Published online 29 Decembe 2015 Nucleic Acids Resea ch, 2016, Vol. 44, No. 8 3643–3658
doi: 10.1093/na /gk 1512
The cellula g ow h a e con ols o e all mRNA
u no e , and modula es ei he ansc ip ion o
deg ada ion a es o pa icula gene egulons
Jos´
eGa c
´
ıa-Ma ´
ınez1,2,†, Lidia Delgado-Ramos3,4,†, Guille mo Ayala5, Vicen Pelechano6,
Daniel A. Medina2,7, Fany Ca asco2,7,Ram´
on Gonz´
alez8, Edua do And ´
es-Le´
on3,
La s S einme z6,9,10, Jonas Wa inge 11, Sebas i´
an Ch´
a ez3,4,* and Jos´
eE.P
´
e ez-O ´
ın2,7,*
1Depa amen o de Gen´
e ica, Facul ad de Ciencias Biol´
ogicas, Uni e si a de Val`
encia. C/D . Moline 50. E46100,
Bu jasso , Spain, 2ERI Bio ecmed, Facul ad de Ciencias Biol´
ogicas, Uni e si a de Valencia. C/D . Moline 50.
E46100, Bu jasso , Spain, 3Ins i u o de Biomedicina de Se illa (IBiS), Hospi al Vi gen del Roc´
ıo-CSIC-Uni e sidad de
Se illa, C/An onio Mau a Mon ane , E41013 Se illa, 4Depa amen o de Gen´
e ica, Uni e sidad de Se illa, A enida
de la Reina Me cedes s/n, E41012, Spain, 5Depa amen o de Es ad´
ıs ica e In es igaci´
on Ope a i a, Facul ad de
Ma em´
a icas, Uni e si a de Val`
encia. C/D . Moline 50. E46100, Bu jasso , Spain, 6Eu opean Molecula Biology
Labo a o y (EMBL), Genome Biology Uni , Meye ho s asse 1, 69117 Heidelbe g, Ge many, 7Depa amen o de
Bioqu´
ımica y Biolog´
ıa Molecula , Facul ad de Ciencias Biol´
ogicas, Uni e si a de Valencia. C/D . Moline 50.
E46100, Bu jasso , Spain, 8Ins i u o de Ciencias de la Vid y del Vino (CSIC, Uni e sidad de La Rioja, Gobie no de La
Rioja), Finca La G aje a LO-20 Salida 13, Au o ´
ıa del Camino de San iago, E26007 Log o˜
no, Spain, 9S an o d
Uni e si y School o Medicine, Depa men o Gene ics, S an o d, CA 94305, USA, 10S an o d Genome Technology
Cen e , 3165 Po e D . Palo Al o, CA 94305, USA and 11Depa men o Chemis y and Molecula Biology, Uni e si y
o Go henbu g, Medicina ega an 9 c, 40530 G¨
o ebo g, Sweden
Recei ed No embe 04, 2015; Re ised No embe 30, 2015; Accep ed Decembe 16, 2015
ABSTRACT
We analyzed 80 di e en genomic expe imen s, and
ound a posi i e co ela ion be ween bo h RNA poly-
me ase II ansc ip ion and mRNA deg ada ion wi h
g ow h a es in yeas . Thus, in spi e o he ma ked
a ia ion in mRNA u no e , he o al mRNA con-
cen a ion emained app oxima ely cons an . Some
genes, howe e , egula ed hei mRNA concen a ion
by uncoupling mRNA s abili y om he ansc ip ion
a e. Ribosome- ela ed genes modula ed hei an-
sc ip ion a es o inc ease mRNA le els unde as
g ow h. In con as , mi ochond ia- ela ed and s ess-
induced genes lowe ed mRNA le els by educing
mRNA s abili y o he ansc ip ion a e, espec i ely.
We also de ec ed hese egula ions wi hin he he -
e ogenei y o a wild- ype cell popula ion g owing in
op imal condi ions. The ansc ip omic analysis o
so ed mic ocolonies con i med ha he g ow h a e
dic a es al e na i e exp ession p og ams by modu-
la ing ansc ip ion and mRNA decay.
The egula ion o o e all mRNA u no e keeps a
cons an a io be ween mRNA decay and he dilu-
ion o [mRNA] caused by cellula g ow h. This eg-
ula ion minimizes he indisc imina e ansmission o
mRNAs om mo he o daugh e cells, and a o s
he esponse capaci y o he la e o physiological
signals and en i onmen al changes. We also con-
clude ha , by uncoupling mRNA syn hesis om de-
cay, cells con ol he mRNA abundance o hose gene
egulons ha cha ac e ize as and slow g ow h.
INTRODUCTION
Cells mus adap o changing en i onmen al condi ions o
main ain i ness and o compe e wi h o he geno ypes du -
ing he na u al selec ion p ocess. As i ness is he ne g ow h
o a geno ype o e ime, one o he mos impo an a i-
ables o single cell o ganisms du ing he cou se o adap a-
ion is he g ow h a e (GR) o a popula ion composed only
o one geno ype in ela ion o he g ow h a e o o he iso-
genic popula ions. I is o en assumed ha highe popula-
ion g ow h a es in mic oo ganisms equi e highe p o ein
syn hesis a es (1–5). This is because p o eins cons i u e a
*To whom co espondence should be add essed. Tel: +34 963543467; Fax +34 963544635; Email: jose.e.pe ez@u .es
Co espondence may also be add essed o Sebas i´
an Ch´
a ez. Tel: +34 955923127; Fax +34 955 923 101; Email: scha [email protected]
†These au ho s con ibu ed equally o his wo k as i s au ho s.
