiedprotein kinase R (PKR) that is activated upon binding to dsRNA and blocks translation by phosphorylatingthealphasubunitofeukaryoticinitiationfactor2(eIF2α)(Meursetal.,1990). Activation of PKR represents a part of a complex response to foreign molecules known as the interferon response (reviewed in Sadler and Williams, 2007), which includes activation oftheNFκBtranscriptionfactorandmanyinterferon-stimulatedgenes(ISGs)(Geissetal., 2001). In addition to PKR, several other proteins recognizing dsRNA induce the interferon response, including helicases RIG-I and MDA5, which sense cytoplasmic dsRNA and activate interferon expression, and the 2’,5’-oligoadenylate synthetase (OAS) , which produces 2’,5’-linked oligoadenylates that induce general degradation of RNAs by activating latent RNaseL,andspecificToll-likereceptors(TLRs)(reviewedinGantierandWilliams,2007; Sadler and Williams, 2007). ThereisanevolutionaryconnectionbetweenRNAiandtheinterferonresponse.Mammalian RNA helicases Ddx58, Dhx58 and Ifih1, which are involved in immune response, are the closest homologs of helicases involved in processing of long dsRNA during RNAi in Caenorhabditis elegans. Notably, DDX58, also known as RIG-I, is an established componentoftheinterferonresponsetolongdsRNA(Yoneyamaetal.,2004).Thissuggests that the interferon response, which has a common trigger and evolved after the RNAi pathway, adopted several components from the latter pathway. Notably, there is also connection between interferon pathway and A-to-I editing; analysis of mutant mice showed mice suggested that Adar1 targets dsRNA and prevents MDA5-mediated interferon response (Liddicoat et al., 2015). Acknowledgement IwouldliketothankmycolleaguesJanPaces,MiloslavNic,andTomasNovotnyforhelp withcollectingliteratureforthereview.ThereviewcontentwasproducedunderacontractOC/EFSA/GMO/2015/01-CT01withEuropeanFoodSafetyAuthority(EFSA);the opinionsexpressedarethoseofthecontractoronlyanddonotrepresentEFSA’sofficial Introduction_to_RNAi.indd 20Introduction_to_RNAi.indd 20 09.07.20 8:3409.07.20 8:34 INTRODuCTION 21 position. 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Scadden,A.D.(2005).TheRISCsubunitTudor-SNbindstohyper-editeddouble-strandedRNAand promotes its cleavage. Nat Struct Mol Biol 12, 489–496. Introduction_to_RNAi.indd 25Introduction_to_RNAi.indd 25 09.07.20 8:3409.07.20 8:34 MAMMALS I 32 Methyl-CpG-bindingproteinMECP2(Chengetal.,2014;Tsujimuraetal.,2015),which is known to stably bind methylated DNA. According to one report, MECP2 promotes the posttranscriptional processing of particular miRNAs including miR-199a, which stimulates mTORsignalling(thekeypathwayregulatingcellmetabolism,growth,andsurvival)by targetinginhibitorsofmTORsignalling(Tsujimuraetal.,2015).Incontrast,Chengetal reported that MECP2 binds directly to DGCR8 and interferes with the assembly of the Microprocessor complex, thus affecting gene expression posttranscriptionally via relieving repression of miRNA targets (Cheng et al., 2014). Microprocessor complex localization and function(s) TheMicroprocessorcomplexshowsapparentnuclearcompartmentalization.Whiletransiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. (Pawlicki and Steitz, 2008), these foci do not appear to be major sites of pri-miRNA processing, which seems to be coupled to transcription(PawlickiandSteitz,2009).Thisisconsistentwithlive-imaging,which revealed that a large fraction of Microprocessor resides with unspliced pri-miRNAs in close proximitytotheirgenes.ThisanalysisalsoprovidedadirectvisualevidencethatDGCR8 and Drosha are targeted to pri-miRNAs as a preformed complex (Bellemer et al., 2012). Importantly,literaturereviewidentifiedalsoreportsdescribingadditionalrolesofthe Microprocessor complex and its components beyond miRNA biogenesis although Microprocessor expression seems to be tuned according to pri-miRNA substrates (Barad et al., 2012). Non-canonical roles of Microprocessor (or Drosha) include: 1) mRNA cleavage (Chongetal.,2010),exemplifiedbyDrosha-dependentcleavageofHoxd4 RNA (Phua et al., 2011) or destabilization of Neurog2 mRNA, which supports neural stem cell maintenance by blocking accumulation of differentiation and determination factors (Knuckles et al., 2012), 2) processing of long non-coding RNAs restricted to the nucleus (Ganesan and Rao, 2008), 3), ribosomal RNA biogenesis (Liang and Crooke, 2011), and 4) cleavage of viral RNA (Shapiro et al., 2014). While immunoprecipitation of the Microprocessor complex followed by next-generation sequencing showed that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor complex (Seong et al., 2014), high-throughput sequencing and cross-linking immunoprecipitation(HITS-CLIP)analysisofRNAsboundtoDGCR8suggestthatmiRNAsmay not be the most abundant targets. DGCR8-bound RNAs also comprised several hundred mRNAs, small nucleolar RNAs (snoRNAs), and long noncoding RNAs (Macias et al., 2012). Interestingly, DGCR8-mediated cleavage of snoRNAs was independent of Drosha, indicating participation of DGCR8 in other RNA processing complexes (Macias et al., 2012). One of such complexes is the exosome (an hRRP6-containing nucleolar form), where DGCR8 is essential for its recruitment to snoRNAs and to the human telomeraseRNAcomponent(hTR/TERC)(Maciasetal.,2015).Thus,DGCR8actsasanadaptor recruiting the exosome complex to structured RNAs and inducing their degradation. (Macias et al., 2015). Introduction_to_RNAi.indd 32Introduction_to_RNAi.indd 32 09.07.20 8:3409.07.20 8:34 MAMMALS I 33 Microprocessor complex crosstalk with other pathways In terms of a crosstalk with other pathways, it has been established that some miRNA precursors are edited by ADARs (Alon et al., 2012; Garcia-Lopez et al., 2013; Peng et al., 2012;Tomasellietal.,2015;Veselyetal.,2014;Veselyetal.,2012;Yangetal.,2006) apparently as early as pri-miRNAs (Bahn et al., 2015; Chen et al., 2015). According to one model, ADAR1 interacts with Drosha and DGCR8 in the nucleus and possibly outcompetesDGCR8inprimarymiRNAbinding,thusenhancingmaturemiRNAexpression.This appears dependent on ADAR1 editing activity, at least for a subset of targets (Bahn et al., 2015). According to the selective elimination model, miRNAs, such as miR-151, are edited andeliminatedbyTudor-SN(aribonucleasespecifictoinosine-containingdsRNAsand a reported component of RISC) during mouse preimplantation development (Garcia-Lopez et al., 2013). Similarly, pri-miR-142 editing results in suppression of its processing by Droshawhiletheeditedpri-miR-142isdegradedbyTudor-SN.Consequently,maturemiRNA-142 expression substantially increases in ADAR1-/- or ADAR2-/- mice (Yang et al., 2006).Accordingtothestimulationmodel,exemplifiedbymiR-497,abundanteditingevent promotesprocessingbyDroshaofthecorrespondingpri-miRNA(Veselyetal.,2014). Dicer – cytoplasmic production of miRNA from pre-miRNA A pre-miRNA produced by the Microprocessor complex is transported to the cytoplasm via Exportin5inaRanGTP-dependentmanner.Thenextpre-miRNAprocessingstepisDicer mediated cleavage, which takes place the cytoplasm. Structure of Dicer ThefulllengthmammalianDicerhasnotbeencrystallized.Thecurrentunderstandingof the mammalian Dicer structure has thus been inferred from several different sources, which can be divided into four groups: (I) Biochemical studies of recombinant Dicer and individual domains (Ma et al., 2008;Parketal.,2011;Provostetal.,2002;Zhangetal.,2002;Zhangetal.,2004). (II) The crystal structure of Giardia intestinalis Dicer (serving as a comparative scaffold) (MacRae et al., 2007; MacRae et al., 2006b). (III) Crystallographic studies on mammalian Dicer fragments (Du et al., 2008; Wilson et al., 2015) or on individual domains(Maetal.,2004;Takeshitaetal.,2007;Tian et al., 2014; Wilson et al., 2015) (IV) Cryo-EM studies of human Dicer and its complexes with other proteins (Lau etal.,2012;Lauetal.,2009;Tayloretal.,2013;Wangetal.,2009;Wilsonetal., 2015). Dicer is an siRNA-producing RNase III enzyme conserved across eukaryotes (Bernstein et al., 2001). Mammalian Dicer proteins are ~220 kDa multidomain proteins, which are composed of domains ordered from the Nto the C-terminus as follows: N-terminal DExD and helicase superfamily C-terminal domains, a domain of unknown function DUF283, Introduction_to_RNAi.indd 33Introduction_to_RNAi.indd 33 09.07.20 8:3409.07.20 8:34 MAMMALS I 34 aPAZdomain,RNaseIIIaandRNaseIIIbdomains,andtheC-terminaldsRBD(Fig.2) (Nicholson and Nicholson, 2002). In contrast to the simplest RNase III family members (exemplifiedbyE.coliRNaseIII),whichcarryonlyoneRNaseIIIdomainanddimerize when cleaving dsRNA (Johanson et al., 2013; Lamontagne et al., 2001). Dicer proteins carrytwoRNaseIIIdomains,whichformanintramoleculardimer(Zhangetal.,2004). Giardia Dicer structure revealed spatial organization of the core part of eukaryotic Dicer proteinsandexplainedhowDicergeneratessmallRNAsofspecificlengths(MacRaeetal., 2006a).Thiscrystalstructurethenservedasaframeworkfordecipheringthestructureof otherDicerproteins,includingmammalianDicers.ThefrontviewoftheGiardia Dicer structureresemblesanaxe.ThebladeisformedofanintramolecularduplexoftwoRNase III domains, which are connected by a bridging domain constituting the back end of the blade.TheplatformdomainisadjacenttotheRNaseIIIadomainandmakesuptheupper partofthehandle.ThePAZdomainisconnectedbyalonghelixtotheRNaseIIIadomain and forms the base of the handle (MacRae et al., 2006b). Altogether, the Giardia Dicer is formedofthreerigidregions,whicharelinkedbyflexiblehinges.Oneregionisformed by RNase III domains and the bridging domain, the second by the platform domain and theconnectorhelix,andthethirdbythePAZdomain.Thesethreepartscanswingrelative to each other and possibly ensure accommodation of Dicer to the structure of its substrate (MacRaeetal.,2006a).ThisconformationalflexibilitylikelyenablesbindingofdsRNAs with non-canonical base pairing as well as imperfect duplexes of pre-miRNAs (MacRae et al., 2006a). In addition, dsRNA binding is presumably stabilized by several positively chargedpatchesonthesurfaceofGiardiaDicerbetweentheprocessingcenterandthePAZ domain, which are in contact with dsRNA (MacRae et al., 2006a; MacRae et al., 2007). Mammalian Dicers are much larger and contain domains absent in the Giardia Dicer butfollowthesameorganizationalandfunctionalprinciples(Fig.2).Thecrystalstructure of GiardiaDicerconfirmedanearlierbiochemicalanalysispredictingthatthetwo RNase III domains of the human Dicer form an intramolecular dimer resulting in a single processingcenterplacedataspecificdistancefromthePAZdomain(Zhangetal.,2004). Figure 2 Domain architectures of Dicers from Giardia and humans. Introduction_to_RNAi.indd 34Introduction_to_RNAi.indd 34 09.07.20 8:3409.07.20 8:34 MAMMALS I 35 Astructuralcomponentdefiningthisdistanceisanαhelix(connectorhelix),whichdirectly linksPAZandRNaseIIIdomains(MacRaeetal.,2006b).Thus,thekeyfunctionalaspect that emerged from Dicer’s structural analysis was that it functions as a molecular ruler, measuringthelengthofthesubstratefromthePAZdomaintoRNaseIIIdomainswhere each domain cleaves one strand. Importantly, the mammalian Dicer (and metazoan Dicers ingeneral)differfromGiardia’sintwomainaspects.Thefirstisthegeneraltopology reflectingthefactthatthetypicalmammalianDicerproductisshorter(21–23nt).Thesecond one is that the mammalian Dicer contains additional functional domains important for substrate recognition and processing. As indicated above, the architecture of the human Dicer and positions of its domains and interacting partners have been inferred by cryo-EM of the full length protein and its mutants (Lauetal.,2012;Lauetal.,2009;Tayloretal.,2013;Wangetal.,2009;Wilsonetal., 2015).TheoverallshapeofthehumanDicerresemblestheletterL;theshapeisfurther dividedintoahead,abodyandabase(Fig.2).ThePAZdomainisadjacenttotheplatform domainintheheadoftheproteinwhiletheRNaseIIIbislocatedinthebody.Thus,thehead of the human Dicer is a topological equivalent of the base of the handle in Giardia’s Dicer. Thehelicasedomainconstitutesthebase,whichhasnoequivalentinGiardia’sDicer.The positionoftheprocessingcenterrelativetothePAZdomainsdiffersbetweenhumanand Giardia Dicers, which explains the fact that the human Dicer produces siRNA about four nucleotides shorter than the Giardia Dicer, which corresponds to ~ one-third of a dsRNA helicalturn(Lauetal.,2012).Therefore,theprocessingcenterhastoaccessthecleavage site of dsRNA from the different angle relative to the dsRNA helical end in comparison with Giardia Dicer (Lau et al., 2012). For understanding substrate selection and processing, two areas of Dicer’s structure deservespecialattention:thePAZandtheN-terminaldomains,whicharedescribedbelow. Thefollowingtextrepresentsexhaustiveliteraturesurveyfocusedonthestructuraland functional aspects of the two domains. The PAZ domain ThePAZdomainfoundinDicerandArgonauteproteinsisadsRNA-terminusbinding module(Maetal.,2004;MacRaeetal.,2006b).ThePAZdomainhasa3’overhangbinding pocketbutonlythePAZdomainofDicerhasanextraloopenrichedinbasicaminoacids, changingelectrostaticpotentialandmolecularsurfaceofthepocket.Thesechangesmay influenceRNAbindingbyDicerandhanding-offthesubstratetootherproteinscomplexes (MacRaeetal.,2006b).ThePAZdomainofmetazoanDicersalsorecognizesphosphorylated 5’ end of a pre-miRNA. A mutation of the 5’ binding pocket leads to dysregulation of miRNA biogenesis in vivo(Parketal.,2011).The5’bindingpocketisconservedin Drosophila DCR-1 and human Dicer but not in Giardia Dicer (Park et al., 2011). Importantly, the 5’ binding pocket appears conserved in Dicer proteins functioning in miRNA biogenesis (human Dicer, Drosophila DCR-1) but not in Dicer proteins dedicated to long dsRNA processing (Giardia, Schizosaccharomyces, Drosophila DCR-2). Accordingly, simultaneousfixingof3’and5’endsemergesasafeatureimportantforfidelityofmiRNA biogenesis but not for siRNAs (Park et al., 2011). Introduction_to_RNAi.indd 35Introduction_to_RNAi.indd 35 09.07.20 8:3409.07.20 8:34 MAMMALS I 36 The N-terminal helicase domain TheN-terminusofmetazoanDicersharboursacomplexhelicasestructure,whichisadjacent to RNase III catalytic domains (Lau et al., 2012). Although the helicase must come intocontactwiththesubstrate,itsfunctionalsignificanceisstillonlypartiallyunderstood. However, it is clear that the N-terminal helicase region is the key for the substrate preference. In mammals (and in most metazoan phyla), a single gene encodes Dicer, which has to process both: miRNA precursors into miRNAs as well as long double-stranded RNAs into small interfering RNAs (siRNAs). Different taxons apparently differ in how much they employ both types of Dicer activities; the mammalian Dicer is mainly dedicated to the miRNA pathway while its natural production is very limited. TheN-terminalhelicasebelongstotheRIG-I-likehelicasefamily(Zouetal.,2009)and consists of a proximal DExD/H domain and an adjacent helicase superfamily c-terminal domain(Fig.2).AconventionalhelicasedomainhasanATPaseactivity.Indeed,invertebrateDicersbindandhydrolyzeATP(Bernsteinetal.,2001;Kettingetal.,2001;Nykanen etal.,2001;Zamoreetal.,2000).However,despitetheN-terminalhelicasewithconserved motifsimportantforATPbindingandhydrolysisispresentinmammalianDicers,there isnoevidenceofATPrequirementforthehumanDiceractivity(Provostetal.,2002; Zhangetal.,2002).ThehumanDicerhasthesameprocessingefficiencyinthepresence orabsenceofATP.Moreover,therateofcleavageisnotinfluencedbyadditionofother nucleotides,non-cleavableATPanaloguesoramutationintheWalkerAmotifofATPase/ helicasedomain(Provostetal.,2002;Zhangetal.,2002).Notably,theseexperimentswere performed using a long dsRNA substrate with blunt ends, whose processing by invertebratesDicersisATP-dependent(Bernsteinetal.,2001;Kettingetal.,2001;Nykanenetal., 2001;Zamoreetal.,2000;Zhangetal.,2002).Remarkably,deletionofthehelicasedomain results in high cleavage rate of long dsRNAs by human Dicer in vitro (Ma et al., 2008) as well as in vivoinmurineandhumancells(Flemretal.,2013;Kennedyetal.,2015).Thus, the N-terminal helicase in mammalian Dicers has a different role in substrate recognition and processing than the helicase in invertebrate Dicers although the overall shapes of human and Drosophila Dicer proteins are similar (Lau et al., 2012). ThecrystalstructureoftheN-terminalhelicasehasnotbeenobtained.Thus,basedon the cryo-EM-based modelling, the N-terminal helicase is composed of three globular subdomains (HEL1, HEL2, HEL2i) where the DExD/H domain corresponds to HEL1 and the helicase superfamily c terminal domain to HEL2 and HEL2i. All three parts of the helicase form a clamp near the RNase III domain active site. Interestingly, the N-terminal helicase was found in two distinct conformations, with respect to the body of the enzyme (Lau et al., 2012), similar to the RIG-I helicase which was used as a template for modelling (Kowalinski et al., 2011). Analysisofsubstrate-specificstructuralrearrangementsproposedthathumanDicerexists inthreestatesdependingonpresenceandtypeofsubstrate(Tayloretal.,2013).unbound Dicerexistingin“canonicalstate”rearrangesuponsubstratebindingthatinvolvesthePAZ domainaswellasthehelicasedomain.Substrate-boundDicerexistseitherinan“open”or closed”state.Theopenstateiscleavage-competentanditistypicalforpre-miRNAbinding. It is characterized by binding of a pre-miRNA along the platform, bending of the helicase Introduction_to_RNAi.indd 36Introduction_to_RNAi.indd 36 09.07.20 8:3409.07.20 8:34 MAMMALS I 37 domain,andaccessofRNaseIIIaandIIIbsitestothesubstrate(Tayloretal.,2013).The closed state has been observed for a 35 bp A-form RNA duplex, which represents a siRNA precursor.Inthisstate,thesubstrateistrappedbetweenthePAZandhelicasedomainsaway fromthecatalyticsites(Tayloretal.,2013).Thisprovidesastructuralexplanationforprevious observations that Dicer poorly processes longer perfect duplexes in vitro and in vivo (Kim et al., 2005; Nejepinska et al., 2012b). Takentogether,itisapparentthatmiRNAbiogenesishasbeenthepreferredroleforDicer duringvertebrateevolution.ThehelicasedomaininmammalianDicersprovidesastructural basisforsubstratespecificity,namelydistinguishingpre-miRNAsasthepreferredsubstrate. In addition, a natural Dicer isoform has been found in mouse oocytes, which lacks the N-terminalhelicasedomain,canefficientlygeneratesiRNAsfromlongdsRNAs,andissufficient forenhancingRNAiinculturedcells.Thisisoformisaconsequenceofarodent-specific retrotransposon insertion and is present in Muridaefamily(Flemretal.,2013).Thisdemonstrates that, while the mammalian Dicer primarily dedicated to the miRNA pathway, a small change in a mammalian Dicer gene can restore RNAi activity. Substrates and their processing by mammalian Dicer proteins Thefirstin vitro studies of recombinant human Dicer showed that substrate cleavage is dependentonMg2+butnotonATPpresence(Provostetal.,2002;Zhangetal.,2002). Subsequently, it was reported that Dicer can cleave long dsRNAs and pre-miRNAs with differentefficiency,whichstemsfromsubstrate’sstructuralproperties(Chakravarthyetal., 2010;Fengetal.,2012;Flores-Jassoetal.,2009;Maetal.,2008).Therefore,cleavageof miRNA precursors and long dsRNAs will be discussed in separate sections. Canonical miRNA substrates Canonical miRNAs of ~22 nt in length (Fig. 3) are the dominant Dicer products in mammalian cells. Dicer mutagenesis showed that inactivation of the RNase IIIA domain results in complete loss of 3p-derived mature miRNAs, but only partial reduction in 5p-derived mature miRNAs (Gurtan et al., 2012). Conversely, inactivation of the RNase IIIB domain by mutation of D1709, a residue mutated in some cancers, produced complete loss of 5p-derived mature miRNAs, but only partial reduction in 3p-derived mature miRNAs (Gurtan etal.,2012).MutationofthePAZdomaincausedglobalreductionofmiRNAprocessing, while mutation of the Walker A motif in the helicase domain of Dicer did not alter miRNA processing(Gurtanetal.,2012).Theseresultsareconsistentwiththeabovementioned structural features of Dicer. Pre-miRNAsarethemostefficientlycleavedDicersubstratesin vitro. In contrast to long dsRNA, a canonical pre-miRNA is cleaved only once and releases a single small RNA duplex. Human Dicer alone cleaves pre-miRNAs much faster than pre-siRNA substrates under both single and multiple turnover conditions; with more than 100-fold difference in maximalcleavagerates(Vmax)undermultipleturnoverconditions(Chakravarthyetal., 2010).ThisindicatesthatthemammalianDicerisoptimizedformiRNAbiogenesisand Introduction_to_RNAi.indd 37Introduction_to_RNAi.indd 37 09.07.20 8:3409.07.20 8:34 MAMMALS I 38 severalspecificstructuraladaptationsdiscussedbelowsupportthisnotion.Dicerseemsto interact directly with the terminal loop region of a pre-miRNA (Feng et al., 2012; Gu et al., 2012b) while a large pre-miRNA terminal loop further enhances pre-miRNA cleavage (Feng et al., 2012). A large-scale in vitro analysis and mutagenesis study of 161 human pre-miRNAs showed that human Dicer tolerates remarkable structural variation in pre-miRNA substrates(Fengetal.,2012).ThedsRNAstructureinthestemregionandthe2-nt3’-overhang structure in a pre-miRNA contribute to binding and cleavage by Dicer (Feng et al., 2012). Acharacteristicfeatureofthepre-miRNAhairpin,whichisaccessedbythePAZdomain of Dicer, is a 2 nt 3’ overhang generated by the nuclear Microprocessor complex (Gregory et al., 2004). Pre-miRNAs with the 2 nt 3’ overhang at the 3’ terminus are bound by Dicer withhigheraffinitythanpre-miRNAswithdifferentends(Fengetal.,2012).Moreover, the 2 nt 3’end overhang leads to a higher substrate processing, which was shown on both, pre-miRNAsandperfectduplexes(Fengetal.,2012;Parketal.,2011;Zhangetal.,2004). Such preference is likely conferred by/due to simultaneous binding of pre-miRNA end by both5’and3’bindingpocketsinthePAZdomain(Parketal.,2011).Importantly,fidelityof miRNA biogenesis is critical for miRNA functionality because a single nucleotide shift at the 5’endofamiRNAwouldredefineitstargetrepertoire.Incontrast,RNAi,whichtypically involves perfect complementarity between a small RNA and its target, would be essentially insensitive to a precise cleavage positioning as long as it would not affect Argonaute loading. Thus,thesimultaneousrecognitionofbothstrandsatthe2nt3’overhangterminusbyDicer can be seen as an adaptation driven by miRNA biogenesis (Park et al., 2011). ThesecondstructuraladaptationofmammalianDicersupportingmiRNAbiogenesisis the N-terminal helicase, which forms a clamp-like structure adjacent to RNase III domains, hence it is positioned to bind the stem loop of a pre-miRNA (Lau et al., 2012). While the loss of the entire N-terminal helicase only slightly increases pre-miRNA processing activity 0 100 200 300 400 500 18 19 20 21 22 23 24 25 26 27 28 number of miRNAs miRNA length miRNA size distribution in Mus musculus Figure 3 Mammalian miRNA size distribution Distribution of mature murine miRNA lengths according to miRNA annotations in miRBase (release 21) Introduction_to_RNAi.indd 38Introduction_to_RNAi.indd 38 09.07.20 8:3409.07.20 8:34 MAMMALS I 39 in vitro (Ma et al., 2008), pre-miRNA-processing by recombinant Dicer in vitro is much faster than that of a perfect duplex (Chakravarthy et al., 2010; Ma et al., 2008). In vivo, a naturally occurring N-terminally truncated Dicer isoform can rescue miRNA biogenesis in Dicer-/-embryonicstemcells(ESCs)(Flemretal.,2013).ThissuggeststhattheN-terminal helicase domain in mammalian Dicers is not important for miRNA biogenesis per se; it rather provides constrains for substrate selectivity favouring pre-miRNAs. Thisisconsistentwiththemodelwherepre-miRNAbindingisassociatedwiththecleavage-competent open conformation. In the open state, a pre-miRNA is bound along the platform, the helicase domain is bent, and RNase IIIa and IIIb sites have access to the substrate(Tayloretal.,2013).Ithasbeenproposedthattheloopofapre-miRNAmayprevent adoption of the closed conformation by Dicer by interacting with HEL1 and HEL2i domains and possibly stabilizing the open conformation of Dicer (Feng et al., 2012; Lau etal.,2012;Maetal.,2012).ThisalsoindicatesthattheN-terminalhelicasehadacquired distinct roles in Dicer function in RNA silencing during evolution. In mammalian cells, the N-terminal helicase has a gatekeeper function where pre-miRNA loops appear to be a key keeping the gate open. Dicer-dependent non-canonical miRNA substrates Apart from canonical miRNA substrates mentioned above, Dicer is processing additional miRNA-like substrates, which are independent of the Microprocessor complex (described in a separate section below). Some non-canonical miRNAs are produced by Dicer in a Microprocessor-independent fashion, including mirtrons, which utilize the splicing machinery to bypass the Microprocessor complex. Mirtrons are substantially longer than Microprocessor-generated pre-miRNAs and exhibit 3’ uridylation and 5’ heterogeneity (Wen et al., 2015). A recent analysis yielded ~500 novel mouse and human introns that generate Dicer-dependentsmallRNAduplexes(Wenetal.,2015).Theserepresentnearly1000loci distributed in four splicing-mediated biogenesis subclasses, with 5’-tailed mirtrons being the dominant subtype (Wen et al., 2015). Another example of non-canonical miRNAs found in the literature are Microprocessor-independent miRNAs which were originally described as small interfering RNAs derived from a unique hairpin formed from short interspersed nuclear elements (SINEs) (Babiarz et al., 2008; Castellano and Stebbing, 2013). While a typical pre-miRNA is a hairpin RNA with 2-nt 3’ overhangs, production of a mature miRNA from an endogenous hairpin RNA with 5’ overhangs has also been reported; mouse pre-mir-1982 is a mirtron with an 11 nt tail at the 5’ end (Babiarz et al., 2008). A possible mechanism for processing such templates has been provided by an in vitro study which showed that Dicer can produce such miRNAs in a two-step cleavage, which releases dsRNAsafterthefirstcleavageandbindsthemagainintheinversedirectionforasecond cleavage (Ando et al., 2011a). Long dsRNA substrates In addition to pre-miRNA, Dicer can process long dsRNAs coming from different sources. Exogenous sources of dsRNA include viral dsRNAs and imply function of RNAi in Introduction_to_RNAi.indd 39Introduction_to_RNAi.indd 39 09.07.20 8:3409.07.20 8:34 MAMMALS I 40 eukaryoticantiviralimmuneresponse(VanceandVaucheret,2001;Wangetal.,2006; Wilkins et al., 2005). Endogenous dsRNAs have variable length and termini, and are generated by transcription of inverted repeats, by convergent transcription or by pairing of complementary RNAs in trans. Importantly, mammals lack an ortholog of RNA-dependent-RNA polymerase (RdRP), which is a conserved component of RNAi-related mechanisms in plants, fungi and invertebrates (see the separate RdRP section). Endogenous RNAi in mouse oocytes, the best documented mammalian endogenous RNAi example, works independently of RdRP activity (Stein et al., 2003). ThehumanDicerbindslongdsRNAbutnotsiRNAsin vitro (Provost et al., 2002). Long dsRNA binding is independent both on Mg2+andATP.ThehumanDicerpreferentially bindsandcleaveslongdsRNAfromtheend,duetoinefficientbindingofinternalregions ofdsRNA(Zhangetal.,2002).Incomparisontopre-miRNAprocessing,humanDicer exhibits lower cleavage activity on perfect dsRNA substrates (Ma et al., 2008). An explanation was proposed that a closed conformation of the N-terminal helicase domain disturbs the RNase III catalytic core and inhibits cleavage of perfect dsRNAs (Lau et al., 2012). As it was mentioned, in vitro deletion of the N-terminal helicase domain increases cleavage activity of human recombinant Dicer (~65-fold). Authors hypothesize that DExD/H-box domain mainly inhibits the functionality of the Dicer active site, but not RNA binding (Ma etal.,2008).Thismodelissupportedbypreviouslymentionedstructuraldata,whereDicer isinaclosedstatewitha35bpA-formRNAduplextrappedbetweenPAZandhelicase domainsawayfromthecatalyticcenter(Tayloretal.,2013). ThecomplexityofthedifferentialsubstrateprocessingbyDicerisillustratedbyaDicer mutant carrying an in-frame 43-amino-acid insertion immediately adjacent to the DExHbox.ThisDicerexhibitsdefectsintheprocessingofmost,butnotall,endogenous pre-miRNAsintomaturemiRNAbutenhancedprocessingefficiencyandconcomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used (Soifer etal.,2008).Thisresultimpliesanimportantfunctionforthehelicasedomainintheprocessing of thermodynamically unstable hairpin structures (Soifer et al., 2008). Dicer-mediated cleavage of dsRNA can be stimulated in vitrobyTARBP2.However,it isnotclearifTARBP2stimulationcouldbesufficienttoinduceendogenousRNAiin vivo (Chakravarthy et al., 2010). So far, the evidence for endogenous RNAi (including attempts to induce RNAi with exogenous substrates) is scarce (reviewed in detail in Nejepinska etal.,2012a;Svoboda,2014).Theonlytissuetype,whereabundantendogenoussiRNAs are present and where long dsRNA readily induces RNAi are mouse oocytes, which express anoocyte-specificDicerisoformlackingapartoftheN-terminalhelicasedomain(Flemr et al., 2013), thus mimicking some of the Dicer mutants tested in vitro (Ma et al., 2008). Takentogether,longdsRNA,thetypicalendogenousRNAisubstrate,ispoorlyprocessed byendogenousfull-lengthDicer.ThisisduetothegatekeeperroleoftheN-terminalhelicase domain, which does not open upon binding long dsRNA. Off note is that the human Dicer can bind 21-nt ssRNAs in vitro, independent of their sequence and secondary structure. Dicer binds ssRNAs having a 5’-phosphate with greater affinityversusthosewitha5’-hydroxyl.(KiniandWalton,2007). Introduction_to_RNAi.indd 40Introduction_to_RNAi.indd 40 09.07.20 8:3409.07.20 8:34 MAMMALS I 41 Dicer-interacting dsRBPs: TARBP2 and PACT A common Dicer interacting partner found across Metazoa is a dsRBP with tandemly arrayed dsRBDs. Mammals have four dsRBP with tandemly arrayed dsRBDs proteins: trans-activationresponsiveRNA-bindingprotein2(TARBP2),proteinactivatorofPKR (PACT),Staufen1(STAu1),andStaufen2(STAu2).However,onlyTARBP2(alsoknown asTRBPorTRBP2)andPACTwereidentifiedasDicerbindingpartners(Chendrimada et al., 2005; Haase et al., 2005). TARBP2andPACTareparalogs,whichevolvedthroughageneduplicationeventinan ancestralchordate(DanielsandGatignol,2012).ThestructureofhumanTARBP2hasbeen partially resolved (Benoit and Plevin, 2013). Each protein consists of three dsRBDs, where thefirsttwodomainscanbinddsRNA(ormiRNA)whilethethirddomainhasapartial homology to dsRBD and does not bind dsRNA. Instead, it mediates protein-protein interactions and is a part of a larger protein-protein interacting C-terminal region referred to asMedipaldomainasitinteractswithMerlin,Dicer,andPACT(reviewedinDanielsand Gatignol,2012).TARBP2andPACTcanalsoformhomodimersandheterodimersthrough the Medipal domain (Laraki et al., 2008). ThebindingsiteofTARBP2andPACTonDicerwasrecentlydeterminedusingcryoEM and crystallography (Wilson et al., 2015). Homology-based modelling showed that Dicer-bindingresiduesareconservedinTARBP2andPACT, implicating that binding of TARBP2andPACTtoDicerismutuallyexclusive(Wilsonetal.,2015). TARBPhasapositiveeffectonDiceractivity.HumanDicerismuchfasteratprocessing a pre-miRNA substrate compared to a pre-siRNA substrate under both single and multiple turnoverconditions.Maximalcleavagerates(Vmax) calculated by Michaelis-Menten analysisdifferedbymorethan100-foldundermultipleturnoverconditions.TARBP2wasfound in vitro to stimulate Dicer-mediated cleavage of both, pre-miRNA and pre-siRNA substrates; this stimulation requires the two N-terminal dsRBDs (Chakravarthy et al., 2010). Thus,whilethestructureofthesubstrateaffects the rate at which Dicer generates small RNAs,TARBP2stimulatesdicingbypresumablyenhancingthestabilityofDicer-substrate complexes (Chakravarthy et al., 2010). WhencomparedtoDicerandDicer:TARBP2complex,PACTinhibitsDicerprocessing ofpre-siRNAsubstrates(Leeetal.,2013).ThetwoN-terminaldsRBDscontributetothe observed differences in dsRNA substrate recognition and processing behaviour of Dicer:dsRNA-bindingproteincomplexes(Leeetal.,2013).Inaddition,PACTandTARBP2have non-redundant effects on the generation of different-sized miRNAs (isomiRs) (Kim et al., 2014;Leeetal.,2013;Wilsonetal.,2015).CellslackingTARBP2exhibitalteredcleavage sites in a subset of miRNAs but no effect on general miRNA abundance or Argonaute loading(Kimetal.,2014).Thus,impactofTARBP2andPACTonmiRNAsbiogenesisin vivo seems to be relatively minor (Kim et al., 2014; Wilson et al., 2015). However, it should be pointed out that any change in the 5’ end position of any miRNA will have a strong effect on itstargetrepertoire.Takentogether,TARBP2andPACTareregulatoryfactorsthatcontributetothesubstratespecificityandcleavagefidelityduringmiRNAandsiRNAproduction. Moreover,TARBP2andPACThaveanadditionalroleinacross-talkoftheinterferon (IFN)responseandsmallRNApathways(reviewedinDanielsandGatignol,2012).The Introduction_to_RNAi.indd 41Introduction_to_RNAi.indd 41 09.07.20 8:3409.07.20 8:34 MAMMALS I 48 Y393 phosphorylation–Tyrosine393(Y393)wasimplicatedinEGFR-mediatedrepression of miRNA biogenesis during hypoxia (Shen et al., 2013). According to the model, Y393 negatively impacts the interaction between AGO2 and Dicer and inhibits maturation of long-loop pre-miRNAs carrying tumour-suppressor-like miRNAs (Shen et al., 2013). Prolyl 4-hydroxylation Prolyl 4hydroxylation has been implicated in AGO stabilization and increased RNAi. Mass spectrometry analysis hydroxylation of the endogenous AGO2 at proline 700 (P700) and P700A mutation resulted in destabilization of AGO2 (Qi et al., 2008). Prolyl hydroxylation was observed under hypoxic conditions, where it lead to increased AGO2 stability (Wu et al., 2011). AGO2 hydroxylation correlated with increased miRNA levels as well as the endonuclease activity of AGO2 (Wu et al., 2011). Conversely, human cells depleted andmouseembryonicfibroblastcellsdepletedofaspecificprolyl-4-hydroxylaseshowed reduced stability of AGO2 and impaired RISC activity (Qi et al., 2008). Hydroxylation of AGO2 was required for its association with HSP90 (see further below), which is implicated in the RISC loading with miRNAs and translocation to stress granules (Wu et al., 2011). SUMOylation Thesmallubiquitin-likemodifier(SuMO)regulatesvariouscellularprocesses.AGO2was identifiedasasubstrateforSuMOE3ligasePIAS3.AGO2wasSuMOylatedinmammalian cells by both SUMO1 and SUMO2 primarily at lysine 402. Mutation of the SUMO consensus site reduced RNAi activity of AGO2, suggesting that SUMOylation might regulate endonucleolytic activity of AGO2 (Josa-Prado et al., 2015) Ubiquitination Ubiquitin-proteasome apparently tunes AGO levels to adjust miRNA, AGO and Dicer stoichiometry (Smibert et al., 2013). It was found that levels of AGO1 are adjusted according to miRNA expression in a ubiquitin-proteasome-dependent manner (Smibert et al., 2013). Similarly, lower stability of AGO2 in Dicer-knockout cells could be rescued by proteasome inhibition or Dicer expression (Smibert et al., 2013). AGO and GW182 protein levels also depend onHSP90availability(Johnstonetal.,2010).Twostudiesshowexamplesofdevelopmentally regulated ubiquitination, which is apparently used to suppress AGO activities during developmental transitions. First, the let-7 target Lin-41geneinmiceisastemcellspecificE3 ubiquitin ligase targeting AGO1, AGO2, and AGO4 proteins (Rybak et al., 2009). Second, AGOproteinsaredownregulatedinaproteasome-dependentmannerduringTcelldifferentiation, presumably as a part of gene expression reprogramming (Bronevetsky et al., 2013). Poly-ADP-ribosylation ThisAGOmodificationsseemstobelinkedtosuppressionofRNAsilencing.Poly(ADP-ribose) has been associated with the assembly of stress granules, which accumulate Introduction_to_RNAi.indd 48Introduction_to_RNAi.indd 48 09.07.20 8:3409.07.20 8:34 MAMMALS I 49 RNA-binding proteins regulating mRNAs stability and translation upon stress. Stress granuleproteinsmodifiedbypoly(ADP-ribose)includeAGO1—4(Leungetal.,2011).Interestingly, poly-ADP-ribosylation of RISC associated with reduced RISC activity has been observed upon viral infection (Seo et al., 2013). According to the model, poly-ADP-ribosylation after viral infection releases miRNA-mediated repression of interferon-stimulated genes, hence boosting innate antiviral pathways (Seo et al., 2013). Other Dicer and AGO interacting proteins ApartfromtheRISC-loadingcomplexandmiRISCcomponentssuchasGW182/TNRC6 or DDX6 and others mentioned above and elsewhere, a large number of AGO-interacting partnershasbeenidentifiedinthepastandreportedindividually(seefurtherbelow)or comprehensively (Meister et al., 2005). Here, I provide an overview of those interacting partners. DDX3 – DEAD-box helicase 3 is one of the helicases sensing viral double-stranded RNAs. DDX3 was also among the P-body components recruited to the West Nile virus replication sites and regulating viral replication (Chahar et al., 2013). DDX3 was also identifiedbyanRNAiscreenasanessentialfactorinvolvedinRNAipathway(Kasimetal., 2013). DDX3 is co-localized with AGO2 and a dominant negative mutant of DDX3 affected the RNAi activity (Kasim et al., 2013). CLIMP-63–Thecytoskeleton-linkingendoplasmicreticulum(ER)membraneprotein of63kDa(CLIMP-63)wasidentifiedasanovelDicer-interactingproteinthroughayeast two-hybrid screening. CLIMP-63 interacts with Dicer to form a high molecular weight complex,whichiscatalyticallyactiveinpre-miRNAprocessing(Pepinetal.,2012).These results are consistent with analysis of Dicer compartmentalization, which showed that loading of small RNAs into RISC, cognate mRNA binding, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (Stalder et al., 2013). While the major RNAi pathway proteins are found in most subcellular compartments, the miRNAand siRNA-loaded AGO2 populations co-sediment almost exclusively with theroughendoplasmicreticulummembranes,togetherwithDicer,TARBP2,andPACT (Stalder et al., 2013). NUP153–ThenuclearporecomplexproteinNuP1wasfoundtoassociatewithhuman Dicerprotein.Theassociationwasdetectedmainlyinthecytoplasmbutwasalsoapparent at the nuclear periphery. Accordingly, it has been suggested that NUP153 plays a role in the nuclear localization of Dicer (Ando et al., 2011b) FMRP – X mental retardation protein (FMRP) is included in the list despite its questionable role in mammalian RNA silencing. In any case, our literature search revealed a number of articles dealing with mammalian FMRPs because FMMRP is a highly conserved protein and its Drosophila ortholog dFXR was implicated in RNAi (Caudy et al., 2002; Ishizuka et al., 2002). According to the available data, FMRP is associated with RNA silencing factors. FMRP co-localized with AGO2 (Goodier et al., 2007) and immunoprecipitation suggested that a portion of Dicer and AGO were associated with each other and with FMRP (Lugli et al., 2005). In vitro data using recombinant proteins, suggested Introduction_to_RNAi.indd 49Introduction_to_RNAi.indd 49 09.07.20 8:3409.07.20 8:34 MAMMALS I 50 that human FMRP can act as a miRNA acceptor protein for Dicer and facilitate the assemblyofmiRNAsonspecifictargetRNAsequences(Planteetal.,2006).Therequirementof FMRPforefficientRNAiwasalsosupportedin vivo by reporter assays supporting the role of FMRP in the mammalian RISC (Plante et al., 2006). However, the loss of mammalian FMRP did not reveal any apparent direct impact on RISC function (Didiot et al., 2009; Madsen et al., 2009). Huntingtin – AGO2 was found as one of the Huntingtin associated proteins by co-immunprecipitation. Furthermore, Huntingtin and AGO2 co-localized in P-bodies and, importantly, depletion of Huntingtin compromised RNA-mediated gene silencing (Savas et al., 2008).However,themolecularmechanismbywhichHuntingtinwouldinfluenceRNA silencing remain unknown. 