C
The Au ho (s) 2015. Published by Ox o d Uni e si y P ess on behal o Nucleic Acids Resea ch.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by-nc/4.0/), which
pe mi s non-comme cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Fo comme cial e-use, please con ac
jou nals.pe [email protected]
3644 Nucleic Acids Resea ch, 2016, Vol. 44, No. 8
la ge ac ion o d y mass in bo h p oka yo es (6) and eu-
ka yo es (e.g. yeas (7)) and, he e o e, hei syn hesis is he
mos ene ge ically demanding p ocess (8). This p ocess is
no exclusi e o unicellula o ganisms and p obably as -
p oli e a ing umo cells a e also go e ned by hese ules
(9,10).
The ansla ion machine y includes he mos abundan
noncoding RNAs: RNA and RNAs. Thus he euka y-
o ic RNA polyme ases (RNA pol) de o ed o he syn he-
sis o RNA and RNA (RNA pol I and III) mus inc ease
hei ansc ip ion a es (TRs, see he explana ion abou he
ac onyms used in M&M) in pa allel o he GR (11). RNA
pol II, howe e , ansc ibes a much la ge se o mRNAs
subjec ed o mul iple egula o y in luences, many o which
lowe in concen a ion wi h he GR (12). So al hough a
la ge se o mRNAs, which a e highly ansc ibed, is also de-
o ed o ibosome biosyn hesis and ansla ion ac o s (8),
he ela ionship be ween he GR and RNA pol II TR is no
ob iously p edic able. Unlike RNAs and RNAs, which
a e s able molecules, mRNAs ha e a sho e hal -li e, and
bo h hei syn hesis and decay signi ican ly con ibu e o
egula e hei abundance (13). The RNA pol II ansc ip-
ion a e, especially o single cell o ganisms, is highly a i-
able and a p oxy o hei physiology and me abolism (3).
The in luence o he cell cycle (14) and cell size (15)on
he ansc ip ion a e has been in es iga ed in he model
yeas Saccha omyces ce e isiae. Cell cycle leng h and cell
size a e ela ed o he GR (15). T ansc ip ome dependence
on he yeas GR has been ho oughly in es iga ed in D. Bo -
s ein’s labo a o y (16–19). These au ho s ha e ound ha
he mRNA amoun /concen a ion (RA) o abou one hi d
o he genes a ies in a GR-dependen manne . In ac based
on an RA signa u e o 72 genes, i is possible o p edic he
ins an aneous GR wi h easonable accu acy (17).
The [mRNA] o a gi en gene is con olled by he ac ion o
wo opposi e o ces: syn hesis by RNA pol II and deg ada-
ion by se e al exonuclease pa hways (20). In he exponen-
ial phase, he GR is a dis inc i e ea u e o any yeas s ain
and en i onmen al condi ion. In his phase, mos mRNAs
a e in a s eady s a e (21), in which syn hesis and deg ada ion
a es (DR) a e iden ical. Syn hesis is a ze o-o de eac ion,
whe eas deg ada ion is a i s -o de eac ion ha depends
on a kine ic deg ada ion cons an (kd) and he mRNA con-
cen a ion. The e o e, TR =DR =kd·RA whe e kdis a
pa icula ea u e o each mRNA species and is in e sely
ela ed wi h he mRNA hal -li e (HL): kd=ln2/HL.
These simple equa ions allow any o he h ee kine ic pa-
ame e s (RA, TR, o kd) o be de i ed om he o he wo
(22).
Gi en he appea ance o se e al genome-wide echniques
o measu e he syn hesis and deg ada ion a es and he
mRNA concen a ions, in se e al model o ganisms, i is
possible o quan i y he espec i e con ibu ions o an-
sc ip ion and deg ada ion o he ac ual concen a ion o
each indi idual mRNA (13,23), and also o he sum o
hem all. To do his, we used mul iple da a se s o di e -
en mu an s and al e ed he key pa hways in gene egu-
la ion and unde di e en g ow h condi ions ob ained by
ou sel es and o he s o es he ela ionship o he h ee ki-
ne ic pa ame e s wi h he g ow h a e a global and single
gene le els. We ound ha TR and DR gene ally ended o
inc ease when g owing as e , and in such a way ha he
o al mRNA abundance o mos genes emained indepen-
den o he GR in a yeas cell. Howe e , some genes, which
co esponded o pa icula p o ein unc ions, showed in-
c eases and/o dec eases exclusi ely on one side o he equi-
lib ium, which p o oked changes in he ne abundance o
speci ic mRNAs. Wi h an inc easing GR, he abundance
o he ansla ion- ela ed mRNAs and he s ess-induced
mRNAs inc eased and dec eased, espec i ely, due o an-
sc ip ion in bo h cases. The abundance o he mi ochond ia-
ela ed mRNAs also dec eased wi h an inc easing g ow h
a e, which led o a highe p opo ion o e men a ion o e
espi a ion a a highe GR, e en in he same high glucose
medium. Howe e , his was caused by inc eased mRNA
des abiliza ion, and no by educed ansc ip ion.
We con i med he la e by a o ally di e en expe imen-
al app oach. We ook ad an age o he in insic he e o-
genei y o he GR wi hin wild- ype yeas popula ions un-
de op imal en i onmen al condi ions (24) ose upanew
in-house de eloped p ocedu e o allow he sepa a ion o
yeas mic ocolonies acco ding o hei GR om a popula-
ion o gene ically iden ical cells. When compa ing mRNA
exp ession le els, we ound ha slow- and as -p oli e a ing
subpopula ions exhibi ed di e en ial ansc ip omic signa-
u es. This sugges s he exis ence o dis inc exp ession p o-
g ams ha a e closely linked o he GR and a e independen
o he en i onmen . The mi ochond ia- and espi a ion-
ela ed genes we e o e exp essed in he cells om slowe
g owing mic ocolonies. The PUF3 gene, which encodes an
RNA-binding p o ein (RBP) ha egula es he s abili y
and ansla ion o many mRNAs ela ed wi h mi ochon-
d ial unc ions, was also o e exp essed a a low g ow h
a e in bo h he mu an collec ion and slow-g owing mic o-
colonies. Slowe g owing mic ocolonies we e speci ically en-
iched in long mRNA iso o ms wi h Pu 3-binding si es a
hei 3UTRs.
We p opose ha he g ow h a e in luences gene exp es-
sion by ac ing on bo h sides o he [mRNA] equilib ium:
syn hesis and deg ada ion. The impo ance o hese wo op-
posi e p ocesses is dis inc o di e en classes o gene unc-
ions. In pa icula o espi a ion- ela ed mRNAs, Pu 3 ex-
e s a s abilizing in luence and p omo es espi a ion a a low
GR in a manne ha is independen o ex e nal glucose con-
cen a ions.
MATERIALS AND METHODS
Me a-analysis o expe imen al da a se s
In he i s me a-analysis we used se e al S. ce e isiae an-
sc ip omic da a se s (published and unpublished) ob ained
by us. These da a included 42 da a se s o nascen an-
sc ip ion a es and 21 da a se s o mRNA amoun s, bo h
ob ained by he GRO p o ocol (25). All hese da a se s
we e ob ained om exponen ially g owing yeas popula-
ions. Di e ences in GRs we e due o dis inc cul u e con-
di ions (ca bon sou ce, empe a u e), and also o he single
dele ions o indi idual genes. In 34 cases, he TR o a e -
e ence sample was simul aneously de e mined. The nascen
TR and RA alues o indi idual genes we e measu ed as
a ios wi h ega d o a ele an e e ence sample: a wild-
ype (mos ly BY4741, see Supplemen a y Table S1) s ain
Nucleic Acids Resea ch, 2016, Vol. 44, No. 8 3645
a 28◦C in YPD (yeas ex ac 1%, pep one 1%, glucose 2%)
medium. In his way, da a can be compa ed ac oss expe i-
men s. Indi idual alues we e summed o ob ain he global
es ima es o all he genes. Fo he o al TR (RNA pol I + II
+ III), he o al adioac i i y inco po a ed du ing a un-on
expe imen was used.
A ecen ly published se o 44 yeas mu an s was also
used, in which he au ho s employed uni o m cul u e con-
di ions: exponen ial phase cells g own a 30◦CinYPD(26)
o which he ma u e syn hesis a e (TR), RA and kdwe e
de e mined acco ding o he cDTA p o ocol. Since di e en
mu an s ha e dis inc cell olumes, he TR and RA alues
we e co ec ed by di iding each TR and RA gene alue by
each mu an /w old o cell olume compa ed o i s wild-
ype s ain. The da a o he di e en yeas mu an s and
hei wild ype we e ob ained om a ious sou ces (27,28).
Nascen TR and ma u e mRNA syn hesis a e ep esen
di e en aspec s o he same phenomenon (22). In his pa-
pe , o he sake o simplici y, we used he ac onym TR o e-
e o he molecula p ocess o RNA syn hesis by any RNA
polyme ase. Mos o he ime, we e e only o he RNA pol
II TR, o he wise we indica e wha we e e o. In GRO ex-
pe imen s TR is calcula ed as nascen ansc ip ion a e bu
in he cDTA me hod (26) a ma u e mRNA syn hesis a e
is de e mined by in i o RNA labeling. The e m syn hesis
a e is used when alking abou chemical equilib ium (as in
he equa ion desc ibed in he In oduc ion sec ion); whe e i
e e s o he a e o change o he ma u e mRNA concen a-
ion in he cy oplasm (22). The syn hesis a e can be in e ed
om nascen TR da a by assuming ha a ixed pe cen age
o nascen mRNA molecules each he cy oplasm.
G ow h a e es ima ions
Fo all ou expe imen al condi ions, we ob ained he GR
by g owing 50 ml o yeas cul u es in 250-ml lasks wi h
shaking (190 pm) a he desi ed empe a u e. Aliquo s we e
aken e e y 30 min in he exponen ial phase and hei OD600
( om 0.05 o 0.7) we e measu ed. The GR (in h−1)in he
exponen ial phase was calcula ed om g ow h cu es (Sup-
plemen a y Table S1A). Simila ly o he 44 yeas mu an s
used om Sun e al. (26), he GRs o 38 o hese s ains
we e calcula ed om he g ow h cu es ob ained by mic o-
cul i a ion in a Biosc een C eade , as desc ibed elsewhe e
(29) (Supplemen a y Table S1B). The exponen ial phase
GRs we e ex ac ed om g ow h cu es and a e aged o e
eplica es (n =2) (Supplemen a y Table S1A), as p e iously
desc ibed (30).