14–3–3 – Cell cycle regulating 14–3–3 proteins were reported to bind the amino terminus of AGO1 and AGO2 (Stoica et al., 2006). Overexpression of the Ago1 amino terminus in yeast resulted in cell cycle delay at the G(2)/M boundary prompting a hypothesis that 14–3–3 proteins contribute to Argonaute protein functions in cell cycle and/or gene-silencing pathways (Stoica et al., 2006). UPF1 – mRNA surveillance protein appears to provide a nexus between three different mechanisms of RNA metabolism: adenosine deamination, mRNA surveillance (non-sense-mediated decay) and RNA silencing. Both, human ADAR1 and UPF1 were found associated within nuclear RNA-splicing complexes (Agranat et al., 2008). At the same time, UPF1 was connected to RNA silencing (Jin et al., 2009). UPF1 interacts with human AGO1 and AGO2 and co-localizes with them into P-bodies. UPF knockdown yielded upregulation of miRNA targets while its overexpression resulted in their downregulation(Jinetal.,2009).ThiswouldsuggestthatuPFmaycontributetoRNA silencing, maybe at the level of RISC binding to its targets and accelerating their decay (Jin et al., 2009). RBM4–TheRNA-bindingmotifprotein4(RBM4)playsmultiplerolesinmRNA metabolism.RBM4 was found during proteomic analysis of AGO-containing miRNPs (i.e. miRISC) and RBM4 knockdown showed that it is required for miRNA-guided gene regulation (Hock et al., 2007). It was also found to co-localize with AGO2 during muscle cell differentiation(LinandTarn,2009).RBM4interactsdirectlywithAGO2andmayselectively enhancemiRISCassociationwithtargetmRNAs(LinandTarn,2009).RBM4wasalso implicateditmiRNA-mediatedrepressionininflammationwhereinflammation-induced miRNA-146promotesafeed-forwardloopthatmodifiesthroughphosphorylationthesubcellular localization RBM4 and promotes its interaction with AGO2 and, subsequently, tamesanexcessiveacuteinflammatoryresponse(Brudeckietal.,2013) TRIM32–TRIM-NHL32proteinregulatesproteindegradationandmiRNAactivityin neural progenitor cells to control the balance between differentiating neurons and daughter cellsretainingtheprogenitorfate.TRIM32wasshowntobindAGO1andincreasethe activityofspecificmiRNAs,suchasLet-7(Schwambornetal.,2009) QKI-6 – QKI-6 is one of the protein isoforms encoded by the qkI gene in mice. QKI-6 was found to interact with AGO2 and to co-localize with AGO2 into stress granules (Wang et al., 2010). At the same time QKI-6 depletion lead to increased miR-7 expression while QKI-6 presence inhibits processing of pri-miR-7 into miR-7 in glioblastoma cells (Wang Introduction_to_RNAi.indd 50Introduction_to_RNAi.indd 50 09.07.20 8:3409.07.20 8:34 MAMMALS I 51 et al., 2013). It has been suggested that OKI-6 mediates selective nuclear retention of primiR-7, hence preventing its processing (Wang et al., 2013). Further research is needed to clarify these two seemingly distant activities of OKI-6. RACK – receptor for activated protein kinase C (RACK1), a constituent of the eukaryotic 40S subunit, was reported to be important for miRNA-mediated gene regulation in C. elegans and humans, essentially linking miRISC with the ribosome (Jannot et al., 2011). RACK1wasalsoidentifiedasagenenecessaryforfullmiRNAfunctionascreenforgenes regulating miRNA function (Otsuka et al., 2011). RACK1 interacts with components of the miRISC in nematodes and mammals; the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes (Jannot et al., 2011). Another study found that RACK1 binds to KH-type splicing regulatory protein (KSRP) and is required for the recruitment of mature miRNAs to RISC (Otsuka et al., 2011) PTB–PolypyrimidineTractBindingProtein(hnRNPI)wasfoundduringasearchfor proteinsinvolvedinlet-7mediatedgeneregulation.(Engelsetal.,2012).PTBinteracts with miRNAs and human AGO2 through RNA and there is a population of cellular targets thatareco-regulatedbyPTBandAGO2(Engelsetal.,2012). LRRK2 – leucine-rich repeat kinase 2 (LRRK2) gain-of-function mutations cause age-dependent degeneration of dopaminergic neurons. the analysis of the molecular mechanism of pathogenesis in Drosophila and humans revealed that LRRK2 associates with Drosophila AGO1 or human AGO2 (Gehrke et al., 2010) and that the gain-of-function LRRK2 mutant antagonizes let-7, causing derepression of Let-7 targets (Gehrke et al., 2010) APOBEC3G – the apolipoprotein-B-mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) is cytidine deaminase. APOBEC3G is an antiviral factor is found in P-bodies (Izumi et al., 2013; Wichroski et al., 2006). APOBEC3H also inhibits miRNA-mediated repression of translation (Huang et al., 2007) by competitively inhibiting bindingofMOV10toAGO2,causingeitherabnormalassemblyorabnormalmaturationof miRISC (Liu et al., 2012a). AGO loading and RISC formation ThenextimportantstepisformationofRISC,theeffectorcomplexofmiRNAandRNAi pathways. It involves formation of the RISC Loading Complex (RLC), transfer of a small RNA on an AGO-protein, and RISC activation. RISC Loading Complex (RLC) RISC assembly was so far explored more in Drosophila (Iwasaki et al., 2010; Pham et al., 2004;Tomarietal.,2004a;Tomarietal.,2004b)thaninmammals(Bernardetal.,2015; Gregory et al., 2005; MacRae et al., 2008) perhaps because of the robust in vitro system of Drosophila embryo lysate. Mammals differ from Drosophila because they do not use different Dicer and Argonaute proteins dedicated to RNAi and miRNA pathway although Introduction_to_RNAi.indd 51Introduction_to_RNAi.indd 51 09.07.20 8:3409.07.20 8:34 MAMMALS I 52 it is assumed that both pathways use a similar if not the same RLC. Our knowledge of the mammalian RLC comes mainly from cells where RLC normally loads miRNAs or from in vitroreconstitutionoftheRLCwithpurifiedproteins.TheminimalRLCiscomposed ofDicer,TARBP2andAGO2(Gregoryetal.,2005;MacRaeetal.,2008).In vitro reconstituted mammalian RLC contains one copy of each protein and has dicing, guide-strand selection, loading, and slicing activities (Bernard et al., 2015; Gregory et al., 2005; MacRae et al., 2008; Martinez et al., 2002). AGO interacts with Dicer through a subregion of the PIWI domain (the PIWI-box), whichbindsdirectlytotheDicerRNaseIIIdomain.(Tahbazetal.,2004).Single-particle EManalysissuggestedthatDicer’sN-terminalDExH/DdomaininteractswithTARBP2, whereas its C-terminal catalytic domains in the main body are proximal to AGO2 (Wang et al., 2009). Interestingly, binding of AGO to Dicer inhibits dicing activity in vitro(Tahbaz et al., 2004). Analysis of individual siRNA positions revealed that RNA sequences atpositions9–12and15–18wereassociatedwithTARBP2whilepositions19–21with AGO.AGObindingwasenhancedbypositions15–18(Takahashietal.,2014).AGO2 was reported to binds primarily to the 5’- and alternatively, to the 3’-end of pre-miRNAs. (Tanetal.,2011).AllfourhumanAGOproteinsshowremarkablysimilarstructuralpreferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3’-mid (guide position 12–15) mismatches facilitate unwinding. All these features of humanAGOproteinsarehighlyreminiscentofflyAGO1butnotflyAGO2.(Yodaetal., 2010).BiochemicalandstructuralanalysissuggeststhatTARBP2isflexiblyboundtothe DicerDExH/Ddomain(Danielsetal.,2009;Wangetal.,2009).TARBP2seemstobridge releaseofthesiRNAbyDicerandloadingoftheduplexontoAGO2.BindingbyTARBP2 may allow the siRNA intermediate to stay associated with the RLC after release from DicerandmayalsohelpinorientationofthesiRNAforAGO2loading.Justasinflies,human RISC assembly is uncoupled from dicing (Yoda et al., 2010). Analysis of miRNA-carrying RISC (miRISC) yielded a similar picture. Since loading of miRNA duplexes to AGO proteins is assisted by HSP70/ HSP90 chaperones (Maniataki and Mourelatos, 2005b; Yoda et al., 2010), HSP90 is sometimes also included as the componentofmiRLC(Liuetal.,2012b).Atthesametime,AGO2andDiceraresufficientfor processingandloadingofmiRNAsintoRISC(Tanetal.,2011). Combination of in vivo studies in Dicer-/- cells reconstituted with wild-type or catalytically inactive Dicer showed that the miRNA loading complex (miRLC) is the primary machinerylinkingpre-miRNAprocessingtomiRNAloadingandleadalsotodefinition of a miRNA Precursor Deposit Complex (miPDC) for Dicer-independent RISC loading exemplifiedbymiR-451(Liuetal.,2012b).miPDCisformedofAGO,pre-miRNA,and HSP chaperone. It functions in Dicer-independent miRNA biogenesis (e.g. miR-451) and also promotes miRNP assembly of certain Dicer-dependent miRNAs (Liu et al., 2012b). Earlierstudiessuggestedadifferencebetweenflyandhumansystemsbecausehuman RISCassemblyusingimmunopurifiedorreconstitutedhumanRLCcontainingAGO2,DicerandTARBP2didnotrequireATPhydrolysis,(Gregoryetal.,2005;MacRaeetal.,2008; ManiatakiandMourelatos,2005b).RecentdatasuggestthatATPfacilitatesalsohuman RISC loading while it is dispensable for unwinding (Yoda et al., 2010). Introduction_to_RNAi.indd 52Introduction_to_RNAi.indd 52 09.07.20 8:3409.07.20 8:34 MAMMALS I 53 Accessory RLC factors Apart from the three established RLC components, several proteins emerged as RLC cofactors, among which stand out HSP70/90 chaperones. Heat shock protein 90 was actually the firstidentifiedAGO-associatedproteinevenbeforeAGOwasassociatedwithRNAsilencing(Tahbazetal.,2001).InhibitionofHSP90reducesAGOlevels(Johnstonetal.,2010; MartinezandGregory,2013;Tahbazetal.,2001)aswellasGW182proteinlevelsand abolishes P-bodies (Johnston et al., 2010). In addition, stable binding between AGO and DicerisdependentontheactivityofHsp90(Tahbazetal.,2004)andassociationofAGO2 with HSP90 involves prolyl-hydroxylation of AGO2 (Wu et al., 2011). HSP90 activity is notrequiredforassociationofAGOwithintracellularmembrane(Tahbazetal.,2001)but appears to chaperon AGO proteins before binding RNA and may facilitate loading of small RNAs (Johnston et al., 2010). Interestingly, miRNA*s (miRNA* is an equivalent of the passenger strand) with fast turnover exhibited different sensitivity to HSP90 inhibition suggesting differential HSP90 requirements for different miRNA*s (Guo et al., 2015). HSP90 is also a negative regulator of PKR; it is able to bind and inhibit PKR phosphorylation andpreventapoptosis(Donzeetal.,2001).Thus,HSP90providesafactorbridgingRNA silencing and innate immunity. Furthermore, HSP90 co-chaperones FKBP4/5 control AGO2 expression and facilitate RISCassembly(Martinezetal.,2013).FKBP4/5wereidentifiedasAGO2-associatedproteins in mouse embryonic stem cells. Inhibition of FKBP4/5 lead to decreased Ago2 protein levels while overexpression stabilized AGO2 expression (Martinez et al., 2013). Another study has found that FKBP4 forms a stable complex with human AGO2 before small RNA loadinginthecytoplasmandisrequiredforefficientRNAi(Pareetal.,2013). Another component reported to function as an RISC-loading factor is RNA helicase A (RHA, also known as DHX9) Dicer (Robb and Rana, 2007). RHA is a conserved protein with two dsRBDs (Nagata et al., 2012) with multiple roles in the gene expression of cellular and viral mRNAs. RHA recognizes highly structured nucleotides and catalytically rearranges the various interactions between RNA, DNA, and protein molecules to provide a platform for the ribonucleoprotein complex. RHA was shown in human cells to function in theRNAipathwayandinteractwithsiRNA,AGO2,TARBP2,andDicer(RobbandRana, 2007). RHA-depleted cells, showed reduced RNAi, apparently as a consequence of lower active RISC suggesting that RHA functions in RISC as an siRNA-loading factor (Robb and Rana, 2007). A later structural analysis of dsRBDs showed that both dsRBDs are required for RISC association, and such association is mediated by dsRNA (Fu and Yuan, 2013). Are mammalian miRNAs sorted? As mentioned above, of the four AGO proteins that can be loaded with small RNAs equally well (Meister et al., 2004). All four mouse AGO proteins seem to be functionally redundant in the miRNA pathway as shown by rescue experiments in ESCs lacking all four Argonaute genes (Su et al., 2009). Consistent with this, all four AGOs are functionally equivalent when accommodating bulged miRNA duplexes, whereas AGO1 and AGO2 appear to be more effective at utilizing perfectly matched siRNAs (Su et al., 2009). Furthermore, AGO2 can Introduction_to_RNAi.indd 53Introduction_to_RNAi.indd 53 09.07.20 8:3409.07.20 8:34 MAMMALS I 54 execute endonucleolytic cleavage of cognate RNAs while all four can mediate translational repression.ThisraisesaquestionwhethersmallRNAsmayundergosomekindofsorting thatwouldresultinpreferentialloadingontospecificAGOhomologs. Structural analysis showed that all four human AGO proteins showed similar structural preferences for small-RNA duplexes, which were highly reminiscent of Drosophila AGO1 but not of AGO2 (Yoda et al., 2010). Human AGO2 and AGO3 immunoprecipitation and subsequent sequencing of small RNAs revealed that both AGOs were associated with 21–23ntRNAs,majorityofwhichweremiRNAs(Azuma-Mukaietal.,2008).Whilefifteen miRNAsshowedmorethan2-foldsignificantdifferenceinloadingontoAGO2orAGO3, it is not clear whether this discrimination occurs also in vivo (Azuma-Mukai et al., 2008). A detailed analysis of small RNAs associated with all four human AGO proteins revealed approximately equivalent amounts of sequence tags derived from miRNA loci associatedwithindividualAGOswithsomeexceptionsthatcouldbecoupledtospecificAGOs (Burroughs et al., 2011). However, further analysis suggested existence of some sorting mechanism affecting a subset of distinct isomiRs that seemed to be differentially associated withdistinctAGOproteins(Burroughsetal.,2011).Thisobservationcontrastswithanother cloning and deep sequencing experiment addressing distribution of endogenous miRNAs associatedwithAGO1–3,whichdidnotfindevidenceformiRNAsortinginhumancells. (Dueck et al., 2012). It is possible that sorting of small RNAs on AGO proteins may not be a general phenomenon while differential presence of small RNAs on AGO proteins can also emerge from selectivemechanismsoperatingafterloading.Thiscanbeillustratedonselectiveprogressive 3’ shortening of AGO2-bound miRNAs observed in the brain (Juvvuna et al., 2012). Furthermore,DuecketalalsoreportedthatAGOidentityappearstoinfluencethelength ofsomemiRNAs,whileothersremainunaffected(Duecketal.,2012).Takentogether,it seems that miRNAs are generally not sorted for loading onto AGO proteins. Notable exceptions include miRNAs with unique biogenesis such as miR-451 whose biogenesis requires AGO2 slicing activity (Dueck et al., 2012). Loading asymmetry While both siRNA strands can guide post-transcriptional silencing in mammals (Wei et al., 2009), selection of the loaded strand exhibits a clear and long-known thermodynamic bias wherethestrandwhose5′-endislessthermodynamicallystableispreferentiallyloaded onto AGO as the guide strand (Khvorova et al., 2003; Schwarz et al., 2003). Selection of the guide strand involves multiple sensors – this includes AGO2 strand selection capability (Noland and Doudna, 2013; Suzuki et al., 2015), which is enhanced in complex with DicerandTARBP2orPACT.Inaddition,strandselectionforsomemiRNAsisenhanced incomplexescontainingPACTbutnotTARBP2(NolandandDoudna,2013).Notably, TARBP2waspredictedtobeasensorofthethermodynamicstabilityof5’siRNAinstrand selectionduringRISCloading,similarlytoDCR-2andR2D2(aTARBP2homolog)in Drosophila (Wang et al., 2009). However, the supporting evidence is inconclusive (Haase etal.,2005)althoughsomearguethatTARBP2canindeedactsasasensor(Gredelletal., 2010).Furthermore,whileTARBP2functionissimilartothatofR2D2,TARBP2sequence Introduction_to_RNAi.indd 54Introduction_to_RNAi.indd 54 09.07.20 8:3409.07.20 8:34 MAMMALS I 55 is more closely related to Loquacious than R2D2 (Murphy et al., 2008). Finally, quantitative analysis of RISC assembly and target silencing activity in the presence or absence of Dicer suggest that the mammalian Dicer is nonessential for asymmetric RISC loading in vivo and in vitro.(BetancurandTomari,2012). RISC activation ThenextstepafterAGOloadingisremovalofthepassengerstrandfromtheloadedduplex RNA. In some cases, the passenger strand can be eliminated by the slicer activity where the RISCcomplexusestheguidesiRNAtocleavethepassengerstrand.Inotherwordsthefirst cleavage actually targets the passenger strand of a loaded siRNA duplex to free the guiding strand,soitcanbasepairtocognatemRNAs(Matrangaetal.,2005).Thecleavage-assisted mechanismistypicalforAGO2-loadedflyandhumansiRNAsintheRNAipathwaywhile passenger strand cleavage is not important for loading miRNAs (Matranga et al., 2005). Slicer-independent mechanism is needed to remove the passenger strands from non-slicing AGO proteins and from miRNA duplexes bound to AGO2 where the passenger strand cannotbecleaved.Asslicer-deficienthAGO1,hAGO3,andhAGO4areabletoejectthe passenger strand of siRNA duplexes at 37°C, it is apparent that AGO1, 3, and 4 can be readily programmed with siRNAs at the physiological temperature (Park and Shin, 2015). Thisimpliesthataslicer-independentmechanism,whichreliesonthethermaldynamics ofthePAZdomain(Guetal.,2012a;ParkandShin,2015),islikelyacommonfeatureof human AGOs. Importantly, RISC activation has been associated with additional factors. One of them is C3PO, an endonuclease that activates RISC (Ye et al., 2011). According to the model of RISC activation that integrates the C3PO crystal structure, Ago2 directly binds duplex siRNA and nicks the passenger strand, and then C3PO activates RISC by degrading the Ago2-nicked passenger strand (Ye et al., 2011)> Another factors is La, Sjogren’s syndrome antigen B (SSB)/autoantigen, which is acting as an activator of the RISC-mediated mRNA cleavageactivity.(Liuetal.,2011).Thus,similarlytoC3PO,Laisaregulatoryfactor helping to remove AGO2-cleaved products in order to promote active RISC formation (Liu et al., 2011). Additional small RNAs associated with AGO proteins Our literature search revealed a heterogeneous group of publications describing small RNAs loaded on AGO proteins that were clearly distinct from canonical miRNAs – small RNAs generated by the mechanism described above. A canonical miRNA is transcribed by polymerase II, the primary transcript contains a ~ 70 nt short hairpin precursor pre-miRNA, which is released by the Microprocessor complex, transported to the cytoplasm where Dicer cleaves of the loop and one of the strands of the miRNA duplex is loaded onto miRISC. However, next generation sequencing revealed existence of AGO-loaded small RNAs that were apparently generated from different substrates and by molecular mechanisms, which deviated from the canonical pathway. Below is an overview of diversity of AGO-bound RNAs, which emerged from the literature search. Introduction_to_RNAi.indd 55Introduction_to_RNAi.indd 55 09.07.20 8:3409.07.20 8:34 MAMMALS I 56 Non-canonical miRNAs can be divided across two axes – (I) according to the RNA precursor and (II) according to the proteins involved in (or omitted from) their biogenesis. Non-canonical miRNAs were discovered during systematic analyses of small RNAs in different model systems, such as disease models (e.g. (Xia et al., 2013) or cultured cells (Babiarz et al., 2011; Babiarz et al., 2008). A good experimental strategy to identify non-canonical miRNAs is a high throughput sequencing analysis of genetic models lacking some of the components of RNA silencing such as Dicer or DGCR8 (Babiarz et al., 2011; Babiarz et al., 2008). Phenotypic difference and differential expression of distinct miRNA-like sequences can indicate biological roles of non-canonical miRNAs while knock-out data offer an insight into the non-canonical biogenesis mechanism Perhaps the best known non-canonical miRNA class, which comes from unique, Microprocessor-independent precursors, are mirtrons, miRNA-like molecules arising from spliced-out introns, which are Microprocessor-independent (Babiarz et al., 2011; Berezikov et al., 2007; Ladewig et al., 2012; Schamberger et al., 2012; Sibley et al., 2012; Westholm et al., 2012). Interestingly, some predicted mirtron-like miRNAs (miR-1225 and miR-1228) are splicing-independent (simtrons) and their biogenesis involves Drosha but neither DGCR8 nor Dicer (Havens et al., 2012). Other non-canonical substrates can be, for example, 5‘-Capped RNAs (Xie et al., 2013), SINE repeat-derived, (Babiarz et al., 2008; Castellano and Stebbing, 2013), small vault RNA (svtRNA2–1a) (Minones-Moyano et al., 2013), or RNase III transcripts (Maurin et al., 2012) including annotated RNAs such as snoRNAs (Burroughs et al., 2011; Ender et al., 2008; Li et al., 2012), 7SL RNA (Ren et al., 2012), tRNA fragments (Burroughs et al., 2011; Haussecker et al., 2010; Kumar et al., 2014; Li et al., 2012; ManiatakiandMourelatos,2005a;Mauteetal.,2013;Venkateshetal.,2016).Non-canonical miRNAs can be also produced from viral RNAs (Bogerd et al., 2010; Kincaid et al., 2014; Lietal.,2009;Xuetal.,2009).Anon-canonicalsmallRNAclassofunclearsignificanceare semi-microRNAs (smiRNAs), which are ~ 12nt short RNA fragments apparently emerging from other miRNAs, such as let-7 or miR-223 (Plante et al., 2012). Non-canonicalmiRNAscanbealsoclassifiedbytheirbiogenesisasMicroprocessor, DGCR8-, or Dicer-independent. For instance, the above-mention mirtrons do not require the Microprocessor complex while simtrons require Drosha but neither DGCR8 nor Dicer. Non-canonicalmiRNAscanbealsoproducedfrombona-fidemiRNAprecursors,which give a rise to a small RNA in a non-canonical way, for example by a dual role of AGO protein (Diederichs and Haber, 2007). A classic example is miR-451, a Dicer-independent miRNAbiogenesispathwaythatrequiresAgocatalysis(Cheloufietal.,2010).Aunique type of non-canonical miRNAs are loop-miRs, which are released from the loop region of a pre-miRNA (Okamura et al., 2013; Winter et al., 2013). Target recognition and modes of silencing Target recognition TargetrecognitionbyRISCismediatedbybasepairingbetweenRISC-loadedsmallRNAs and cognate RNAs. Consistently with the structural analysis of AGO proteins, target Introduction_to_RNAi.indd 56Introduction_to_RNAi.indd 56 09.07.20 8:3409.07.20 8:34 MAMMALS I 57 recognition by siRNAs exhibits a distinct 5’ bias. Analysis of miRNA-targeted mRNAs in DrosophilaandmammalsrevealedthatmiRNAbases2–8formadistinct„seed“,which basepairsperfectlytothetargettranscript(Enrightetal.,2003;Lewisetal.,2003).Thisis consistent with the fact that the 5’ half of a small RNA provides most of the binding energy thattethersRISCtoatargetRNA(Doenchetal.,2003;HaleyandZamore,2004).Structural features of the target site are only important for RISC binding, while sequence features suchastheA/ucontentofthe3’uTRareimportantformRNAdegradation.(Hausseretal., 2009). According to analyses of RISC kinetics, small RNAs loaded onto AGO proteins are actuallycomposedoffivedistinctdomains(Fig.5):theanchor,seed,central,3’supplementary, and tail (Wee et al., 2012). Biochemical analysis of target recognition by mammalian RISC showed that the RISC is apparently not systematically scanning transcripts. RISC is unable to unfold structured RNA.Thus,RISCrandomlytransientlycontactssingle-strandedRNAandpromotessiRNA-target base pairing where the 5’end of the loaded siRNA creates a thermodynamic threshold for stable association of RISC with its target (Ameres et al., 2007). Thefactthat5’and3’endsofasiRNAareboundbydistinctbindingpocketsandthat both ends contribute differently to binding to the target lead to a „two-state model of Argonaute function proposed based on the Drosophilamodel(Tomarietal.,2004b).Inthis model,the3’endisboundinthePAZdomainandthe5’endtoinapocketattheinterface betweentheMIDandthePIWIdomains.The5’endispre-organizedtointeractwiththe cognate mRNA and, upon binding, the 3’ end is dislodged from the binding pocket to allow for base pairing of the 3’ end. Importantly, kinetics of silencing is critical for understanding target recognition and silencing by of small RNAs. A kinetic study of Drosophila and mouse AGO2 found that mouse AGO2, which mainly mediates miRNA-directed repression in vivo, dissociates rapidly and with similar rates for fully paired and seed-matched targets (Wee et al., 2012). An important conclusion from this study is that low-abundant miRNAs are unlikely to contribute much biologically meaningful regulation because they are present at a concentration less than their KD for seed-matching targets (Wee et al., 2012). Theseresultsweresubsequentlycorroboratedbysinglemoleculeanalysis.Single-moleculefluorescenceexperimentsusingaminimalRISC(asmallRNAandAGO2)showed that target binding starts at the seed region of the guide RNA (Chandradoss et al., 2015; Jo et al., 2015a; Jo et al., 2015b). AGO2 initially scans for complementarity to nucleotides 2–4 of the miRNA and this interaction propagates into a stable association when target complementarity extends across the seed (Chandradoss et al., 2015). Stable RISC binding is thusefficientlyestablishedwiththeseedmatchonly,providingapotentialexplanationfor the seed-match rule of miRNA target selection (Chandradoss et al., 2015; Jo et al., 2015a; Jo et al., 2015b). 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Introduction_to_RNAi.indd 80Introduction_to_RNAi.indd 80 09.07.20 8:3409.07.20 8:34 https://doi.org/10.14712/9788024643724.4 81 RNAi AND miRNA PATHWAYS IN MAMMALS II – BIOLOGICAL ROLES Mammals II Keywords:dsRNA,siRNA,miRNA,Dicer,TARBP2,PACT,Argonaute PETRSVOBODA Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska1083,14220Prague4,CzechRepublic Correspondence to: Petr Svoboda, Institute of Molecular Genetics ASCR, Videnska1083,14220Prague4,CzechRepublic,tel.#+420241063147, e-mail: [email protected]. ABSTRACT RNAsilencingdenotessequence-specificrepressionmediatedbysmallRNAs.Inmammals,therearetwoclosely related pathways, which share several protein factors: RNA interference (RNAi) and microRNA (miRNA) pathway.ThemiRNApathwayregulatesendogenousprotein-codinggeneexpression.Ithasbeenimplicated in many biological processes and majority of mammalian genes appear to be directly or indirectly exposed to miRNA-mediated regulations. RNAi generally serves as a form of innate immunity targeting viruses and mobile elements,althoughitoccasionallyalsoacquiredfunctioninprotein-codinggeneregulation.Thefunctionof RNAi in mammals is still poorly understood but it is clear that proteins supporting RNAi are also involved in miRNA biogenesis and function. Because of the large volume of the existing literature, the review of mammalian miRNAandRNAipathwayswasdividedintotwoparts,wherefirstonereviewedcomponentsofthepathways andthesecondone,presentedhere,reviewsrolesandsignificanceofthepathways. Introduction InthefirstpartofthereviewoffmammalianRNAiandmiRNApathways,Ifocusedon mechanistic description of the pathways. Here, I will provide an overview of biological roles and biological phenomena associated with mammalian RNAi and miRNA pathways (Fig. 1). miRNA-mediated control of gene expression – important functional aspects ThecurrentmiRBase(KozomaraandGriffiths-Jones,2014)edition22.1annotates1917humanmiRNAlocithatgiveriseto2654annotatedmiRNAs.Thereare1234precursorsand Introduction_to_RNAi.indd 81Introduction_to_RNAi.indd 81 09.07.20 8:3409.07.20 8:34 MAMMALS II 82 1978 mature miRNAs annotated in mouse. A simple connection of these counts with the factthatonlynucleotides2–8ofamiRNAaresufficientfortargetrecognitionandsuppression implies that miRNA-mediated repression is a widespread and extremely evolvable regulatory system for gene expression. At the same time, one should not forget the above-mentioned stoichiometry between miRNAs and their target sites that is needed for efficientsilencing. Evolution of miRNAs is fast – there are only a few miRNAs conserved between Drosophila and mammals. Given the diversity of canonical and non-canonical miRNAs, it is conceivable that miRNAs to emerge from random formation of Drosha/Dicer substrates. Newly evolving miRNAs likely form a considerable portion of annotated miRNAs, especially inspecieswheremiRNAsaredeeplysequencedandlow-abundantmiRNAsareidentified. According to the evolutionary theory, new miRNAs would either acquire function and becomefixedduringevolutionortheywouldbelost.Inaddition,thetargetrepertoireof existing miRNAs can also rapidly evolve since a single point mutation can weaken an existingregulationorcreateanewone.Thisideaisconsistentwiththedatashowingthat mammalianmRNAsareunderselectivepressuretomaintainand/oravoidspecific7-nucleotideseedingregions(Farhetal.,2005).ItcanbenicelyexemplifiedontheTexelsheep phenotype where a single mutation creating a novel miRNA target site in myostatin causes the exceptional meatiness of this breed (Clop et al., 2006). Figure 1 Mechanistical merging of miRNA and RNAi pathways in mammals Introduction_to_RNAi.indd 82Introduction_to_RNAi.indd 82 09.07.20 8:3409.07.20 8:34 MAMMALS II 83 ThesetofmiRNAsineachcelltypeformsacombinatorialpost-transcriptionalregulation system stabilizing gene expression pattern. miRNAs have widespread impact onexpressionandevolutionofprotein-codinggenes(Farhetal.,2005).Thenumberof mRNAs that have functionally important interaction with miRNAs (i.e. suppression of this interaction yields a phenotype) in a studied model system is presumably small and certainlydifficulttodiscernamongthepossibleinteractions.Thus,everysearchforfunctionally important interactions between miRNAs and their targets has to face the fact that miRNAs represent a dynamically evolving system with countless random interactions, which are not biologically relevant. Extracellular microRNAs AninterestingresearchfielddevelopedaroundthereleaseofmiRNAsfromcells,detection of extracellular miRNAs, and transfer between cells. Importantly, the vast majority of the references provided descriptive and correlative data documenting presence of circulating miRNAs under different conditions (e.g. (Arroyo et al., 2011; Bellingham et al., 2012; Huangetal.,2013b;Luoetal.,2009;Novellinoetal.,2012;Turchinovichetal.,2011). I will not review the bulk of the circulating RNA literature, which provides data concerning biomarker potential of circulating miRNAs, undoubtedly of extreme clinical relevance but of minimal relevance for this review. Below, I summarize results, which admittedly raise more questions than provide satisfactory answers. Small RNAs can be transmitted from one cell to another under physiological conditions, as evidenced, for example, by systemic RNAi in arthropods or plants. Small RNAs can utilize dedicated transporters, common communication channels, or secretory vehicles. It was also reported that Gap junctions can serve for miRNA transfer from microvascular endothelialcellstocoloncancercells(Thuringeretal.,2016).Circulatingmammalian miRNAs were reported 2008 when they were found in serum of lymphoma patients; they were immediately recognized as potential non-invasive biomarkers for cancer diagnostics andtreatment(Lawrieetal.,2008).Thesameyear,placentalmiRNAswerefoundcirculating in maternal plasma (Chim et al., 2008), which was one of the discoveries leading to the notion that miRNAs could be a mobile regulating molecule (Iguchi et al., 2010) and that could even mediate transgenerational epigenetic heritance (Sharma, 2015) or be transmitted acrossspecies(Bucketal.,2014;Zhangetal.,2012).Sincethen,extracellularmiRNAs wereidentifiedinabroadrangeofbiologicalfluids,includingplasma,aqueoushumour, cerebrospinalfluid,nasalmucus,ormilk(Baglioetal.,2015;Dismukeetal.,2015;Huang et al., 2013b; Izumi et al., 2015; Kropp et al., 2014; Pegtel et al., 2011; Wu et al., 2015a). miRNAswereidentifiedinthecargoofexosomes,membranousvesicles40to100nmin diameter, which are constitutively released by almost all cell type and are found essentially ineverybiologicalfluid(reviewed,forexample,inRak,2013;Yoonetal.,2014) However, extracellular miRNAs do not need to be necessarily encapsulated in extracellular vesicles, as two studies showed that 95–99% of extracellular miRNA are not in extracellular vesicles but associated with AGO proteins in serum and cell culture media (Arroyo etal.,2011;Turchinovichetal.,2011).Furthermore,mostindividualexosomesinstandard Introduction_to_RNAi.indd 83Introduction_to_RNAi.indd 83 09.07.20 8:3409.07.20 8:34 MAMMALS II 84 preparationsdonotseemtocontainbiologicallysignificantnumbersofmiRNAs(Chevillet etal.,2014).ThemolecularmechanismofmiRNArelease,eitherasacargoinavesicleor free, is poorly understood and the current knowledge does not allow for building a coherent model as the literature is scarce. Non-templated nucleotide additions were found to distinguish between cellular miRNAs, which were 3’ end adenylated in cells whereas 3’ end uridylated isoforms appeared overrepresented in exosomes suggesting a possible role of3’terminalmodificationsinsortingmiRNAsintoextracellularvesicles(Koppers-Lalic et al., 2014). Recently, ALIX, an accessory protein of the endosomal sorting complex, it has been implicated in sorting miRNAs into extracellular vesicles based on its interaction with AGO2 and reduced miRNAs levels in extracellular vesicles upon Alix knock-down (Iavello et al., 2016). Importantly, any model where miRNAs would be carried over to regulate gene expression by the canonical miRNA activity must face the kinetic data mentioned above (Wee et al., 2012). While one cannot exclude a non-canonical signalling function of circulating miRNAs (which has not been conclusively demonstrated yet), the literature on circulating RNAs may include misleading statements, which are unsupported by experimental evidence. Takentogether,whileexistenceofcirculatingmiRNAshasbeendemonstratedbeyond a doubt, experimental evidence for their function (if any) is not conclusive. Exosomal vesicles can carry miRNAs and siRNAs – in the latter case, exosomes were adapted for a delivery tool for siRNAs, which has a good potential for further development of siRNA therapy (El-Andaloussi et al., 2012; Kumar et al., 2015; Lasser, 2012; Lee et al., 2012; Nguyen and Szoka, 2012; Shtam et al., 2013; Wahlgren et al., 2012; Wahlgren et al., 2016). RNAi pathway in mammals– important functional aspects Itshouldreiteratedthatthe,“so-called”RNAiknock-downwithsiRNAsinmammalian cells is essentially using the miRNA pathway with retained the ability to cleave perfectlycomplementarytargetsbyAGO2.ThemammaliancanonicalRNAi(i.e.longdsRNA-driven) is a dormant pathway, at best. By that is meant that the protein factors present ineverymammaliancells(Dicer,TARBP2,andAGO2)arecompetenttosupportRNAi butlongdsRNAdoesnotefficientlyinduceRNAiinmostmammaliancells(Nejepinska etal.,2012).ThisnotionissupportedbythereconstitutionofhumanRNAinterferencein buddingyeastdemonstratesthatDicer,TARBP2,andAGO2aresufficienttofunctionally reconstituteRNAi(Suketal.,2011).Thisdemonstratesthatthesethreeproteinsconstitute the essential core of RNAi mechanism although RNAi is not properly reconstituted whenbonafideRNAiprecursorswereco-expressed(Wangetal.,2013).Theproblemis apparently at the level of Dicer processing as the human slicer AGO2 RNAi role is so conserved that it could function in RNAi in the early divergent protozoan Trypanosoma brucei, demonstrating conservation of basic features of the RNAi mechanism (Shi et al., 2006). In an analogous experiment, human AGO2 could not replace Arabidopsis thaliana AGO1 in the miRNA pathway (Deveson et al., 2013). In a sense, these different results are not that surprising considering the minimal requirements for RNAi and the complexity of the miRNA pathway, which provides a larger space for evolution of incompatible adaptations. Introduction_to_RNAi.indd 84Introduction_to_RNAi.indd 84 09.07.20 8:3409.07.20 8:34 MAMMALS II 85 However, there are some cases indicating that RNAi is still active in mammals and, under unique circumstances, may be even an essential pathway. ThemainbottleneckforcanonicalRNAiinmammalsisefficientproductionofsiRNAs from long dsRNA, which is poor in most mammalian cells (Flemr et al., 2013; Nejepinska et al., 2012). However, several reports showed that induction of RNAi with intracellular expression of long dsRNA can be achieved in transformed and primary somatic cells (Dialloetal.,2003;Elbashiretal.,2001;Ganetal.,2002;ShinagawaandIshii,2003;Tran etal.,2004;Yietal.,2003).ThesedataimplythatRNAicanoccurifthereisasufficient amount of long dsRNA, which is directed preferentially to RNAi but not into other dsRNA pathways. Under these circumstances, the limiting factor is just Dicer’s ability to produce siRNA (Flemr et al., 2013). Endogenous RNAi in the germline Retrotransposon repression in mouse oocytes RNAi-mediated mobile element silencing has also been documented in the mouse germline (Tametal.,2008;Watanabeetal.,2006;Watanabeetal.,2008).MutationsinthepiRNA pathway components are detrimental to sperm development, suggesting that piRNAs are the dominant class of small RNAs controlling mobile element activity in the male germline (reviewedinTothetal.,2016).Incontrast,femalemicelackingfunctionalpiRNApathway are fertile with no obvious defects in oocytes (Carmell et al., 2007). Endo-siRNAs suppress TEssilencinginmammalianoocytesasdocumentedbyderepressionofsomeretrotransposons in oocytes depleted of Dicer or AGO2 (Murchison et al., 2007; Watanabe et al., 2008). As already proposed for invertebrates, the piRNA and endo-siRNA pathways likely cooperate in creating a complex silencing network against mobile elements in the mammalian germline.LongterminalrepeatMTelementsandSINEelementsarestronglyupregulated in Dicer-/- oocytes, while the levels of IAP transposon are elevated in the absence of MILI protein but not in Dicer-/- oocytes (Murchison et al., 2007; Watanabe et al., 2008). Still many locicomposedofothertypesofTEs,e.g.LINEretrotransposons,giverisetobothpiRNAs and endo-siRNAs, again suggesting that the biogenesis of these small RNAs is interdependent.TheroleofendogenousRNAiinTEsilencingextendsfromgermcellstopreimplantation embryo stages. Apart from maternally derived piRNAs and endo-siRNAs, which persist in the embryos for a large part of preimplantation development, zygotic endo-siRNAs are generated de novomainlytocontroltheactivityofzygoticallyactivatedMuERV-L retrotransposon (Ohnishi et al., 2010; Svoboda et al., 2004). SINE-derived endo-siRNAs also increase in abundance in early embryo stages, which is consistent with the observation that B1/Alu SINE endo-siRNAs account for a vast majority of endo-siRNAs sequenced from mouse ES cells (mESCs) (Babiarz et al., 2008). Whether these SINE endo-siRNAs playanactiveroleinTEsilencinginmESCssimilarlytootherTE-derivedendo-siRNAs in oocytes remains to be determined. RNAi-dependent silencing of LINE transposons has also been described in cultured HeLa cells, where endo-siRNAs derived from bidirectional transcripts of sense and antisense L1 promoter were proposed to control L1 activity (Yang and Kazazian, 2006). Although some evidence for retrotransposon-derived endo-siRNAs Introduction_to_RNAi.indd 85Introduction_to_RNAi.indd 85 09.07.20 8:3409.07.20 8:34 MAMMALS II 86 from mammalian somatic cells was obtained from deep sequencing data (Kawaji et al., 2008),aconvincingsupportforthefunctionofendo-siRNAsinTEsilencinginmammalian somatic tissues, has yet to be provided. Control of endogenous genes in mouse oocytes In mice, perturbation of the endo-siRNA pathway in oocytes is responsible for severe meioticdefectsandresultingfemaleinfertility.Targetedoocyte-specificknockoutofbothDicer and Ago2 lead to similar phenotypes including chromosome misalignment and defective spindle(Kanedaetal.,2009;Murchisonetal.,2007;Tangetal.,2007).Theseeffects were originally attributed to the loss of maternal miRNAs. However, miRNA pathway is suppressed in mouse oocytes and oocytes lacking Dgcr8, which is required for canonical miRNAbiogenesis,canbefertilizedanddonotshowanysignificantdisturbanceofthe transcriptome(Maetal.,2010;Suhetal.,2010).ThismeansthatthecanonicalmiRNA pathway is non-essential and largely inactive in mouse oocytes despite intact biogenesis of miRNAs (Fig. 2). In fact, the spindle phenotype is caused by the loss of a highly active RNAi pathway in mouse oocytes. High-throughput analysis of small RNAs in mouse oocytesrevealedauniqueclassofendo-siRNAsderivedfromprocessedpseudogenes(Tam etal.,2008;Watanabeetal.,2008).TranscriptomesofoocyteslackingDicer and Ago2 (including oocytes expressing catalytically-dead AGO2) are similarly affected (Kaneda et al., 2009; Stein et al., 2015). At the same time, genes matching pseudogene-derived endo-siRNAs are enriched in the group of upregulated genes in both knockouts (Kaneda etal.,2009;Steinetal.,2015;Tametal.,2008;Watanabeetal.,2008). In addition, putative endo-siRNA targets are enriched in cell cycle regulators and genes involvedinmicrotubuleorganizationanddynamics(Tametal.,2008).Thesefindingssuggest that regulation of protein-coding genes by endo-siRNAs controls the equilibrium of protein factors required for proper spindle formation, chromosome segregation and meiosis progression in mouse oocytes. As pseudogenes are rapidly evolving source of dsRNA for endo-siRNA production, it will be interesting to investigate whether the role