Expe imen se and da a no maliza ion
We employed da a se s om many di e en expe imen s ob-
ained om a ious pla o ms. The exp ession alues ac-
qui ed om hese expe imen s we e no malized by means o
quan ile no maliza ion (31), and we e implemen ed in o he
unc ion No malizeBe weenA ays o he Limma R package
(32).
Co ela ion analysis be ween pa ame e s and he g ow h a e:
global alues
The global endencies be ween di e en pa ame e s and he
GR o ou da a se s and o hose o Sun e al. (26)we ecal-
cula ed by plo ing he o al RNA pol II ansc ip ion, and
also o he mRNA concen a ion and he mRNA deg ada-
ion cons an (kd). They we e all ep esen ed as being ela-
i e o hei wild- ype o e e ence condi ion. The obse ed
Pea son’s co ela ion coe icien , , and he P- alue o he
s a is ically signi ican de ia ion om he null hypo hesis o
no co ela ion ( =0) we e calcula ed. A mul iple eg ession
model was also applied (see s a is ical appendix in Supple-
men a y In o ma ion).
En ichmen analyses o gene ca ego ies
Fo each sample in hese expe imen s, we had he co-
a iable GR and he co esponding exp ession p o ile pe
gene. The GR-gene exp ession co a ia ion was quan i ied
using a modi ied e sion o Pea son’s co ela ion ( o de-
ails, see R. Tibshi ani, G. Chu, B. Na asimhan and J.
Li, 2011, SAM, Signi icance Analysis o Mic oa ays. R
package e sion 2.0. h p://CRAN.R-p ojec .o g/package=
sam ) be ween bo h alues. A gene- o-gene analysis o he
di e en ial exp ession was pe o med by he Signi icance
Analysis o Mic oa ays (SAM) me hod (33,34) wi h a alse
disco e y a e o q <0.05. A gene se en ichmen analysis
was applied o p e iously de ec ed se s o di e en ially ex-
p essed genes. A unila e al Fishe ’s exac es was applied
whe e he gene se s o be compa ed we e he Gene On ology
g oups. Analyses we e un wi h R packages (R. Gen leman,
wi h con ibu ions om S. Falcon and D. Sa ka , 2014, Ca -
ego y Analysis. R package e sion 2.32.0 and M. Ca lson,
2014), o g.Sc.sgd.db: Genome wide anno a ion o Yeas .
R package e sion 3.0.0). The whole R code used in his
pape is ound as Supplemen a y Ma e ial. A second en-
ichmen analysis was un by he Gene Se Analysis (GSA)
me hod (B. E on and R. Tibshi ani, 2010 GSA:, Gene se
analysis. R package e sion 1.03. h p://CRAN.R-p ojec .
o g/package=GSA)(35). A de ailed s a is ical p o ocol is
gi en as a s a is ical appendix in Supplemen a y In o ma-
ion.
Gene on ology ep esen a ions
The genes om he global expe imen se s o all he pa-
ame e s (TR, RA, and kd) om bo h da a sou ces we e
anked acco ding o hei co ela ion wi h he GR. The
o e - ep esen ed unc ional ca ego ies (Gene On ology) ob-
ained in he SAM and GSA analyses we e educed and i-
sualized wi h he Re iGO web se e ((36)h p:// e igo.i b.
h /).
Calcula ing he espi a o y quo ien (RQ) index
The p o ocol desc ibed in Qui ´
os e al. (37) was used wi h
he ollowing modi ica ions. RQ de e mina ions we e made
in MiniBio bio eac o s (250 ml nominal olume) (App-
likon, Schiedam, The Ne he lands) equipped wi h Pel ie -
e ige a ed gas condense s, illed wi h YPD b o h (200 ml).
Cul u es we e spa ged wi h ai a a gas low o 600 ml/h.
3646 Nucleic Acids Resea ch, 2016, Vol. 44, No. 8
The gas low was con olled wi h MFC17 mass low con-
olle s (Aalbo g, O angebu g, NY, USA), whose calib a-
ion was e i ied egula ly by a soap bubble lowme e . The
ins an CO2and O2concen a ions in he exhaus gas we e
eco ded e e y minu e in a BlueInOne gas analyze (Blue-
sens, He en, Ge many). The measu ed pe cen ages o bo h
gases we e con e ed in o ins an p oduc ion o consump-
ion a es (exp essed in moles o gas pe li e o cul u e
hou ly) by aking in o accoun he con ibu ion o CO2p o-
duc ion and O2consump ion o he o al gas low. The accu-
mula ed p oduc ion o consump ion o each gas was de e -
mined as he in eg al o e ime o he ins an alues. The
ime poin ha co esponded o 0.15 moles o CO2p o-
duced pe li e o cul u e was chosen as a way o s anda d-
ize he RQ alues o compa isons. A his ime poin , mos
o he ini ial suga was al eady consumed, hus he co e-
sponding RQ alues (RQ 0.15) would easonably summa-
ize all he cul u e s ages. RQ 0.15 was calcula ed as he a io
be ween he accumula ed amoun s (exp essed in moles) o
he p oduced CO2and he consumed O2 o his ime poin .
Cell mic oencapsula ion and mic ocolony assay
The mic oencapsula ion o indi idual yeas cells in algi-
na e mic osphe es (no mo e han one cell pe mic opa icle)
was pe o med using a Cellena mic oencapsula o (Ingeni-
a ics), as desc ibed elsewhe e (38). Encapsula ed cells we e
incuba ed oge he in YPD medium unde s anda d cul u -
ing condi ions. A e e hanol ixa ion, mic ocolonies we e
analyzed and so ed using a BioSo e la ge-pa icle low
cy ome e (Union Biome ica). A de ailed p o ocol o he
so ing p ocedu es is desc ibed in (39).
T ansc ip omic analysis o mic ocolonies
The o al RNA ex ac ed om he so ed mic ocolonies and
om he non-so ed con ols was sequenced ollowing he
3T- ill p o ocol as p e iously desc ibed (40). The s a ing
ma e ial used was 500–3000 ng o o al RNA.
The aw nex gene a ion sequencing eads o be i s as-
sessed o hei quali y wi h he FASTQC ool ki Bad qual-
i y eads (ph ed sco e <20) we e immed. The genome se-
quence o S. ce e isiae S288c ( e sion R64) and i s anno-
a ions we e e ie ed om SGD (Saccha omyces Genome
Da abase, h p://yeas genome.o g/) and used o all he
analyses. Raw eads we e aligned agains he e e ence
genome using BWA/Bow ie2. We quan i ied he gene ex-
p ession om he mapped eads using he HTSeq-coun
package (41) in o de o ob ain coun s o mapped eads pe
gene (in he ‘in e sec ion-nonemp y’ mode). As he coun
p ocess was dependen on sequencing dep h, hose samples
wi h a small numbe o eads we e emo ed, whe eas hose
samples wi h a huge numbe o eads we e down-sampled
using he Fas qSample u ili y om he Sho ead package
(42).
The s a is ical en i onmen R ( e sion 3.1; (43)), was
used o pe o m he s a is ical analysis. Hie a chical clus e -
ing, boxplo s, and Mul idimensional Scaling (MDS) analy-
ses we e pe o med be o e and a e no maliza ion o mea-
su e he di e ences among samples. The edgeR package
om R/BioConduc o (44) was used o no malize and i
coun da a o he di e en ial gene exp ession analysis. In
o de o a oid a bias in poo ly exp essed genes, hose genes
wi h less han i e mapped eads pe million in a leas wo
samples we e emo ed. An iso o m-speci ic analysis was
pe o med and an assignmen was made o ansc ip s us-
ing GSNAP ( e sion 2012–01–11) as p e iously desc ibed
(40).
RESULTS
T ansc ip ion a e scales wi h he yeas g ow h a e
To analyze he ela ionship be ween mRNA u no e and
g ow h a e, we analyzed a se o 42 genome-wide ansc ip-
ion a e expe imen s (25,45–48)(P
´
e ez-O ´
ın, Medina and
An ´unez, unpublished), pe o med by he genomic un-on
(GRO) me hod ha p o ides nascen TR da a (25). These
expe imen s co esponded o he yeas s ains (wild- ypes
and mu an s) ha g ow in di e en ca bon sou ces (YPD,
YPGal, YPRa ) and a dis inc empe a u es ( om 23◦C o
37◦C; see Supplemen a y Table S1A). In each expe imen ,
we ob ained indi idual mRNA amoun s, nascen TR and kd
o mos p o ein-coding genes, as well as he o al TR ( o all
h ee nuclea RNA polyme ases). All he expe imen s we e
compa able because he da a we e no malized by cell num-
be s and cell olumes (48).
We i s analyzed he global RNA pol II nascen TR and
RA da a by summing all he p o ein-coding genes wi h a
leas 32 alid da a poin s (5411 genes). Figu e 1A shows a
clea posi i e co ela ion be ween he o al RNA pol II TR
and he g ow h a e (in ed). Wi hin he analyzed GR ange,
an inc ease in he TR was app oxima ely p opo ional o
an inc ease in he GR. This dependence was no caused by
he genes ha encoded he ansla ion machine y because
he o e all endency emained almos iden ical when hey
we e excluded (Figu e 1A in blue). The o al TR (including
RNA pol I, II and III), which was also ob ained in he un-
on expe imen , co ela ed posi i ely wi h he g ow h a e
(Supplemen a y Figu e S1). This was expec ed because ap-
p oxima ely a 75% o he o al TR came om RNA pol I +
III ansc ip ion (8), which was done o ansc ibe he non-
coding componen s o he ansla ion machine y ( RNAs
and RNAs).
Gi en he e y di e se se o s ains and g ow h condi-
ions (see Supplemen a y Table S1A), hey all e ealed hei
own pa icula in luence on he o al TR. Thus he obse ed
g ow h a e dependence o he TR mus be ega ded as e-
ma kably obus . None heless, all he da a we e ob ained
by a pa icula echnique, GRO, which could be subjec o
echnique-speci ic sys ema ic biases. To independen ly con-
i m ou esul s, we used an independen published da a se
based on me abolic mRNA labeling, which di ec ly mea-
su es he ma u e mRNAs ha appea in he cy oplasm. We
analyzed he da a ha co esponded o 44 yeas mu an
popula ions ha g ew exponen ially a 30◦C in YPD om
which he TR, RA and kd o mos o he p o ein-coding
genes we e ex ac ed (26). To complemen his da a se , we
measu ed he GRs o mos o hese mu an s (38 s ains, see
Supplemen a y Table S1B) by mic o-cul i a ion (30). As he
TR should be measu ed as a change in mRNA concen a-
ion o e ime (22), and he o iginal da a we e no co ec ed,
we used he olume es ima es o hese mu an s ob ained by
Nucleic Acids Resea ch, 2016, Vol. 44, No. 8 3647
Figu e 1. The mRNA u no e co ela ed posi i ely wi h he yeas g ow h a e. (A) We plo ed 34 o he 42 di e en g ow h a ia ions (mu an s ains,
empe a u e o ca bon sou ce a ia ions) o which we had ela i e ansc ip ion a es o a e e ence sample. A clea dependence o he o al nascen
ansc ip ion a e (TR) o RNA pol II is obse ed, as measu ed by GRO (23) wi h he GR o he cul u e in ae a ed lasks. Values a e ela i e o he
co esponding e e ence s ain. Red symbols co espond o he a e age o all he da a, whe eas blue symbols co espond o he a e age o all he da a
excep genes RP and RiBi. Pea son’s co ela ion coe icien ( ) and he associa ed P- alue a e shown. (B–D). We used he da a o he a e age o al mRNA
syn hesis a e (TR), mRNA le els (RA) o mRNA deg ada ion cons an (kd) om 38 mu an s ains (26) and he GR de e mined by mic o-cul i a ion.