of RNAi in spindle formation during meiotic maturation of oocytes is conserved in mammals. Thereasonforhighlevelsofendo-siRNAsandthehighRNAiactivityinmouseoocytes is the aforementioned truncated Dicer isoform that lacks the N-terminal helicase domain (Flemretal.,2013)(Fig.2).ItefficientlygeneratessiRNAsfromlongdsRNAs,andissufficientforenhancingRNAiinculturedcellswhileitslossinmouseoocytesyieldsthesame phenotype as conditional knock-outs of Dicer or Ago2 (Flemr et al., 2013). Endo-siRNAs have also been proposed to contribute to the self-renewal and proliferation of mouse embryonic stem cells (mESCs), since the proliferation and differentiation defects observed in Dicer-/- mESCs are more dramatic than in Dgcr8-/- mESCs (Kanellopoulou et al., 2005; Murchison et al., 2005; Wang et al., 2007). A population of endo-siRNAs derivedmostlyfromhairpinformingB1/AlusubclassofSINEelementswasidentified in mESCs (Babiarz et al., 2008). Fragments of SINE elements are commonly present in untranslated regions of protein-coding transcripts and it is therefore possible that SINE-derived endo-siRNAs participate in posttranscriptional gene silencing in mESCs. However, this hypothesis has not been tested experimentally. Introduction_to_RNAi.indd 86Introduction_to_RNAi.indd 86 09.07.20 8:3409.07.20 8:34 MAMMALS II 87 Endogenous RNAi in the soma Little evidence is available for potential role of endo-siRNAs in the regulation of protein-codingmRNAsinmammaliansomatictissues.Thenaturalantisensetranscriptionin somatic cells, which has a potential to generate dsRNA, yields low levels of endo-siRNAs, whose biological relevance is questionable. At the same time, endo-siRNAs derived from natural antisense transcripts of Slc34agenewereidentifiedinmousekidney,where Na/phosphate cotransporter exerts its physiological function (Carlile et al., 2009). However, changes in expression levels of Slc34a upon suppression of the endo-siRNA pathway have not been addressed. In mouse hippocampus, deep sequencing revealed a set of potential endo-siRNAs generated from overlapping sense/antisense transcripts and from hairpinstructureswithinintronsofprotein-codinggenes(Smalheiseretal.,2011).The most abundant endo-siRNAs from SynGAP1 gene locus were also found in complexes with AGO proteins and FMRP in vivo. Interestingly, a large part of potential hippocampal endo-siRNA targets encode for proteins involved in the control of synaptic plasticity and thenumberofendo-siRNAsderivedfromthesegenelociincreasedsignificantlyduring olfactory discrimination training (Smalheiser et al., 2011). Given the fact that vast majority ofidentifiedendo-siRNAsequencesmappedtointronicregions,theendo-siRNAscould Figure 2 miRNA & RNA arrangement in mouse oocytes Introduction_to_RNAi.indd 87Introduction_to_RNAi.indd 87 09.07.20 8:3409.07.20 8:34 MAMMALS II 88 act co-transcriptionally on nuclear pre-mRNAs, perhaps similarly to the mechanism of RNAi-mediated inhibition of RNA Pol II elongation described in C. elegans (Guang et al., 2010). Alternatively, endo-siRNAs could control correct distribution of target mRNAs as unspliced pre-mRNA can be exported from the neuronal nucleus and transported to dendritesforprocessing(Glanzeretal.,2005).Inanycase,thesefindingsopenanattractive hypothesis that endo-siRNAs participate in synaptic plasticity during learning process and the neuronal endo-siRNA pathway might be also linked to various neurodegenerative disorders (Smalheiser et al., 2011). Antiviral RNAi In contrast to nematodes and insects, data supporting involvement of mammalian RNAi in antiviral defense is weak (reviewed in detail in Cullen, 2006; Cullen et al., 2013). It is unlikely that RNAi substantially acts as an antiviral mechanism in mammals where long dsRNA induces a complex sequence-independent antiviral response, commonly known as the interferon response (reviewed in Gantier and Williams, 2007). Consistent with this, no siRNAs of viral origin have been found in human cells infected with a wide range of viruses (Pfeffer et al., 2005). Occasional observations, such as detection of a single siRNA inHIV-1infectedcells(Bennasseretal.,2005)doesnotprovideanyconclusiveevidence that RNAi is processing viral dsRNA and suppresses viruses under physiological conditions in vivo. It must be stressed that circumstantial evidence suggesting the role of RNAi in viral suppression must be critically examined and interpreted. One has to keep in mind, for example, thatdata,whichappearasevidenceforviralsuppressionbyRNAi,couldreflectmiRNA-mediated effects. Since viruses co-evolve with different hosts and explore all possible strategiestomaintainandincreasetheirfitness,itisnotsurprisingthatviralreproductive strategies come into contact with mammalian RNA silencing pathways, particularly the miRNA pathway, which shares components with the RNAi pathway. For example, EpsteinBarrvirus(EBV)andseveralothervirusesencodetheirownmiRNAs(Parameswaran et al., 2010; Pfeffer et al., 2005; Pfeffer et al., 2004; Sullivan et al., 2005) or take advantage of host cell miRNAs to enhance their replication (Jopling et al., 2005). Another evidence for an interaction between viruses and RNA silencing is the presence of putative suppressors of RNA silencing (SRS) in various viruses. As viral genomes rapidly evolve, SRS should be functionally relevant. For example, B2 protein in Nodaviruses (e.g.FHV)isessentialforreplication,inhibitsDicerfunction,andB2-deficientFHVcanbe rescuedbyartificialinhibitionofRNAiresponse(Lietal.,2002).B2proteinalsoenhances the accumulation of Nodaviral RNA in infected mammalian cells (Fenner et al., 2006; Johnsonetal.,2004).OtherpotentialSRSmoleculeshavebeenidentifiedinvirusesinfecting vertebrates,suchasAdenovirusVA1noncodingRNA(LuandCullen,2004),Influenza NS1protein(Lietal.,2004),VacciniavirusE3Lprotein(Lietal.,2004),EbolavirusVP35 protein(Haasnootetal.,2007),TASproteininprimatefoamyvirus(Lecellieretal.,2005), orHIV-1TATprotein(Bennasseretal.,2005). TheexistenceofSRSinvirusesinfectingmammalsdoesnotprovethatthesevirusesare targeted by mammalian RNAi. First, viruses may have a broader range of hosts (or vectors), Introduction_to_RNAi.indd 88Introduction_to_RNAi.indd 88 09.07.20 8:3409.07.20 8:34 MAMMALS II 89 including,e.g.bloodsuckinginsects.Thus,aviruscanbetargetedbyRNAiinonehost and by another defense mechanism in another one. For example, the Dengue virus, whose life cycle takes place in humans and mosquitoes, is targeted by RNAi in mosquitoes and it likelyevolvedanadaptationtocircumventRNAi(Sanchez-Vargasetal.,2009).Second, viral SRS in mammalian cells may have other purpose than counteracting viral suppression by RNAi. Since biogenesis and mechanism of action of mammalian miRNAs overlaps with RNAi, it is possible that the role of such SRS is to modify cellular gene expression by suppressingtheactivityofmiRNAs.Third,themaineffectofSRSmaybeaimedatother defense mechanisms recognizing and responding to dsRNA and, as a consequence, SRS effects on RNAi are observed. Systemic RNAi in mammals Non-cell autonomous RNA with an extent similar to that of C. elegans or in some insects is highly unlikely to function in vertebrates. However, a limited environmental or systemic RNAi may exist there as the homologs of sid-1 have been found in all sequenced vertebrate genomes(JoseandHunter,2007).Twosid-1homologs(SidT1andSidT2)arepresentin miceandhumanswithadocumentedroleforSidT1indsRNAuptakeinhumans(Duxbury et al., 2005; Wolfrum et al., 2007). Furthermore, experimental overexpression of human SidT1significantlyfacilitatedcellularuptakeofsiRNAsandresultedinincreasedRNAi efficacy(Duxburyetal.,2005).Asitwillbediscussedlater,themammalianimmunesystem employs a number of proteins responding to dsRNA independently of RNAi (Gantier and Williams, 2007), while RNAi does not seem to participate in the innate immunity (Cullen,2006;Cullenetal.,2013).Thus,theprimaryroleofadsRNAuptakemechanism in mammals is likely not involving RNAi even though it could have served such a role in an ancestral organism. Nuclear function of small RNAs Theliteraturesearchyieldedalargeheterogeneousgroupofpublicationsconcerningnuclear localization of Dicer and AGO proteins as well as nuclear effects, including transcriptional gene silencing. Some of these observations might come from physiologically relevant nuclear silencing mechanisms. However, when critically evaluating published studies, not enough evidence was found, to establish a model for transcriptional silencing in mammals; except of the PIWI-induced transcriptional silencing in the germline (REF). Here, I will provide an overview of nuclear aspects of RNA silencing and highlight those observations which might be related to the miRNA pathway or long dsRNA response. Homology-dependentphenomenaandobservationsthatmayreflectnuclearmechanisms involving small RNAs can be sorted into several areas, which will be discussed further below: Indirect effects of miRNAs on chromatin Nuclear RNAi (nuclear post-transcriptional silencing) Transcriptional regulations (stimulation/repression) by exogenous small RNAs Introduction_to_RNAi.indd 89Introduction_to_RNAi.indd 89 09.07.20 8:3409.07.20 8:34 MAMMALS II 96 2012; Wei et al., 2012). What is somewhat confusing in DNA-repair associated small RNAsistheroleofmiRNA-specificfactorsDrosha(Franciaetal.,2012)orDGCR8 (Swahari et al., 2016). Despite the heterogeneity of the nuclear effects and many unknowns, some common themes emerged, allowing for formulating testable hypotheses that could be critically evaluated. First, nuclear effects can be mediated by small RNAs provided in trans. Second, smallRNAsrecruitAGOproteinsinasequence-specificmanner,mostlikelyrecognizing alocaltranscript(perhapsanncRNA).Third,theeffectinvolvesachangeinthechromatin structure.Thus,bycarefullyexaminingessentialexogenoussiRNApropertiesinpreviously reported nuclear effects, one should be able to demonstrate that the silencing phenomenon truly involves an AGO-loaded small RNA engaging another nuclear RNA and whether theeffectrequiresthe“slicer”activity.Detailedexaminationoftheseedsequencewould alsodiscernbetweenspecificnucleareffectsandoff-targeting.Furthermore,shouldthe effect involve small RNA loaded AGO protein, the kinetics of the phenomenon should be in agreement with known RISC kinetics discussed above. Finally, if the aforementioned phenomena rely on localized recruitment of AGO-loaded small RNAs, one should be able tomimicthoseeffectsbytetheringAGOproteinsthroughsequence-specificDNAbinding modulessuchasthoseemployedbyTALENorCRISPRnucleases.Theseresearchdirections should be combined with validated antibodies for chromatin immunoprecipitation andimmunofluorescence(orepitopeknock-inintocandidategenes),moreextensiveuseof mutants defective in RNA silencing, detailed quantitative analysis of cellular fractionation andidentificationsofinteractingpartner,studiesofputativenuclearimportandexportsignals of Dicer and AGO proteins, and advanced imaging techniques. Other dsRNA–associated mechanisms I – dsRNA sensing in the interferon pathway Long dsRNA is not a usual RNA molecule in eukaryotic cells while RNA viruses produce dsRNA during replication. A common mechanism repressing viruses in non-vertebrate species is RNA silencing (Wang et al., 2006; Wilkins et al., 2005). However, response to foreign long dsRNA in mammals is much more complex and involves a set of sequence-independentsensorstriggeringexpressionofadefinedsetofgenesknownasinterferon-stimulatedgenes(ISGs).Theinterferonpathwayisthemostubiquitoussequence-independent pathwayinducedbydsRNAinmammaliancells(reviewedindetailindeVeeretal.,2005). Among the relevant sensors recognizing cytoplasmic dsRNA are protein kinase R (PKR), thehelicaseRIG-I,MDA5,2’,5’-oligoadenylatesynthetase(2’,5’-OAS),orToll-likereceptors(TLR3,7,8)(reviewedinGantierandWilliams,2007;SadlerandWilliams,2007). Notably, there are also dsRNA-independent mechanisms that can activate interferons in mammalian cells. Altogether, different stimuli are being sensed and converge on activation ofoverlappingbutdistinctsetsofISGs(Geissetal.,2001).Thesituationisevenmore convoluted by cellular diversity as some cell types, particularly immune cells, can elicit theinterferonresponsebyadditional,cell-type-specificpathways(reviewedinSchleeand Hartmann, 2010). Introduction_to_RNAi.indd 96Introduction_to_RNAi.indd 96 09.07.20 8:3409.07.20 8:34 MAMMALS II 97 PKR PKR is the oldest known mammalian dsRNA sensing protein. A pioneering work by Hunter et al. showed that different types of dsRNA can block translation in reticulocyte lysates (Hunteretal.,1975).AnalysisofthephenomenonidentifiedPKRthatisactivatedupon binding to dsRNA and blocks translation by phosphorylating the alpha subunit of eukaryoticinitiationfactor2(eIF-2α)(Meursetal.,1990).ActivationofPKRalsoincludes activationoftheNFκBtranscriptionfactorandalargenumberofinterferon-stimulated genes (ISGs) (Geiss et al., 2001). PKR response to viral dsRNA can be coordinated with other dsRNA sensors, such as RIG-I and MDA5 (Sen et al., 2011). PKR can also respond to endogenous RNAs in unique physiological regulations (Bevilacqua et al., 1998; Bommer et al., 2002). However, endogenously expressed long dsRNA does not necessarily induce canonical PKR response with interferon activation, although PKR binding to dsRNA and restricted translational repression can be observed (Nejepinska et al., 2012; Nejepinska et al., 2014). It was believed that dsRNA <30-bp in length does not induce PKR. However, Marques et al. reported that, siRNAs can bind and activate PKR in vitro regardless of siRNA termini (Marques et al., 2006) arguing against the long-established 30-bp length as the minimal size-limit for PKR activation. Therearealsootherdataindicatingsensitivityof PKR to dsRNA motifs shorter than 30-bp (Puthenveetil et al., 2006; Reynolds et al., 2006; ZhengandBevilacqua,2004). RIG-I-like receptors (RIG-I, MDA5, LGP2) Mammalian somatic cells can respond to dsRNA in a sequence-independent manner.. In addition to PKR, several other proteins recognizing dsRNA are integrated to the interferon response, including helicases RIG-I (retinoic-acid-inducible gene-I, also known as DDX58), MDA5 (IFIH1), and LGP2 (DHX58), which sense cytoplasmic dsRNA and activate interferon expression. RIG-I is a cytoplasmic sensor differentiating between endogenous and foreign RNAs structures. In particular, RIG-I is activated by blunt-ended dsRNAs with or without a 5’-triphosphate, by single-stranded RNA marked by a 5’- triphosphate, and by polyuridine sequences. RIG-I domains organize into a ring around dsRNA, capping one end, while contacting both strands; the structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA (Jiang et al., 2011a; Jiang et al., 2011b). Like RIG-I and LGP2, MDA5 preferentially binds dsRNA with blunt ends (Li et al., 2009a). RIG-I,MDA5,andLGP2exhibitdifferencesinrecognizingspecificRNAstructuresand different types of viruses providing a broader range of coordinated sensitivity do different potential threats (Kato et al., 2006; Li et al., 2009b; McCartney et al., 2008; Sen et al., 2011; Slater et al., 2010; Wu et al., 2015b). Interestingly, RIG-I can become activated also withsiRNAslacking2-nt3’overhangs(Marquesetal.,2006).Thesedataimplythat2-nt 3’ overhangs generated by Dicer are the structural basis for discriminating between Dicer products and other short dsRNA. Roles of MDA5 and LGP2 in siRNA-mediated interferon response remains to be addressed. Furthermore, recognition 5’ triphosphate RNA ends RIG-I (Hornung et al., 2006; Pichlmair et al., 2006) highlights importance of appropriate Introduction_to_RNAi.indd 97Introduction_to_RNAi.indd 97 09.07.20 8:3409.07.20 8:34 MAMMALS II 98 processing of 5’ termini of RNAs produced by phage polymerases when such RNAs are used in mammalian cells. It is not clear how PKR and RIG-I pathways are integrated. RIG-I binds siRNAs (with or without 2-nt 3’ overhangs) in vitro and it shows greater unwinding of blunt-ended siRNAs. Unwinding is then translated into the interferon activation mediated via IRF-3. Toll-like Receptor 3 (TLR3) TLR3isamemberoftheToll-likereceptor(TLR)familyandfunctionsasasensorof extracellular, intracellular and viral dsRNAs (Amarante et al., 2011; Seo et al., 2013; Wang etal.,2015b;Wuetal.,2015b;Yangetal.,2006b).TLR3hasdistinctorcomplementary roles to RIG-I and related helicases in sensing foreign molecules and activating downstream responses (Livengood et al., 2007; McCartney et al., 2009; Slater et al., 2010; Wu et al., 2015b). Oligoadenylate Synthetase (OAS) Interferon and dsRNA also activate 2’,5’-oligoadenylate synthetase (2’,5’-OAS) that produces 2’,5’ oligoadenylates with 5’-terminal triphosphate residues that subsequently induce activationofRNAseL;aproteinresponsibleforgeneralRNAdegradation(deVeeretal., 2005). TARBP2 and PACT InteractionsbetweenRNAi,miRNA,andinterferonresponsearepoorlyunderstood.There aretwoclearmechanisticconnectionsbetweenthesetwopathways.First,TARBP2and PACT,twodsRNAbindingproteins,whichwerementionedearlierasDicer-interacting proteins,interactalsowithPKR.Notably,whileTARBP2inhibitsPKR(Cosentinoetal., 1995;Parketal.,1994),PACThastheoppositerole(PatelandSen,1998).Whilecytoplasmic long dsRNA in somatic cells apparently triggers the interferon response, it is not clear if the same dsRNA is also routed into the RNAi pathways. Experiments in oocytes and undifferentiated embryonic stem cells (Stein et al., 2005; Yang et al., 2001) suggest that RNAi dominates response to cytoplasmic long dsRNA in the absence of a strong interferon response and that the interferon pathway dominates when its relevant components are present. On the other hand, this view may be too simplistic as it does not explain the lack of both, RNAi and interferon response, in somatic cells expressing long dsRNA (Nejepinska et al., 2012; Nejepinska et al., 2014). In any case, understanding the role of TARBP2andPACTisoformsinroutinglongdsRNAintoRNAiandinterferonpathways requires further studies. ThereisaclearevolutionaryconnectionbetweenRNAiandinterferonresponse.The above-mentioned mammalian RNA helicases RIG-I, LGP2 and MDA5 are the closest homologs of helicases involved in processing of long dsRNA during RNAi in C. elegans. Notably, RIG-I is an established component of the interferon response to long dsRNA (Yoneyamaetal.,2004).Thissuggeststhattheinterferonresponse,whichhasacommon Introduction_to_RNAi.indd 98Introduction_to_RNAi.indd 98 09.07.20 8:3409.07.20 8:34 MAMMALS II 99 trigger and evolved after the RNAi pathway, adopted several components from the latter pathway. It remains to be determined whether these and other components of RNAi lost their function in RNAi entirely or mediate some form of a cross-talk between RNAi and interferon response. Finally, there is also a complex relationship between miRNA and interferon pathways (Ingle et al., 2015; Ostermann et al., 2012; Shapiro et al., 2014; Xu et al., 2011). One connectionisexemplifiedbyviralmiRNAs,whichvirusesusetoregulatethehostresponse,in particular factors of the interferon pathway (Ostermann et al., 2012) or other cellular signalling (Xu et al., 2011). However endogenous cellular miRNAs may also act to suppress the interferon response factors, such as the case mir-485, which has a dual role in targeting RIG-Iaswellastheinfluenza virus H5N1 (Ingle et al., 2015). Other dsRNA-associated mechanisms II – Adenosine deamination A-to-IeditingisacovalentRNAmodificationsystemofbroadsignificance(reviewed in Nishikura, 2016). It is mediated by adenosine deaminases acting on RNA (ADARs), enzymes that carry two or three dsRBD and recognize both interand intramolecular dsRNAs longer than 20–30 bp (Nishikura et al., 1991). ADARs convert adenosines to inosines, which base pair with cytosines, which are interpreted as guanosines during translation. Thus,RNAeditingaffectscodingpotential, fidelityofRNAreplicationreversetranscription, or formation/stability of RNA secondary structures where a change of a single base in a sequence may result either in dsRNA destabilization (inosine-uridine pair) or stabilization (inosine-cytidine pair) (Nishikura, 2010). Such transition in the local and global stability