The o iginal TR and RA da a we e co ec ed by cell olume using da a om (27,28). Once again, we obse e a posi i e dependence o TR and in kd,which
kep he mRNA le els app oxima ely cons an . Pea son’s co ela ion coe icien s ( ) and associa ed P- alues a e shown. (E) Mul iple Reg ession Model
shows ha he RA can be p edic ed om he TR and he kdwi h highly signi ican P- alues bu i canno be p edic ed om GR (see S a is ical Appendix
in Supplemen a y In o ma ion o u he explana ion).

3648 Nucleic Acids Resea ch, 2016, Vol. 44, No. 8
Jo gensen e al. (27) and T uong e al. (28) o co ec hem.
The plo s o he h ee pa ame e s a e shown in Figu e 1B–
D. He e we see ha he TR s eadily and p opo ionally in-
c eased wi h an inc easing GR (Figu e 1B), whe eas mRNA
s abili y dec eased (kdinc eased, Figu e 1D). The e e se
dependencies o he TR and mRNA s abili y on g ow h a e
ensu ed ha he s eady-s a e mRNA le els, which e lec ed
he join consequence o mRNA syn hesis and deg ada ion
a es, we e la gely independen o he GR (Figu e 1C). A
Mul iple Reg ession Model con i ms ha RA can be p e-
dic ed om he TR and om he kd, as expec ed, wi h highly
signi ican P- alues bu i canno be p edic ed om he GR
(Figu e 1E). Simila esul s o he kdand RA dependence
on GR we e ob ained o ou GRO expe imen s da a se
om a subse o expe imen s (see Supplemen a y Table S4).
Toge he hese esul s compellingly suppo ha he
RNA pol II ansc ip ion a es, measu ed as bo h he
nascen o ma u e TR, we e s ongly associa ed wi h he
g ow h a e. The deg ada ion a e cons an (kd,seeIn o-
duc ion sec ion) also inc eased wi h a ising g ow h a e,
and o al [mRNA] emained cons an . Thus he mRNA
u no e a e o yeas cells co ela ed di ec ly wi h he pop-
ula ion g ow h a es. Mo eo e , he inc ease in mRNA
u no e was quan i a i ely simila o ha o he GR, which
sugges s ha he p opo ionali y be ween bo h should be
main ained.
P o ein syn hesis genes a e mo e ansc ibed and espi a o y
gene ansc ip s a e des abilized a as g ow h
The gene al endency o he RNA pol II TR ep esen s he
a e age o e all p o ein-coding genes. The pa icula en-
dencies o indi idual genes may di e ge subs an ially om
his a e age and can be s udied om ou o iginal da a se s
o om ha published by P. C ame ’s g oup (26). To in-
es iga e he dominan biological ends among indi idual
genes o each TR, kdand RA, we o de ed all he no mal-
ized da a acco ding o hei co ela ion wi h he g ow h a e.
Two di e en s a is ical analyses, he SAM and GSA me h-
ods (see M&M), we e hen applied o iden i y he gene unc-
ions (GO ca ego ies) en iched in he op (highes posi i e
) o he bo om (lowes posi i e o highes nega i e , de-
pending on he plo ) o each anking lis . Bo h me hods and
bo h da a se s ga e simila esul s. As he gene al TR and
kd endencies esul ed in s ong posi i e co ela ions wi h
he GR (Figu e 1), e y ew genes showed nega i e co ela-
ions. Thus o he TR, and kddesc ibed below, ‘ op’ means
he genes wi h he s onges posi i e co ela ion, whe eas
‘bo om’ gene ally indica es he genes wi h he lowes posi-
i e (a ound ze o) co ela ion. In con as , he RA da a se s
a e cen e ed on ze o gi en he gene al absence o a change
wi haGR(Figu e1C). The e o e he ‘bo om’ g oup con-
ains he genes ha uly and nega i ely co ela e wi h he
g ow h a e. As all he da a se s we e no malized by hei
median, he ound endencies ep esen biases o he unc-
ional ca ego ies wi h ega d o he ypical beha io o he
popula ion.
The SAM s a is ical analyses o TR-GR dependence
ound RiBi (Ribosome Biogenesis), and o he p o ein-
syn hesis- ela ed genes, o be en iched among he genes
whose nascen TRs co ela ed posi i ely wi h he GRs (Fig-
u e 2A, Supplemen a y Table S2). In con as , he TRs o
he espi a ion- and mi ochond ia- ela ed genes ended o
be he leas posi i ely co ela ed wi h GRs (Figu e 2B).