ofdsRNAstructurecaninfluencefurtherprocessingofdsRNA,suchastheselectionofthe effectivemiRNAstrand(Bartel,2004;MeisterandTuschl,2004). Mammals (and vertebrates in general) have three ADAR genes (reviewed in Nishikura,2016)(Fig.3).Twoencodeproteinscarryingdeaminaseactivity:ADAR1,whichis interferon-inducible, and ADAR2, which is constitutively expressed. ADAR3 is mostly expressedinthebrainbutitseditingactivityhasnotbeenshownyet.Thespecificityof the ADAR1 and ADAR2 deaminases ranges from highly site-selective to non-selective, dependent on the duplex structure of the substrate RNA. ThecompleteADARstructurehasnotbeensolvedyetbutstructureofseveraldomains isknown–theZalphadomainofthehumaneditingenzymeADAR1(Schwartzetal., 1999)anddsRBDsofADAR2(Stefletal.,2010).TheanalysisofdsRBDsprovidedan insightintoeditingofaspecificsubstrateandrevealedthatdsRBDsofADARnotonly recognizetheshapebutalsothesequenceofthedsRNA(Stefletal.,2010).Theunexpected direct readout of the RNA primary sequence by dsRBDs is achieved via the minor grooveofthedsRNAandthisrecognitioniscriticalforbotheditingandbindingaffinity ofeditedRNA(Stefletal.,2010).ItwasalsoshownthatADAR2formsdimersin vivo andthatdsRBDsarenecessaryandsufficientfordimerizationoftheenzyme(Poulsen et al., 2006). ADARs exhibit complex regulation of localization. For example, it was shown that mouse ADAR1 isoforms are differentially localized in cellular compartments and that their Introduction_to_RNAi.indd 99Introduction_to_RNAi.indd 99 09.07.20 8:3409.07.20 8:34 MAMMALS II 100 localization is controlled by several independent signals, which include a nuclear localization signal (NLS), the nucleolar localization signal (NoLS), the nuclear exporter signal (NES)neartheNterminus(Nieetal.,2004).ADAR1interactswithTuDOR-SNnuclease (Nishikura, 2010; Scadden, 2005; Weissbach and Scadden, 2012; Yang et al., 2006a) and localizes to stress granules upon stress induction (Weissbach and Scadden, 2012) while tudor-SN degrades hyperedited dsRNA (Scadden, 2005). RNA editing concerns a broad range of RNAs including viral and cellular RNAs. Many long perfect dsRNAs (>100 bp) undergo extensive editing with a conversion of approximately 50 % of adenosines to inosines (Nishikura et al., 1991; Polson and Bass, 1994). Extensive editing (hyperediting) is linked with nuclear retention (reviewed in DeCerbo and Carmichael, 2005). On the other hand, short RNAs (~20–30 bp) or imperfect long dsRNAsareeditedselectively;usuallyonlyafewadeninesatspecificsitesaredeaminated (Lehmann and Bass, 1999). High throughput analyses revealed the extent of RNA editing of mammalian RNAs in terms of substrate diversity and frequency of editing in the transcriptome (Carmi et al., 2011; Peng et al., 2012). Edited endogenous RNAs (Dawson et al., 2004; Hundley et al., 2008; Morse et al., 2002; Salameh et al., 2015) include mRNAs, repetitive sequences (mainly Alu (Athanasiadis et al., 2004)), and miRNAs. It was predicted that more than 85% of pre-mRNAs may be edited, predominantly in the non-coding regions (Athanasiadis et al., 2004). Several pri-miRNAs (e.g. miR-142) are known to undergo editing, which inhibits Droshacleavageorevencausesdegradationofpri-miRNAbyTudorSN(Nishikura,2010; Scadden,2005;Yangetal.,2006a).Inothercases,pri-miRNAeditingdoesnotinfluence Drosha activity but inhibits processing of pre-miRNA by Dicer (e.g. miR-151) (Kawahara et al., 2007a). Last but not least, RNA editing might also inhibit export of miRNAs from the nucleus (Nishikura, 2010). A systematic analysis of edited miRNAs in the human brain showed that editing of miRNAs affects several miRNAs but it is not widespread (Alon et al., 2012). A similar picture was obtained from analysis of embryonic miRNAs (Garcia-Lopezetal.,2013;Veselyetal.,2012) One of the roles of ADARs in immunity is to prevent innate immune sensing of self-RNA (Heraud-Farlow and Walkley, 2016). ADARs also affect viral RNAs in various ways – ADARs are both antiviral and proviral; the effect on virus growth and persistence depends Figure 3 Domain composition of mammalian ADAR proteins NES, nuclear export signal, NLS, nuclear localization signal; dsRBD, dsRNA binding domain. Introduction_to_RNAi.indd 100Introduction_to_RNAi.indd 100 09.07.20 8:3409.07.20 8:34 MAMMALS II 101 uponthespecificvirus.(Samuel,2011).VirusestargetedbyADARsinmammalsinclude HIV(Clerziusetal.,2009),herpesvirus(Gandyetal.,2007),HRSV(MartinezandMelero,2002),HCMV(Nachmanietal.,2014),VSV(Nieetal.,2007),andHDV(Wongand Lazinski, 2002). Crosstalk between RNA editing and other dsRNA pathways. ADARs affect other dsRNA pathways in several ways. In RNA silencing, ADARs can competewithRNAifordsRNAsubstrates(includingsiRNAs).TheADAR1isoform (ADAR1p150) strongly binds siRNA and reduces thus the availability of dsRNA for RNAi, resultinginlessefficientRNAiinnormalcellscomparedtoAdar1-/- cells (Yang et al., 2005). Interestingly, injection of high doses of siRNAs enhances ADAR1 expression, suggesting a role of ADAR1 in a cellular feedback mechanism in response to siRNA (Hong et al., 2005). Editing affects base pairing quality of dsRNA substrates as well as target recognition since a single nucleotide mismatch between siRNA and target mRNA can reduce RNAi efficacy(ScaddenandSmith,2001)ormodifytargetspecificity,especiallywhenoccurring in the seed sequence (Kawahara et al., 2007b). MiRNAs would be affected in a similar way. A moderate deamination (one I-U pair per siRNA) does not prevent Dicer processing tosiRNAs(Zamoreetal.,2000)but,hyperediting(~50%ofdeaminatedadenosines)can make dsRNA resistant to Dicer processing (Scadden and Smith, 2001). Thus,ADARsarefactorsconferringtoformationofRNAiresistance(Hongetal.,2005), which may be one of the viral strategies to avoid being targeted through a dsRNA-respondingpathway(Zhengetal.,2005).ADARsinfluencetheinnateimmunityeitherindirectlyby preventing sensing of self-RNA (Heraud-Farlow and Walkley, 2016) or by interacting with innateimmunityfactors,suchasPKR(Clerziusetal.,2009).Theimmunosuppressiverole ofADAR1couldexplainthephenotypeoftheAicardi-Goutieressyndrome(AGS,OMIM# 225750), an autoimmune disorder caused by ADAR1 mutations (Rice et al., 2012). It has been proposed that in the absence of ADAR1, accumulation of cytoplasmic dsRNA may provoke interferon signalling and cause upregulation of interferon-stimulated genes, which is observed in AGS (Rice et al., 2012). Summary In mammals (Fig. 4), the miRNA pathway seems to be the dominant small RNA pathway in the soma while the existence and functionality of endogenous RNAi remains unclear. Theonlycelltypewithwelldocumentedrobustandmechanisticallyexplainedendo-RNAi is the mouse oocyte. Somatic cells typically respond to long dsRNA with a sequence-independent interferon response, which is employing multiple dsRNA sensors, which trigger a complex interferon response. Introduction_to_RNAi.indd 101Introduction_to_RNAi.indd 101 09.07.20 8:3409.07.20 8:34 MAMMALS II 102 Acknowledgement IwouldliketothankmycolleaguesJanPaces,MiloslavNic,andTomasNovotnyforhelp withcollectingliteratureforthereview.ThereviewcontentwasproducedunderacontractOC/EFSA/GMO/2015/01-CT01withEuropeanFoodSafetyAuthority(EFSA);the opinionsexpressedarethoseofthecontractoronlyanddonotrepresentEFSA’sofficial position. Publication of the review was funded by LO1220 and LM2015063 by the Ministry of Education, Youth and Sports. References Adilakshmi,T.,Sudol,I.,andTapinos,N.(2012).CombinatorialActionofmiRNAsRegulatesTranscriptionalandPost-TranscriptionalGeneSilencingfollowinginvivoPNSInjury.PlosOne 7, e39674-e39674. 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Introduction_to_RNAi.indd 118Introduction_to_RNAi.indd 118 09.07.20 8:3409.07.20 8:34 https://doi.org/10.14712/9788024643724.5 119 RNAi AND miRNA PATHWAYS IN BIRDS Birds Keywords:dsRNA,siRNA,miRNA,Dicer,TARBP2,PACT,Argonaute PETRSVOBODA Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska1083,14220Prague4,CzechRepublic Correspondence to: Petr Svoboda, Institute of Molecular Genetics ASCR, Videnska1083,14220Prague4,CzechRepublic,tel.#+420241063147, e-mail: [email protected]. ABSTRACT RNAsilencingdenotessequence-specificrepressionmediatedbysmallRNAs.Invertebrates,therearetwoclosely related pathways, which share several protein factors: RNA interference (RNAi) and microRNA (miRNA) pathway.ThemiRNApathwayregulatesendogenousprotein-codinggeneexpressionandhasbeenimplicatedinmany biological processes. RNAi generally serves as a form of innate immunity targeting viruses and mobile elements. ThistextreviewsmiRNAandRNAipathwaysinbirds.AlthoughtheavailableliteratureonRNAsilencingin birds is very limited, many features can be deduced from the genomic data in the public domain. miRNA, RNAi andotherdsRNA-respondingpathwaysinbirdsappearverymuchlikethoseinmammals,importantbird-specific featuresofRNAsilencingpathwaysareyettobeidentified.ThemiRNApathwayislikelythedominantsmall RNA pathway while the existence and functionality of endogenous RNAi remains unclear. Some variations may be present in the main bird antiviral interferon system. Introduction Birds (Aves)belongtogetherwithmammalsandfishestothegroupCraniata within chordates. Some of the birds are of high economic importance (food industry) or medical relevance (viral vectors causing zoonoses). Bird ancestors branched of mammalian ancestors over 300 MYA when the synapsid lineage leading to mammals branched of the sauropsid lineageleadingtodinosaursandbirds.Thereare~9000extantbirdspecies(Margulisand Schwartz, 1998). During their evolution, birds evolved numerous physiological adaptations in which they differ from mammals, including feathers, shelled eggs with external development, or different sex chromosome system, to name a few. At the same time, they are the closest mammal-related group covered in this series, in terms of synteny and sequence similarity.ThisisusefulforassessingfeaturesofdsRNAandmiRNApathwaysbecause the available literature on RNA silencing in birds is very limited. However, many features can be deduced from the genomic data in the public domain. miRNA, RNAi and other Introduction_to_RNAi.indd 119Introduction_to_RNAi.indd 119 09.07.20 8:3409.07.20 8:34 BIRDS 120 dsRNA-responding pathways in birds are very much like those in mammals and the literature doesnotreportanimportantbird-specificfeatureinRNAsilencingpathways.SincemechanisticalprinciplesofvertebratemiRNAandRNAipathwayswereintroducedinthefirst two reviews of this series (Svoboda, 2019a, b) and in further detail elsewhere (Bartel, 2018; Svoboda, 2014), I will focus here directly on features of these pathways described for birds. Dicer AccordingtothecompletegenomesequencesofchickenandZebraFinch,birdshaveone Dicer protein. Chicken Dicer has been assigned to the chromosome 5 according to the radiationhybridmapping(Tianetal.,2007)whichisinagreementwiththecurrentchicken genomemap.ThereisnodetailedanalysisofavianDicerspecificityandactivity,whichhave to be inferred indirectly from other results. Chicken Dicer can process both, long dsRNA and miRNA precursors, as evidenced by induction of RNAi with long dsRNA (Mauti et al., 2008; Pekarik et al., 2003) and hundreds of avian miRNAs in the miRBase. ThecommonDicerproductsizeseemstobe21–23ntwithatypicalsizeof22nt.This informationcanbeinferredfromavailablemiRBasedata(Fig.1).Thus,theavianDicer produces small RNAs with the same sizes as the mammalian Dicer (Fig. 1). Another possiblesubstrateofDicerinbirdsmightbesnoRNAs,althoughthebiologicalsignificanceof thisobservationremainsunclear(Taftetal.,2009). It is unclear if there are functionally different avian Dicer isoforms as is the case in murineoocytesandsomaticcells(Flemretal.,2013).ThereisonereportofdifferentDicer splice variant in goose (Anser cygnoides) where one variant lacks a linker between DEAD boxandhelicaseCdomainsattheN-terminus(gDicer-b)(Huetal.,2014).TheshorterisoformgDicer-bispresentinmultipletissues,howeveritsfunctionalsignificanceisunclear. ThetruncationisfoundintheN-terminus,whichisassociatedwithsubstrateselectivityand efficientprocessing.Therefore,onemightspeculateaboutsomefunctionaldivergencein substrate processing between the two isoforms. However, there is no experimental evidence atthemoment.Theonlyavailabledata,sofar,concerncloningoftheshortisoformand expressionanalysisofseveraltissuesandfollicularstagesbyRT-PCR(Huetal.,2014). dsRBPs dsRBP binding partners of Dicer have not been studied, so far. Interestingly, the chickengenomecontainsadsRBP,whichisrelatedtoTARBP2andPACT,suggestingamore ancestral vertebrate state and a reduced crosstalk between RNAi and the interferon pathway. Argonaute proteins ArgonautefamilyproteinsareeffectorsofRNAsilencingmechanisms.Theyaredivided into two subfamilies: AGO proteins, which accommodate miRNAs and siRNAs, and PIWI Introduction_to_RNAi.indd 120Introduction_to_RNAi.indd 120 09.07.20 8:3409.07.20 8:34 BIRDS 121 proteins, which accommodate piRNAs. Avian AGO proteins have not been characterized in a published report but public chicken genome data show that the setup is the same as in mammals: Studies in chicken revealed four AGO proteins, where AGO1, 3, and 4 are encoded within one locus on chromosome 23 and AGO2 is encoded separately on chromosome2.Thisarrangementappearstobesharedwithinmammalsandbirds(Zhouetal., 2010). Additional information about avian AGOs can be inferred indirectly from the existence of functional RNAi and miRNA pathways (discussed below), which implies that at leastoneAGOproteinisa“slicer”(presumablyAGO2,givenitsconservedroleasaslicer from Drosophila to mammals). Avian AGO proteins can also mediate post-transcriptional silencing guided by imperfectly base paired miRNAs. In addition, there were two publications found, which mention avian PIWI proteins, which primarily control genome integrity in the germline and are not within the scope of this report (Kim et al., 2012; Lim et al., 2013). Other factors Birds have additional proteins involved in other dsRNA responses, which are either associated with adenosine deamination (Herbert et al., 1995) or interferon response. Interferon response factors, which recognize some form of dsRNA and are also found in mammals, include MDA5 (Hayashi et al., 2014; Lee et al., 2012, 2014), RIG-I (Chen et al., 2015; Li et al., 2014a; Xu et al., 2015), and PKR (Gonzalez-Lopez et al., 2003; Lostale-Seijo et al., 2016;Zhangetal.,2014).Interestingly,chickenlacktheRHA/DHX9homolog(Satoetal., 2015).TheantiviralresponsetodsRNAwillbediscussedfurtherbelow. miRNA pathway AccordingtomiRBase(KozomaraandGriffiths-Jones,2014),birdgenomesencodehundredsofmiRNAs(Table1)Duringthesystematicliteraturereview,miRNA-relatedpublications lacking a mechanistic molecular insight into the miRNA pathway were the most commonclassofannotatedpublicationsforbirds(~50%ofallselectedpublications).These publications fall into four basic categories: a) annotations of novel miRNAs, including high-throughput expression analyses (for example(Godnicetal.,2013;Luoetal.,2012;Taftetal.,2009)andmanyothers).This categoryalsoincludestheoriginalchickenandZebraFinchgenomeannotationpapers (International Chicken Genome Sequencing, 2004; Warren et al., 2010). b) studies of miRNAs in different biological contexts, including reproduction (Lee et al., 2015; Lee et al., 2011), skeletomuscular apparatus (Chen et al., 2009a), bird song physiology (Gunaratne et al., 2011), growth/weight gain (Li et al., 2013), and many others; their comprehensive listing would be beyond the scope of this report. c) studies of relationship between miRNAs and the immune system, especially antiviral – these will be discussed further below in the section 3.1.2.7. 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Introduction_to_RNAi.indd 131Introduction_to_RNAi.indd 131 09.07.20 8:3409.07.20 8:34 Introduction_to_RNAi.indd 132Introduction_to_RNAi.indd 132 09.07.20 8:3409.07.20 8:34 https://doi.org/10.14712/9788024643724.6 133 RNAi AND miRNA PATHWAYS IN FISH Fish Keywords: dsRNA,siRNA,miRNA,Dicer,TARBP2,PACT,Argonaute PETRSVOBODA Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska1083,14220Prague4,CzechRepublic Correspondence to: Petr Svoboda, Institute of Molecular Genetics ASCR, Videnska1083,14220Prague4,CzechRepublic,tel.#+420241063147, e-mail: [email protected]. ABSTRACT RNAsilencingdenotessequence-specificrepressionmediatedbysmallRNAs.Invertebrates,therearetwoclosely related pathways, which share several protein factors: RNA interference (RNAi) and microRNA (miRNA) pathway.ThemiRNApathwayregulatesendogenousprotein-codinggeneexpressionandhasbeenimplicatedinmany biological processes. RNAi generally serves as a form of innate immunity targeting viruses and mobile elements. ThistextreviewsmiRNAandRNAipathwaysinfish.RNAiandotherdsRNA-respondingpathwaysinfishare verymuchlikethoseinmammals,whichisremarkableconsideringthemammalianandfishlineagesseparatedin Paleozoicumsome400millionyearsago.ThemiRNApathwayislikelythedominantsmallRNApathwaywhile, similarly to other vertebrates, the existence and functionality of endogenous RNAi remains unclear. Introduction Fish are an aquatic heterogeneous paraphyletic group with the majority of the species belongingtothebonyfishclass(Osteichthyes) group, which has ~25 000 species, the highest species diversity than any other vertebrate group (Margulis and Schwartz, 1998). Overall,theorganizationofsmallRNApathwaysinfishisverysimilartothatofmammals (Fig.1),whichisnotableconsideringthecommonancestoroffishandmammalsexisted 3602–4500millionyearsago(Volff,2005). Since mechanistical principles of vertebrate miRNA and RNAi pathways were introducedinthefirsttworeviewsofthisseries(Svoboda,2019a,b)andinfurtherdetailelsewhere (Bartel, 2018; Svoboda, 2014), I will focus here directly on features of these pathwaysreportedfromfishmodels.Nextgenerationsequencinganalysisofzebrafishsmall RNAsidentifiedmiRNAsandgermlinepiRNAsascommonsmallRNAs.Thedominant smallRNApathwayinfishisthemiRNApathway.StudiesofrolesofmiRNAsaccount forthevastmajorityoftheliteratureonsmallRNAinfish.Atthesametime,themolecular Introduction_to_RNAi.indd 133Introduction_to_RNAi.indd 133 09.07.20 8:3409.07.20 8:34 FISH 134 mechanismofmiRNAandRNAipathwayswasseldomdrectlystudiedinthefishmodel. ThesecondmoststudiedstudiedsmallRNApathwayinfishisthepiRNApathway,which protects the germline from mobile elements (Houwing et al., 2007; Huang et al., 2011; Kamminga et al., 2010) and differs from miRNA and RNAi pathways in small RNA biogenesis, which does not require Dicer. Dicer Fish genomes carry a single gene for Dicer, which is an ortholog of the mammalian Dicer and Dicer-1 in Drosophila(Murphyetal.,2008).Thisnotionissupportedbyannotatedfish genome data in the UCSC genome browser (https://genome.ucsc.edu/), and tblastn search offishsequencesatNCBI(e.g. Salmo salar, Danio rerio, Takifugu rubripes, Gasterosteus aculeatus (stickleback), Oryzias latipes (medaka)). Existence of a single Dicer gene in fishgenomesisremarkableinteleostfishspecies,whichunderwentgenomeduplication (Howeetal.,2013;MeyerandSchartl,1999).Thissuggestsselectivepressurecouldexist againstDicergeneduplication.ZebrafishDicerisessentialfordevelopmentanditsprimaryroleseemstobemiRNAbiogenesis(Wienholdsetal.,2003).TheroleofDicerin endogenousRNAiinfishhasnotbeenaddressedinmuchdetail.AstudyofDiceringrass carp Ctenopharyngodon idellar revealed a CDS encoding Dicer protein carrying all known functional domains found typically in other Dicers (Shen et al., 2013). Ctenopharyngodon idellar Dicer is abundantly expressed in brain, gill, head kidney, liver, spleen, heart, muscle and intestine. A positive correlation was found between Ctenopharyngodon idellar Dicer mRNAexpressionandinfectionwithgrasscarpreovirus(GCRV)infectionincultured kidney cells and in the liver (8.46-fold, P < 0.01, 12 h post-infection) and spleen in vivo (Shenetal.,2013).Thissuggeststhat Ctenopharyngodon idellar Dicer is an inducible gene responding to viral infection although evidence for virus-derived endogenous siRNAs has not been provided. dsRBPs FishgenomestypicallycontainorthologsofTRBP2andPACT,whicharemammalian Dicer-interacting dsRBPs (Murphy et al., 2008). However, their function in small RNA silencingwasnotstudiedinthefishmodel,sofar. Argonaute proteins