The s a is ical analyses o RA-GR dependence also ound
ha he s eady-s a e mRNA abundance o RiBi- and he
o he p o ein syn hesis- ela ed genes ended o inc ease
wi h ising g ow h a es (Figu e 2C, Supplemen a y Ta-
ble S2). In con as , he s eady-s a e mRNA abundance
o he espi a ion- and mi ochond ia- ela ed genes dimin-
ished wi h as e g ow h (Figu e 2D). We did no ana-
lyze he mRNA s abili y endencies in his da a se because
e y ew da a se s we e a ailable. Once again, he analy-
sis o he TR-GR dependence in he al e na i e da a se
(26) ound ha he mRNA syn hesis a e o he RiBi- and
amino acid biosyn hesis- ela ed genes co ela ed s ongly
and posi i ely wi h g ow h a e, whe eas he TRs o he
cell cycle and egula ion genes ended o decouple om i
(see Supplemen a y Figu e S2 and Table S3). Gi en he
la ge numbe o es ima es in his case, we also analyzed kd-
GR dependence o ind ha he mRNA deg ada ion o he
RiBi and Glycosyla ion genes did no co ela e wi h g ow h
a e, whe eas he mRNA deg ada ion o he acuole- and
mi ochond ia- ela ed genes ended o inc ease wi h ising
GR (see Figu e 2E–F and Supplemen a y Table S2). O he
al e na i e sea ches be ween ou TR and RA da a and he
TRandRAda ao Sune al. (26) we e made by he GSA
me hod. Simila esul s we e ob ained.
D. Bo s ein’s lab made a sys ema ic sea ch o genes
whose RA co ela ed ei he posi i ely o nega i ely wi h
he g ow h a e (16,17,49). In hese s udies, a signa u e o
72 genes was ob ained, which s ongly depended on he
GR. We u he analyzed he esponse o hese genes and
ound ha some 50 genes showed essen ially iden ical (de-
c easing o inc easing) endencies o hei RA and TR p o-
iles (Supplemen a y Figu e S3). This indica es ha mos
g ow h a e- egula ed ansc ip s a e con olled manly a
he syn hesis le el. Howe e , we also ound se en genes o
which mRNA abundance and he TR a e da a co ela ed
in e sely, which sugges s a s ong egula ion a he s abili y
le el.
In conclusion, apa om he gene al endency o cells
o inc ease he o al mRNA syn hesis a e wi h as e
g ow h, he inc eased ela i e ansc ip ion o some unc-
ional classes o genes was pa icula ly p onounced. As a
esul o he highe TRs, he s eady-s a e mRNA abun-
dance o hese pa icula ypes o genes, unlike ha ob-
se ed o he o e all ansc ip ome (Figu e 1C), seemed
o also inc ease wi h as e g ow h. In con as , he s eady-
s a e mRNA concen a ion o o he unc ional classes o
genes ac ually dec eased as a esul o as e g ow h, caused
appa en ly by a pa icula ela i e dec ease in hei mRNA
s abili y (inc ease in kd).
Ribosome biogenesis, mi ochond ia- ela ed and s ess-
induced genes use di e en mechanisms o adjus mRNA
le els o he g ow h a e
We p e iously ound ha some GO ca ego ies di e ed om
he gene al endency o no change in mRNA abundance
by displaying changes in he GR. To shed u he ligh
on o hese opposing ends, we sepa a ely conside ed he i-
Nucleic Acids Resea ch, 2016, Vol. 44, No. 8 3649
Figu e 2. Sea ch o he Gene On ology ca ego ies ha show enhanced dependence on he g ow h a e. GO en ichmen sea ches o he highes posi i e
and nega i e GR dependences on he nascen TR, mRNA le els and deg ada ion a es a e shown. Apa om he gene al endency o he nTR and RA
showninFigu e1, ce ain unc ional ca ego ies a e especially en iched among he highes posi i e co ela ions ( op) wi h he GR, o among he genes
wi h he lowes co ela ion (bo om). We show he e (A–D) he analyses done wi h he da a se s used in Figu e 1 o he nTR and RA, and om he da a
se o Sun e al. (26) o kd(E–F) using an SAM analysis and summa ized by he ReViGO p og am. Simila esul s o he TR and RA we e ob ained wi h
he da a se s used in Figu e 1B–C (26); see Supplemen a y Figu e S3.
3650 Nucleic Acids Resea ch, 2016, Vol. 44, No. 8
bosome biogenesis (GO:0042254) and mi ochond ial ibo-
some (GO:0005761) unc ional ca ego ies. Figu e 3A illus-
a es how he TR o he RiBi genes inc eased o esul in
highe mRNA abundance and he eby, p esumably, main-
ained he ibosome numbe s le el wi h GR demands. This
is he clea es example o a egulon con olled a he an-
sc ip ional le el. RiBi mRNA s abili ies did no change in a
GR-dependen manne (Figu e 3B). This beha io was sim-
ila in ibosomal p o ein (RP) genes (Supplemen a y Figu e
S4). In con as , he mi ochond ial ibosome genes showed
no egula ion a he ansc ip ional le el (Figu e 3C). Ne -
e heless, s eady-s a e mRNA abundance diminished wi h
he GR, which was achie ed by a ma ked educ ion in he
s abili y o hese mRNAs (Figu e 3D). This is he clea es
example o a pos - ansc ip ional egulon (50).