Fish AGO proteins are orthologs of AGO proteins in other vertebrates (Murphy et al., 2008).However,teleostfishcladecontainsanadditionalAGOparalog,whichemerged fromafish-specificgenomeduplicationeventthatoccurredapproximately350million yearsago(McFarlaneetal.,2011).AllfiveAgogenomiclociinteleostscontainspecific, conserved sequence elements in non-coding regions indicating that the teleost AGO Introduction_to_RNAi.indd 134Introduction_to_RNAi.indd 134 09.07.20 8:3409.07.20 8:34 FISH 135 paralogs are differentially regulated, which is consistent with expression analysis in the zebrafishmodel.Multiplesequencealignmentsshowthatteleosthomologspossesscritical aminoacidresiduesforAGOfunctionaswellasortholog-specificfeaturesretainedthroughout the vertebrate lineage (McFarlane et al., 2011). miRNA pathway IntermsofsmallRNAresearch,fishmodelsaretypicallyusedforexploringmiRNApopulations and studying biological roles of different miRNAs. Articles annotating miRNAs andanalyzingtheirexpressionandfunctionrepresentthebulkofthefish-relatedreferences. We have found 348 articles, which annotated and/or analyzed expression and function of miRNAsinfish.However,almostnoneofthesearticlesbroughtanyspecificmechanistic insightintothemolecularmechanismofmiRNAinfish. AccordingtothemiRBase(release22.1),miRNApopulationinfishappearslesscomplexthaninmammals–fishmodelshavelessannotatedmiRNAs(Table1)althoughthere are dozens of reports on next generation sequencing analysis and miRNA annotation. However,zebrafishisanexperimentallyeasilyaccessiblemodelforexploringconservedrolesof miRNAsindifferenttissues,whichisalsoreflectedinthenumberofreferences. Table 1 Numbers of annotated miRNAs in selected fishes in miRBase 22.1 species miRNA precursors mature miRNA Cyprinus carpio 134 146 Danio rerio 355 373 Fugu rubripes 131 108 Ictalurus punctatus 281 205 Oryzias latipes 168 146 Salmo salar 371 498 Tetraodon nigroviridis 132 109 IntermsofthemolecularmechanismofRNAsilencing,studiesinthezebrafishandother fishmodelsbroughtseveralinterestingdiscoveriesconcerningspecificmiRNAfunctions anduniqueadaptationsinfish.Severalexampleshaverelevancefortheresearchofthe molecular mechanism of miRNA pathway: ThefirstexampleisthebiologyofthemiR-430familyofmiRNAs.WhilemiRNAs in mice are essentially irrelevant for the oocyte-to-embryo transition (Suh et al., 2010), thezebrafishoocyte-to-embryotransitionincorporateszygotically-expressedmiR-430 family in maternal mRNA degradation (Giraldez et al., 2006; Mishima et al., 2006). Furthermore,theonsetofmiR-430activityinthezebrafishzygoteallowedforaddressing the relationship between miRNA-induced translational repression and mRNA degradation (Bazzinietal.,2012;Mishimaetal.,2012).usingribosomeprofilingofzygoticstages,it was showed that miR-430 reduces translation before causing mRNA decay (Bazzini et al., Introduction_to_RNAi.indd 135Introduction_to_RNAi.indd 135 09.07.20 8:3409.07.20 8:34 FISH 136 2012).Asignificanttechnologicaloutcomeofthesestudieswasdevelopmentoftargetprotectors,morpholinooligonucleotidesspecificallydisruptingmiRNA-mediatedrepression via hybridizing to and masking miRNA-binding sites (Choi et al., 2007). AnothercontributiontosmallRNAbiologycomingfromfishmodelwasdiscoveryofone of the non-canonical miRNA biogenesis mechanisms, namely Dicer-independent miRNA biogenesis of miR-451, which uses AGO2 slicing activity followed by uridylation and trimming(Cifuentesetal.,2010).TheprocessalsoemploystranslationinitiationfactoreIF1A, which directly interacts with AGO2 and promotes miR-451 biogenesis (Yi et al., 2015). DatafromzebrafishalsocontributedtounderstandingothermiRNAregulations.Two relatedterminaluridyltransferases(TuTases),Zcchc6(TuT7)andZcchc11(TuT4),selectively3‘monouridylateasubsetofmiRNAs(Thorntonetal.,2014).TuTaseinhibitionin zebrafishembryoscausesdevelopmentaldefectsandaberrantHoxgeneexpression(Thornton et al., 2014). AnothermiRNAregulatordiscoveredinthezebrafishisdeadend1(DND1),whichis negatively regulating miRNA targeting. DND1 is an evolutionary conserved RNA-binding protein(RBP)thatcounteractsthefunctionofseveralmiRNAsinzebrafishprimordialgerm cells as well as in human cells. DND1 binds mRNAs and prohibits miRNAs from binding cognate mRNAs. DND1 effects involve uridine-rich regions present in the miRNA-targeted mRNAs (Kedde et al., 2007) TakentogetherstudiesofmolecularmechanismofmiRNA-mediatedrepressioninfish did not reveal any notable deviation from what has been observed in mammals. As the protein machinery appears to be well-conserved, miRNA pathways among vertebrate taxons primarily differ in sets of miRNAs and their targets, which dynamically evolve over time.ThiswasforexampledemonstratedforzebrafishmiR-430andmurinemiR-290–295 miRNA clusters, which share common ancestry, both are associated with early development but do not regulate the same genes although some targets seem to be conserved (Svoboda and Flemr, 2010). RNAi ThepresenceofRNAiresponsewasexaminedinzebrafishatthesameasinotheranimal modelsduringtheturnofthecentury.However,unlikespecificRNAiobservedinmouse oocytesandearlyembryos(Svobodaetal.,2000;WiannyandZernicka-Goetz,2000),long dsRNAinjectionintozebrafishhadbeenyieldinginconsistentresults(Lietal.,2000;Mangosetal.,2001;Oatesetal.,2000;Zhaoetal.,2001).Whilesomereportedspecificknockdowneffects(Lietal.,2000;Mangosetal.,2001),othersobservednon-specificeffects (Oatesetal.,2000;Zhaoetal.,2001).Non-specificeffectsremainedarecurringtheme alsoinlaterstudies(Wangetal.,2010;Zhaoetal.,2008)althoughsomeauthorswereable toachievespecificRNAieffects(DeRienzoetal.,2012;Dongetal.,2013;Yingetal., 2010).Non-specificeffectsinzebrafishembryoswerenotremediedbytheuseofsiRNAs anditwaslatershownthatthebasisofthenon-specificeffectsisinterferencewithmiRNA function(Zhaoetal.,2008).InjectionofzebrafishzygoteswithsiRNAcausedasignificant reductioninmiR-430levelsleadingtounspecificdevelopmentaldefects(Zhaoetal.,2008). Introduction_to_RNAi.indd 136Introduction_to_RNAi.indd 136 09.07.20 8:3409.07.20 8:34 FISH 137 Interestingly, literature survey revealed that experiments with the parasitic sea lamprey (Petromyzon marinus),ajawlessfishrelative,showedthatuptakeoffreesiRNAat5μg/ml did not trigger an RNAi response (Heath et al., 2014). In any case, RNAi did not become afavouriteknock-downstrategytostudygenesduringzebrafishdevelopment;microinjection of morpholino oligonucleotides (Blum et al., 2015; Eisen and Smith, 2008) became the preferred approach instead. Takentogether,successfulRNAiexperimentswithlongdsRNAdemonstratethatzebrafishholdsthemolecularmachineryforexecutingRNAi:Dicer,TARBP2,andAGO2.However,itscapacityformediatingspecificknock-downeffectsislimitedbecausethesame machinery is being simultaneously utilized by the miRNA pathway. Importantly, the availability of the machinery above the minimum capacity sustaining the miRNA pathway functionality likely differs during development and among different cell types. WhilezebrafishholdsthemolecularmachineryforexecutingRNAi,thequestionremains whethertheendogenousRNAihasanysignificantroleinfish.Availabledatadonotprovide unequivocalevidenceforsignificantendogenousRNAiinfish.Nextgenerationsequencing of small RNAs contains fractions of non-miRNA small RNAs of endo-siRNA size, yet it is not clear if these fragments truly represent bona fide endo-siRNAs. Some other data indirectlypointtoapossibleantiviralrole,namelyGCRV-inducedtransientupregulation of Ago2 in rare minnow (Gobiocypris rarus) and Dicer upregulation in grass carp (Ctenopharyngodon idella)(Guoetal.,2012;Shenetal.,2013).InteractionofGCRVwiththe small RNA machinery (the miRNA pathway should not be excluded) has been suggested basedontheobservationingrasscarpkidneycellsthatGCRVdsRNAcouldbeprocessed intosiRNAsbutGCRVinfectiondidnotyieldGCRV-derivedsiRNAswhileDicerupregulationoccurred(Gotesmanetal.,2014).IthasbeenthusproposedthatanunidentifiedRNAi suppressormightcontributetothesurvivaloftheviralgenomeandefficientviralreplication(Gotesmanetal.,2014).Thepresenceofavirus-derivedinhibitorofRNAsilencing inafishRNAviruswouldbeindicativeofanexistingantiviralroleofsmallRNAs,which is being suppressed. However, an alternative scenario that should be considered as well is that dsRNA formed during viral replication is not accessible for Dicer-mediated cleavage. Therefore,furtherresearchisneededtoaddressthisissue. Other notable silencing phenomena Therearetwophenomena,whichclearlyoverlapwithRNAsilencingbuttheirunderlying molecular mechanism remains unclear and will require further investigation. Andrews et al found that introduction of transgenes containing convergent transcription unitsinzebrafishembryosinducedstabletranscriptionalgenesilencingincis and trans. ThesilencingwassuppresseduponDicerknockdown,indicatingprocessingofdouble stranded RNA. ChIP revealed that silencing was accompanied by enrichment of the constitutive heterochromatin mark H3K9me3 (Andrews et al., 2014). While small RNA-induced transcriptional silencing is well established in fungi and plants (and seems to be a product of convergent evolution), the molecular mechanism underlying seemingly related observations in vertebrates is unclear. Introduction_to_RNAi.indd 137Introduction_to_RNAi.indd 137 09.07.20 8:3409.07.20 8:34 ARTHROPODS 144 are with, some exceptions (e.g. armadillo bug an relatives known as woodlice), aquatic andhavedifferentiatedsegmentedbodyandbiramousappendages.Theyincludeshrimp, crayfish,lobsters,crabs,barnacles,prawnsandothers.Hexapoda comprise insects and insect-like animals with six thoracic legs. ThekeymodelorganismforarthropodsisDrosophila, which has been a workhorse of biology for over hundred years. miRNA, RNAi and other dsRNA pathways in Drosophila are well understood and will serve as benchmarks for the entire phylum. Drosophila evolved an extensive genetic separation of miRNA and RNAi pathways where each pathway has a dedicated Dicer, dsRBP, and Argonaute protein. Given the complexity of the phylum and evolutionary time, one could question how representative of arthropods is the Drosophila model. However, analysis of Dicer and AGO indicates that Drosophila isamore-or-lessacceptablemodelformostarthropodsasthe“twoDicersystem”can be recognized within phylogeny of Dicer and AGOs also in Chelicerata (whose common ancestors with Drosophila branched in the most distant past), Myriapoda, and Crustacea (Palmer and Jiggins, 2015). However, it should be kept in mind that some variability could emerge during half a billion years of arthropod evolution. Since mechanistical principles of vertebrate miRNA and RNAi pathways were introducedinthefirstreviewofthisseries(Svoboda,2019)andinfurtherdetailelsewhere(Bartel, 2018), I will focus here directly on features of these pathways discovered in Arthropods. Theformalstructureofthereportwillbeasinotheranimaltaxons–uponmiRNAdna RNAi molecular features of key individual components of reviewed mechanisms, I will discuss the silencing mechanisms and their biological roles. Importantly, to provide an overview of miRNA and dsRNA mechanisms in arthropods, I will focus on description of molecularmechanismsidentifiedinDrosophilaandwillhighlightanddiscusssignificant deviations observed elsewhere in arthropods, especially in more studied organisms, such as mosquitos,flowerbeetle,silkmoth,andshrimps. The Microprocessor complex Drosophila utilizes the same Microprocessor complex as the earlier discussed Metazoa, i.e. a complex of Drosha and DGCR8 homologs, the latter being named Pasha (partner of Figure 1 Simplifi ed division of Arthropoda used in the text The scheme refl ects the Mandibulata model of arthropod phylogenetics described in (Regier et al., 2010) Introduction_to_RNAi.indd 144Introduction_to_RNAi.indd 144 09.07.20 8:3409.07.20 8:34 ARTHROPODS 145 Drosha)(Denlietal.,2004;Filippovetal.,2000;Landthaleretal.,2004).Thecomplex cleaves the pri-miRNA into pre-miRNA in the nucleus. Suppression of Pasha in Drosophila interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs (Denli et al., 2004; Landthaler et al., 2004). Like in other animals, Pasha is essential for processing of canonical miRNAs but is dispensable for mirtrons (Flynt et al., 2010; Martin et al., 2009; Smibert et al., 2011). Drosophila Pasha is possibly phosphorylated by ERK/MAPK, as suggested by phosphorylation of human DGCR8 in insect cells; the phosphorylation appears to increase protein stability without altering miRNA processing activity (Herbert et al., 2013). miRNA biogenesis in Drosophila also involves SmD1, a component of the Drosophila small nuclear ribonucleoprotein particle (snRNP), which interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis (Xiong et al., 2015). AnalysisoftranscriptomechangesuponDroshaknock-downinS2cellsidentified137 Drosha-regulated RNAs, including 11 relatively long (>10 kb) pri-miRNAs (Kadener et al., 2009). Interestingly, >100 RNAs not annotated as miRNAs could be direct targets of Drosha action (Kadener et al., 2009), which is consistent with other model systems where Drosha is having roles beyond miRNA biogenesis. Drosha-regulated RNAs contain conserved hairpins similar to those recognized by the Drosha-Pasha/DGCR8 complex in pri-miRNAs, one of such hairpins is found also in Pasha suggesting a negative feedback loop regulating miRNA-biogenesis (Kadener et al., 2009). miRNA-independent roles of the Microprocessorcomplexcomponentsseemtobereflectedinphenotypesofsomeoftheirmutants (Luhur et al., 2014). In terms of evolutionary diversity of the Microprocessor complex in arthropods, the miRNA pathway seemed to expand in the pea aphid (insect, Hemiptera), whose genome carries four expressed copies of Pasha (Jaubert-Possamai et al., 2010). At the same time, the brown planthopper (insect, Hemiptera), the fall armyworm (insect, Lepidoptera) or the desert locust (insect, Orthoptera) all have a single Pasha (Ghosh et al., 2014; Wynant et al., 2015; Xu et al., 2013), which appears the common case among arthropods when browsing available genome databases. Analysis of Pasha in Litopenaeus vannamei (shrimp) revealed high sequence conservation and nuclear localization, suggesting a well-conserved role in miRNA biogenesis (Chen et al., 2012). Conservation of miRNA pathway in shrimps is further supported by requirement for Drosha, Dicer1 and Ago1 for production of viral RNAs ininfectedshrimps(HeandZhang,2012;Huangetal.,2012). Dicer Drosophila utilizes two Dicer proteins (Fig. 2), Dicer-1 (DCR-1) and Dicer-2 (DCR-2), whicharededicatedtomiRNAandRNAipathways,respectively(Leeetal.,2004).This makes Drosophila (and arthropods in general) unique among the reviewed metazoan model systems (Fig. 3), which employ a single Dicer protein producing multiple classes of small RNAs (miRNAs, endo-siRNAs, exo-siRNAs). Separation of miRNA and RNAi at Dicer level could have an advantage in terms of uncoupling antagonistic evolutionary forces acting on Dicer, i.e. (i) selective pressure on conservation of the miRNA pathway machinery Introduction_to_RNAi.indd 145Introduction_to_RNAi.indd 145 09.07.20 8:3409.07.20 8:34 ARTHROPODS 146 and (ii) host-pathogen arms race where Dicer evolves to avoid viral proteins interfering with its function. ThedomainorganizationofDrosophila Dicer proteins is generally the same as in other metazoan Dicer proteins – they are composed of domains ordered from the Nto the C-terminus as follows: N-terminal helicase domains, a domain of unknown function DUF283, PAZdomain,RNaseIIIaandRNaseIIIbdomains,andtheC-terminaldsRBD(Fig.2).As for other metazoan Dicers, Drosophila Dicer proteins have not been crystallized yet but their structure can be inferred from biochemical studies of recombinant Dicer and individualdomains(Tsutsumietal.,2011;Yeetal.,2007),thecrystalstructureofGiardia intestinalis Dicer (MacRae et al., 2007; MacRae et al., 2006), domain modelling or cryo-EM studies (Lau et al., 2012). Dicer-1 Dicer-1wasoriginallyidentifiedasoneoftwohomologsinDrosophila, which was able to produce siRNAs in vitro and participated in RNAi (Bernstein et al., 2001). Subsequent analysis of Dicer mutants showed that mutation in dicer-1 blocked processing of miRNA precursors while dicer-2 mutants were defective for processing siRNA precursors (Lee et al., 2004). However, consistent with the initial study, Dicer-1 was also implicated in RNAi (Lee et al., 2004). Biochemical analysis of Dicer-1 showed that its functional core consistsofaDuF283domain,aPAZdomain,andtwoRIIIdomains(Yeetal.,2007). With respect to the size of cleavage products, Dicer-1 apparently does not differ from other metazoan Dicers, as the typical product size is 22 nt long (Fig. 4). DCR-1 also functions in biogenesis of mirtron class of miRNAs (Okamura et al., 2007). Dicer-1differsfromDicer-2insubstratespecificitiesandATPrequirements(Jiangetal., 2005).LikehumanDicer,Dicer-1generatessmallRNAsinanATP-independentmanner (Jiangetal.,2005),whereasDicer-2orDicer-2/R2D2requiredATPhydrolysisforefficient siRNA production (Liu et al., 2003). Dicer-1 shows a preference for pre-miRNAs (Jiang etal.,2005;Tsutsumietal.,2011).Itrecognizesthesingle-strandedterminalloopstructure of pre-miRNAs through its N-terminal helicase domain, checks the loop size and measures thedistancebetweenthe3′overhangandtheterminalloop–thisallowsDicer-1toinspect theauthenticityofpre-miRNAstructures(Tsutsumietal.,2011). Figure 2 Comparison of C. elegans, human and Drosophila Dicer proteins Domain composition was adopted from (Jaskiewicz and Filipowicz, 2008). Introduction_to_RNAi.indd 146Introduction_to_RNAi.indd 146 09.07.20 8:3409.07.20 8:34 ARTHROPODS 147 In terms of evolutionary diversity of Dicer-1 in arthropods, as mentioned above, the miRNA pathway seemed to expand in pea aphid (insect, Hemiptera) which utilizes two active copies of Dicer 1 (Jaubert-Possamai et al., 2010; Ortiz-Rivas et al., 2012). However, thisduplicationisarelativelyrecenteventwhilesingleDicer-1wasalsoidentifiedelsewhere among arthropods (Jaubert-Possamai et al., 2010; Ortiz-Rivas et al., 2012), including shrimp (Su et al., 2008), mosquito (Bernhardt et al., 2012), cockroach (Gomez-Orte and Belles, 2009) or locust (Wynant et al., 2015) species. Dicer-2 Dicer-2 in Drosophila is mainly producing siRNAs from long dsRNA and functions in RNAi and antiviral defense (Galiana-Arnoux et al., 2006; Kim et al., 2006). Dicer-2 has actually a dual role in antiviral defense – apart from RNAi, it has an RNAi-independent roleinpromotingTollsignalling(Wangetal.,2015b),butbiologicalaspectsofDicer-2role will be covered later in the text. unlikemammalianDicerorDicer-1paralog,Dicer-2requiresATPforprocessivecleavage of dsRNA (Liu et al., 2003; Nykanen et al., 2001; Provost et al., 2002; Welker et al., 2011;Zhangetal.,2002).Remarkably,analysisofshapesofamammalianDicerandDicer-2 by cryo-EM yielded an L-shaped reconstruction with dimensions strikingly similar to thoseofthehumanenzyme(Lauetal.,2012).Therefore,despitestrikingfunctionaldifferencesinATPrequirementandsubstratepreference,theoverallthree-dimensionalarchitecture of Dicer is well conserved (Lau et al., 2012). Dicer-2containsanN-terminalhelicasemotifandhydrolyzesATP;ATPhydrolysisis required for Dicer-2 to process long dsRNA, but not pre-miRNA (Cenik et al., 2011). Dicer-2worksasadsRNA-stimulatedATPasethathydrolyzesATPtoADP;anditwassuggestedthatDicer-2helicasedomainusesATPtogeneratemanysiRNAsfromasinglemolecule of dsRNA before dissociating from its substrate. (Cenik et al., 2011). ThehelicasedomainofDicer-2alsogovernssubstraterecognitionandcleavageefficiency through discriminating among dsRNA ends. First, it was shown that the helicase domain is essential for cleaving dsRNA with blunt or 5’-overhanging termini, but not those with 3’ overhangs, as in pre-miRNAs (Welker et al., 2011). Subsequently, it was found that the discrimination of termini takes place during initial binding (Sinha et al., 2015). In the absenceofATP,Dicer-2binds3′overhanging(pre-miRNA-like),butnotblunttermini. inthepresenceofATP,Dicer-2bindsbothtypesoftermini,withhighest-affinitybinding observed with blunt dsRNA (Sinha et al., 2015). An important factor in substrate discrimination and processing is inorganic phosphate, which inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs (Cenik et al., 2011). ItwasproposedthattheinorganicphosphateoccupiesaPAZdomain5’phosphatebinding pocket required to bind the 5’ terminal phosphate of short substrates, blocking their use and restrictingpre-miRNAprocessinginfliestoDicer-1(Fukunagaetal.,2014).Bindingof longdsRNAisnotinhibitedwhentheinorganicphosphateoccupiesthePAZdomainbinding pocket because it also involves the helicase domain and/or the central dsRNA-binding domain, which might be combined with displacement of the inorganic phosphate from its binding pocket (Fukunaga et al., 2014) Introduction_to_RNAi.indd 147Introduction_to_RNAi.indd 147 09.07.20 8:3409.07.20 8:34 ARTHROPODS 148 In terms of evolutionary diversity of Dicer-2 in arthropods, most species seem to use only one Dicer-2 but some underwent duplication, such as Daphnia (Crustacea, two Dicer-2 paralogs) or Metaseiulus (Chelicerata,fiveDicer-5paralogs)(PalmerandJiggins,2015). Among the experimentally approached species, one Dicer-2 was reported in experimental results from silk moth (Kolliopoulou and Swevers, 2013), mosquito (Leger et al., 2013), cockroach(Lozanoetal.,2012),Hessianfly(KolliopoulouandSwevers,2013),planthopper(Zhangetal.,2013),emeraldashborer(Zhaoetal.,2015),mite(Hoyetal.,2016), bumble bee (Niu et al., 2016), or shrimp (Niu et al., 2016). dsRBPs in arthropods – R2D2 and LOQS homologs Drosophila also utilizes Dicer partner dsRBPs with tandemly arranged dsRBDs – Loquacious(LOQS)andR2D2.ThefirstDicerpartnerdsRBPinDrosophila is Loquacious, which was found to associate with Dicer-1, suggesting that the miRNA pathway in Drosophila employs a distinct dsRBP in substrate routing (Forstemann et al., 2005; Saito et al., 2005). However, it was also found that Dicer-2-generated siRNAs in the endogenous RNAi pathway depend preferentially on Loquacious and not on R2D2, the canonical Dicer-2 partner Figure 3 Metazoan Dicer phylogeny The unrooted tree shows phylogenetic relationships of Dicer proteins in Metazoa. The blue frame depicts Dicer2 homologs in arthropods. As the length of each branch indicates evolutionary distance (or sequence divergence), it is apparent that arthropod’s Dicer 2 proteins acting in RNAi are evolving at much faster pace than Dicer 1 protein, which function in the miRNA pathway. This is consistent with the above-mentioned notion of antagonistic evolutionary forces acting on Dicer where the miRNA pathway functionality is being conserved while the RNAi functionality is evolving during the host-pathogen arms race where Dicer evolves to avoid viral proteins interfering with its function. Introduction_to_RNAi.indd 148Introduction_to_RNAi.indd 148 09.07.20 8:3409.07.20 8:34 ARTHROPODS 149 (Czech et al., 2008). it turned out that Loquacious gene actually produces three protein isoforms , which associate with Dicer-1 and miRNA pathway (LOQS-PA and LOQS-PB isoform) and Dicer-2 and RNAi (LOQS-PD isoform) (Fukunaga et al., 2012; Hartig et al., 2009;Miyoshietal.,2010a;Zhouetal.,2009). LOQS-PB uses the second dsRNA-binding domain to bind pre-miRNA and the third dsRNA-binding domain to interact with Dcr-1. Both domains of LOQS-PB are required forefficientmiRNAproductionbyenhancingtheaffinityofDcr-1forpre-miRNA(Ye et al., 2007). LOQS-PD and R2D2 function sequentially and non-redundantly in the endogenous RNAi pathway. LOQS-PD stimulates DCR-2-mediated processing of dsRNA whereas R2D2 acts downstream during RISC loading (Hartig and Forstemann, 2011; Marques et al., 2010;Miyoshietal.,2010a).Takentogether,LOQSandR2D2contributetotheprofound mechanistic separation of miRNA and RNAi pathways, which evolved in Drosophila (and presumably in arthropods in general). R2D2associateswithDicer-2andactsinRNAi;itwasco-purifiedwithDicer-2during purifying siRNA-generating activity from Drosophila S2 cell lysates (Liu et al., 2003). Although R2D2 bears 33% similarity to RDE-4 (see the section Nematoda) its role is different.R2D2doesnotinfluenceDCR-2enzymaticactivity(Liuetal.,2003)butrestricts Dicer-2 function to processing of long dsRNAs (Cenik et al., 2011; Fukunaga et al., 2014). It also facilitates passing the cleavage product to AGO2 excluding miRNA-like duplexes with imperfectbasepairing(Tomarietal.,2004a).R2D2hastworoles–itissensingsiRNAthermodynamic asymmetry for strand selection and it is a licensing factor for entry of authentic siRNAsintotheRNAipathway(Nishidaetal.,2013;Tomarietal.,2004b). Unlike the Microprocessor complex, Dicer or Argonautes, dsRBPs seem undergo various functionaladaptationsbetweendifferenttaxons(compare,forexampleTARBP2,RDE-4, R2D2orLOQS).Thispossiblyalsohappensamongarthropods.Anexampleisthelackof the RNAi-associated LOQS-PD isoform outside Drosophila (Haac et al., 2015). Analysis of dsRBPs in the mosquito Aedes aegypti revealed absence of LOQS-PD isoform, conserved roles of R2D2 and LOQS-PB, and LOQS-PA role in biogenesis of both, miRNAs and endo-siRNAs (Haac et al., 2015). Argonaute proteins AGO proteins of arthropods are conserved, i.e. their domain composition is the same as that of mammalian proteins, which was discussed in detail (Peters and Meister, 2007). Arthropods have varying number of Argonaute proteins of the AGO and PIWI clade but it seems that their archetypal state is two AGO proteins, each being associated with one Dicer and one type of small RNA (Palmer and Jiggins, 2015). AGO1 and its loading with miRNAs AGO1 RISC loading is similar to that of human RISC assembly described earlier (Yoda et al., 2010). Dicer-1/LOQS-PB heterodimer functions in assembling AGO1 RISC, which Introduction_to_RNAi.indd 149Introduction_to_RNAi.indd 149 09.07.20 8:3409.07.20 8:34 ARTHROPODS 150 is preferentially loaded with miRNA/miRNA* duplexes while siRNAs are being excluded fromtheassembly(Tomarietal.,2007). AGO1requiresATPformiRISCloading,presumablytotriggerthedynamicconformational opening of AGO proteins so that they can accept small-RNA duplexes (Kawamata et al., 2009). Unwinding of miRNA-miRNA* duplexes is a passive process that does not requireATPorsliceractivityofAgo1(Kawamataetal.,2009). TwodistinctAGOcomplexeswereidentified(Miyoshietal.,2009):(i)AGO1-Dicer-1 complex with pre-miRNA processing activity where the resultant mature RNA was loaded onto AGO1 within the complex – this complex corresponds to miRLC (miRISC loading complex) (ii) the AGO1-GW182 complex with excluded DCR-1, containing mature miRNA no pre-miRNA processing activity – this complex corresponds to miRISC. AGO1 loading also involves R3D1-L, a dsRBP that functions as a cofactor interacting with Dicer-1 and AGO1 (Jiang et al., 2005). AGO1 might also have miRNA-independent role in translational repression where AGO1 is recruited to mRNA via an RNA-binding protein SMAUG and not through miRNA:mRNA interaction (Pinder and Smibert, 2013). AGO2 and its loading with siRNAs ThemodelofRNAiRISCloadinginDrosophila suggests that RISC assembly occurs in severalsteps,whichinvolveaseveralcomplexes(TomariandZamore,2005).Thefirst complex is formed by siRNA, R2D2 and DCR-2, also known as R1 or R2/D2/DCR-2 initiator (RDI) complex (Kim et al., 2007; Pham et al., 2004), which develops into a mature formoftheRISCloadingcomplexRLC(TomariandZamore,2005).TheRLCdetermines strand selection and recruits AGO2 (and other proteins) to form pre-RISC (Kim et al., 2007), which contains duplex siRNA. Finally, the release of the passenger strand from the duplex produces holo-RISC, which can base pair with complementary mRNA substrates. TheloadingisassistedbyHsc70/Hsp90chaperones(Iwasakietal.,2015;Miyoshietal., 2010b). ThecouplingofdsRNAcleavageandRISCassemblyisamatterofdebate.Itwassuggested that, after cleavage, small-RNA duplexes need to dissociate from Dicer and then rebind to a sensor of the thermodynamic asymmetry of the duplex, because the guide strand ofansiRNAwillbeatrandomorientation(Tomarietal.,2004b). AGO2requiresATPforRISCloading(Kawamataetal.,2009;Phametal.,2004;Tomari etal.,2004a).ATPispresumablyusedtotriggerthedynamicconformationalopeningof AGO proteins so that they can accept small-RNA duplexes (Kawamata et al., 2009). StrandselectioninflyRLCiscontrolledbyR2D2.AnalysisoftheinteractionofDCR2/R2D2 complex with siRNA duplexes showed that R2D2 orients the complex according to thermodynamic stabilities of siRNA strands and binds the 5’ phosphate of the passengerstrandatthethermodynamicallymorestableend(Tomarietal.,2004b).Thus,R2D2 functions as a licensing factor for routing siRNAs into the RNAi pathway. Interestingly, a thorough analysis of AGO2 complexes revealed that, unlike mature miRNAs, which are loadedonAGO1,complementarystrandsofmaturemiRNAs(miRNA*)areefficiently loaded on AGO2 in DCR2/R2D2-dependent manner (Ghildiyal et al., 2010; Okamura et al., Introduction_to_RNAi.indd 150Introduction_to_RNAi.indd 150 09.07.20 8:3409.07.20 8:34 ARTHROPODS 151 2011).Thus,theroleofR2D2insortingsmallRNAsiswiderandextendsintothemiRNA pathway. ThefinalstepinassemblyofanactiveRISCisthereleaseofthepassengerstrandfrom the siRNA duplex. Drosophila is Armitage helicase is a candidate for a mechanism separatingthetwosiRNAstrandswhiletheguideremainsboundtoAGO2(Tomarietal.,2004a). However, experimental data support a simple solution where passenger strand cleavage by AGO2 slicer activity liberates the single-stranded guide siRNA strand from the pre-RISC complex (Kim et al., 2007; Matranga et al., 2005; Miyoshi et al., 2005). Removal of siRNA passenger strand cleavage products is assisted by C3PO endoribonuclease, which was identifiedasaRISC-enhancingfactorthatpromotesRISCactivation(Liuetal.,2009). Thecleavage-assistedmechanismistypicalforAGO2-loadedflyandhumansiRNAsin the RNAi pathway while passenger strand cleavage is not important for loading miRNAs (Matranga et al., 2005). Small RNA sorting and mRNA targeting by AGO1 and AGO2 Drosophila sorts Dicer-produced small RNAs onto functionally distinct AGO proteins where AGO1 is dedicated to the miRNA pathway while AGO2 served for RNAi. Small RNA sorting is initiated by substrate recognition and continues through sorted loading onto the AGO proteins. Small-RNA duplexes are actively sorted into AGO-containing complexesaccordingtotheirintrinsicstructures(Forstemannetal.,2007;Tomarietal.,2007). Importantly, separation of miRNA and RNAi at the level of small RNA sorting onto AGO1 andAGO2isnotcompletelypre-determinedbysmallRNAorigins(Tomarietal.,2007).It was found that miRNA*s are often loaded as functional species into AGO2 (Czech et al., 2009; Ghildiyal et al., 2010; Okamura et al., 2009). Furthermore, miRNAs produced by Dicer-1 and LOQS can be loaded by Dicer-2 and R2D2 into an AGO2 RISC (Forstemann et al., 2007). Finally, siRNAs derived from long hairpin RNA genes (hpRNA) also show a hybrid biogenesis combining RNAi factors DIcer-2 and AGO2 and Loquacious isoform (Okamura et al., 2008c). Subsequently, AGO2-RISC mediates RNAi while only AGO1 is able to repress mRNAs with central mismatches in miRNA-binding sites (Forstemann et al., 2007). At the same time,AGO1cannotmediateRNAi,becauseitisaninefficientnucleasewithacatalytic rate limited by the dissociation of reaction products (Forstemann et al., 2007). AGO1 and AGO2 RISCs also differ in mechanisms of translational repression – AGO1-RISC repressestranslationprimarilybyATP-dependentdeadenylationwhileAgo2-RISCcompetitively blocks the interaction of eIF4E with eIF4G and inhibits the cap function (Fukaya et al., 2014; Iwasaki et al., 2009). AGO1-mediated translational repression involves GW182 in the same manner as in mammals (GW182 is separately described further below). miRNA-mediatedsilencinginvolvesrecruitmentofPABP,CCR4-NOTdeadenylaseand decapping complex to RISC (Behm-Ansmant et al., 2006; Chekulaeva et al., 2011; Eulalio etal.,2008;FukayaandTomari,2011;Huntzingeretal.,2010;Huntzingeretal.,2013; Moretti et al., 2012; Rehwinkel et al., 2005). miRNA-mediated repression occurs on ribosome complexes but is independent of ribosomal scanning(Antic et al., 2015; Kuzuoglu-Ozturk et al., 2016). Introduction_to_RNAi.indd 151Introduction_to_RNAi.indd 151 09.07.20 8:3409.07.20 8:34 ARTHROPODS 152 Evolutionary perspective As mentioned above, the archetypal state arthropod state is two AGO proteins, AGO1 and AGO2. Apart from Drosophila, such a situation is found in Daphnia (Crustacea) and Metaseiulus (mite, Chelicerata) (Palmer and Jiggins, 2015). However, upon detailed inspection,onefrequentlyfindsvariabilityinthenumberofAGOproteinsacrossthe phylumorevenacrosssmallertaxonomicunits.ThemiRNApathwayseemedtoexpand in pea aphid (insect, Hemiptera), whose genome two expressed copies of ago1, one of which (ago1b) shows signs of positive selection (Jaubert-Possamai et al., 2010). At the same time, a single AGO1 but duplications of AGO2 were found Ixodes (tick, Chelicerata, three AGO2 paralogs), Strigamia (centipede, Myriapoda, two AGO2 paralogs), Mesobuthus (scorpion, Chelicerata, six AGO2 paralogs) or Tetranychus (spider mite, Chelicerata, six AGO2 paralogs) (Palmer and Jiggins, 2015). Penaeus monodon (black tigershrimp)hasfourfunctionallydiversifiedAGOparalogs(Dechklaretal.,2008;Leebonoi et al., 2015; Phetrungnapha et al., 2013; Yang et al., 2014b). Analysis of Argonaute genes across 86 Dipteran species showed that variation in copy number can occur rapidly, andthatthereisconstantfluxinsomeRNAimechanisms;thissuggeststhatArgonautes undergo frequent evolutionary expansions that facilitate functional divergence (Lewis et al., 2016). Additional miRNA and RNAi factors Thereisalargenumberofaccessoryfactorsbeyondthosedescribedabove.Forexample, a systematic screen of 40% of the genome for genes acting in the miRNA pathway yielded 45 mutations in 24 genes and an estimate of ~100 genes are required to execute the miRNA program (Pressman et al., 2012). Here, we will describe several additional factors, which have been associated with miRNA or RNAi pathways. Nibbler – Nibbler is a 3’-5; exoribonuclease involved in trimming 3’ ends of miRNAs and piRNAs (Feltzin et al., 2015; Han et al., 2011; Liu et al., 2011; Wang et al., 2016; Yang et al., 2014a). In the miRNA pathway, Nibbler shortens distinct longer miRNAs during RISC assembly, yielding miRNA isoforms that are compatible with the preferred length of AGO1-bound small RNAs (Han et al., 2011; Liu et al., 2011). It has been estimated that about a quarter of miRNAs undergoes such a trimming (Han et al., 2011). HEN1 – HEN1 (Pimet, Dmhen1)is an enzyme catalyzing addition of a 2’-O-methyl group at the 3’ end of small RNAs (Horwich et al., 2007; Saito et al., 2007). While this modificationispredominantlyfoundonpiRNAsinDrosophila, it was also found on siRNAs and miRNAs (Abe et al., 2014; Horwich et al., 2007; Yang et al., 2014a). Functionally, 2’-O-methylation of siRNAs loaded on AGO2 prevents tailing and trimming of siRNAs (Ameres et al., 2010). Generally HEN1 and Nibbler thus have antagonistic activities at the 3’ end of small RNAs where Nibbler promotes small RNA trimming while Hen1 prevents it (Ameres et al., 2010; Yang et al., 2014a). 2’-O-methylation is also found on select miRNA isoforms and appeared to increase with age while its reduction was associated with neurodegeneration and shorter life span (Abe et al., 2014). Introduction_to_RNAi.indd 152Introduction_to_RNAi.indd 152 09.07.20 8:3409.07.20 8:34 ARTHROPODS 153 nucleotidyltransferases – tailing of short RNAs is mediated by terminal nucleotidyltransferases, which produce 3’ uridylation or adenylation. PAPD4hasbeenidentifiedas a primary miRNA adenylating enzyme in Drosophila, adenylation did not appear to affect miRNA stability on a genome-wide scale (Burroughs et al., 2010). Another non-canonical adenylase is Wispy, which is responsible for adenylation of miRNAs and biologically it may facilitate clearance of maternal miRNAs in the embryo (Lee et al., 2014). Uridylation is mediated by Tailor, which is a uridylyltransferase that is required for the majority of 3’ endmodificationsofmicroRNAsinDrosophila and predominantly targets mirtron hairpins (Reimao-Pinto et al., 2015; Westholm et al., 2012). GW182 – GW182 is the key co-factor of AGO1 in miRISC. Its role has been described indetailinthemammaliansection,hewewillbrieflynoteitskeyfeatureswithrespect to arthropods. GW182 and its interaction with AGO1 were found to be required for miRNA-mediated repression in Drosophila cells (Behm-Ansmant et al., 2006; Eulalio et al., 2008; Rehwinkel et al., 2005). miRNA-mediated repression also required the decapping complexDCP1:DCP2andCCR4-NOTdeadenylase(Behm-Ansmantetal.,2006;Rehwinkel et al., 2005). Multiple domains of GW182 contribute to miRNA-mediated repression (Chekulaeva et al., 2009; Chekulaeva et al., 2011; Chekulaeva et al., 2010; Eulalio et al., 2009). Similarly to mammals, Drosophila GW182 directly interacts with PABP and CCR4-NOT(Chekulaevaetal.,2011;FukayaandTomari,2011;Huntzingeretal.,2010; Huntzinger et al., 2013; Moretti et al., 2012). Armitage–RNAhelicase,whichwasidentifiedasamaternaleffectgenerequiredfor RNAi(Tomarietal.,2004a).ArmitageisprobablynotrequiredforRISCactivity.Instead, itwasproposedtofacilitateremovalofthepassengerstrandduringRISCformation(Tomari et al., 2004a). Armitage was also implicated in piRNA biogenesis (Huang et al., 2014; Murota et al., 2014; Nagao et al., 2010; Qi et al., 2011; Saito et al., 2010). dFMR1 – Drosophila ortholog of human fragile X mental retardation protein (FMRP) wasidentifiedasaRISCcomponent(Caudyetal.,2002;Ishizukaetal.,2002;Phametal., 2004). dFMR1 is associated with ribosomes through interaction with ribosomal proteins L5 and L1 and with complexes containing miRNAs (Ishizuka et al., 2002). dFMR1 is not a conserved RISC component involved in RNAi as depletion of dFMR1 reduces RNAi efficiencyinDrosophila S2 cells but not in mammals (Caudy et al., 2002). dFMR has been also implicated in the piRNA pathway (Bozzetti et al., 2015; Jiang et al., 2016). VIG–VasaIntronicGene(Caudyetal.,2002;Phametal.,2004).VIGisaconserved protein, which encodes a putative RNA binding protein, whose depletion reduces RNAi efficiency(Caudyetal.,2002).Vig mutants are more susceptible to viral infections in Drosophila(Zambonetal.,2006).WhetherthisroleofVIGiscoupledwithitspresencein theRISCcomplexisnotknown.ThereisnoevidencethatSERBP1,theclosestmammalian VIGhomolog,wouldbeassociatedwithRISC.VIGwasalsoimplicatedinheterochromatin formation (Gracheva et al., 2009). Tudor-SN –TudorStaphylococcalNucleaseisaproteincontainingfivestaphylococcal/micrococcalnucleasedomainsandaTudordomain.ItisacomponentoftheRISCin C. elegans, Drosophilaandmammals(Caudyetal.,2003;Phametal.,2004).Therole ofTudor-SNinRISCRNAiremainsenigmatic.TSNisnotthe„slicer“(Schwarzetal., 2004)anditsknock-downinsilkmothcellshadnoteffectonRNAiefficiency(Zhuetal., Introduction_to_RNAi.indd 153Introduction_to_RNAi.indd 153 09.07.20 8:3409.07.20 8:34 PLANTSII 256 etal.,2015).ThecomplexityofsmallRNAbiologywasreviewedinthefirstplantRNA silencing review (Svoboda, 2019). Here, I will focus on mobility of small RNAs in plants. Plant anatomy and features relevant for movement of molecules Plants are unique in several aspects when compared to cells of other eukaryotic organisms. Plant cell features important for intercellular exchange of molecules include a polysaccharidecellwallandplasmodesmata.Thelatteraremicroscopicchannelstraversingcellwalls of plant cells allowing for movement (symplastic movement or symplast) of molecules between adjacent cells (reviewed in Maule, 2008; Maule et al., 2011). An alternative to the symplastic movement, which involves cell cytoplasm, is apoplastic movement (apoplast) where molecules move through cell walls and intercellular space. Afloweringplant(Fig.2)consistsofthreeanatomicalsystems–root,shoot,andinflorescence. It is anchored by a root, absorbs water and minerals from the grounds and transports them through the xylem vasculature through the plant and into leaves, which are the main photosynthetic organs. Sugars produced in leaves are transported around through the phloem vasculature. Figure 1 Complexity of small RNAs in plants. The scheme was adapted from (Borges and Martienssen, 2015) Introduction_to_RNAi.indd 256Introduction_to_RNAi.indd 256 09.07.20 8:3409.07.20 8:34 [Document text truncated for crawler view.]