Gi en ha he mRNA le els o he espi a o y- and
o he mi ochond ia- ela ed genes lowe ed wi h as e
g ow h, and by assuming no addi ional compensa o y eg-
ula ion o he ansla ion o pos - ansla ional le els, we
expec ed espi a ion o dec ease acco dingly. To es his
p edic ion, we selec ed a se o nine s ains (mu an s and
wild ypes) om ou da a se (see Figu e 1A and Sup-
plemen a y Table S1A) and de e mined hei espi a o y
quo ien ( e men a ion/ espi a ion, RQ) in a mic o e men-
o (37). As seen in Supplemen a y Figu e S5, he lowe
he mRNA le els o he genes ela ed wi h espi a ion
(GO:0009060) o mi ochond ial ibosome (GO:0005761),
he highe he RQs and, hus, he ela i e in luence o es-
pi a ion on me abolism. This shows ha he educ ion in
mRNA le els o he espi a o y and o he mi ochond ial
genes wi h highe g ow h a es is physiologically ele an
and se es o educe espi a ion. This s ongly suppo s he
dependence o as e -g owing yeas cells on he e men a-
i e me abolism.
Finally, we paid special a en ion o he equen ly s ud-
ied s ess-induced genes o he so-called ‘en i onmen al
s ess esponse’ (ESR) (51). These genes ha e been shown o
be up- egula ed in he RA in slow-g owing mu an s (12,16),
which implies a nega i e co ela ion be ween hei mRNA
abundance and g ow h. I was ou in en ion o es ablish
whe he his was indeed he case, and i so, whe he o
no i was achie ed by a change in he TR o DR. Indeed
he mRNA abundance o he s ess-induced sec ion o he
ESR diminished a as e g ow h (Figu e 3E). This dec ease
was achie ed exclusi ely by lowe ing TRs, and no gene al
change ook place in mRNA deg ada ion (Figu e 3F). Thus
he s ess-induced pa o he ESR exempli ied a hi d egu-
la o y s a egy: a nega i e e ec o g ow h a e on ansc ip-
ion a es.
mRNA dependence on he g ow h a e can be de ec ed wi hin
he he e ogenei y o a wild- ype cell popula ion
So a we analyzed he dependence o mRNA concen a-
ions, and hei syn hesis and deg ada ion a es on cellula
g ow h a e ac oss di e en geno ypes and en i onmen al
condi ions, including mu an s ains wi h impai ed p oli -
e a ion capaci y o wild- ype cells ha g ow in di e en en-
i onmen s. Simila ly, p e ious expe imen s ha ha e de-
sc ibed he in luence o he GR on gene exp ession ha e
also been based on he compa ison o cells popula ions in
di e en ca bon sou ces o unde chemos a - egula ed con-
di ions (18,52). These expe imen al designs canno ule ou
he ac ha he seeming dependence o TR, RA and kdon
he GR migh be a con ounding e ec o bo h depending
on a sha ed hi d ac o in he o m o speci ic mu a ions
and speci ic ad e se en i onmen al e ec s. The connec ion
be ween he g ow h a e and RNA me abolism should hap-
pen a he single cell le el. In o de o ule ou ha any o
hose e ec s occu ed only a he popula ion le el, we mea-
su ed he GR o single cells as hei abili y o p oduce mi-
c ocolonies, and we analyzed hei GR ansc ip omes.
To do his, we analyzed he mic ocolonies gene a ed a -
e he encapsula ion o he single cells ha belonged o a
clonal wild- ype popula ion in algina e mic osphe es (Sup-
plemen a y Figu e S6). Algina e encapsula ion allows he
ee di usion o nu ien s, is ully compa ible wi h yeas p o-
li e a ion, and helps so cells acco ding o hei GR po-
en ial (38). No dec eased iabili y was de ec ed a e he
encapsula ion p ocedu e (Supplemen a y Figu e S7A). En-
capsula ed cells we e incuba ed in ich liquid medium un-
de s anda d cul u e condi ions and we e allowed o p oli -
e a e. A he di e en ime poin s, samples we e aken and
obse ed by op ical mic oscopy. The e y di e en size ob-
se ed in mic ocolonies indica ed an e iden a ia ion in he
p oli e a ion capaci y wi hin his clonal popula ion (Figu e
4A). Quan i ica ion o he mic ocolony size by la ge pa -
icle low cy ome y con i med an in insic he e ogenei y
ha inc eased wi h incuba ion ime (Figu e 4B). We es ed
whe he he di e en mic ocolony size was due o a dis inc
size o he mic opa icle i sel o i i was a biased localiza-
ion wi hin he mic opa icle. We ound no di e ence o
ei he (Supplemen a y Figu e S7B and S7C). Mic ocolony
size he e ogenei y could also be due o an unequal lag phase.
The inc easing s a is ical dispe sion shown by mic ocolony
size du ing he ime-cou se expe imen s, by main aining
symme ical dis ibu ion, was ha dly compa ible wi h his
possibili y (Figu e 4B). Ou esul s a e in ag eemen wi h
p e ious epo s on p oli e a ion he e ogenei y conduc ed
unde op imal cul u e condi ions in S. ce e isiae, de ec ed
wi h echnical app oaches ha did no in ol e encapsula-
ion (24).
The combina ion o mic oencapsula ion and la ge pa i-
cle low cy ome y allows sis e mic ocolonies o be physi-
cally sepa a ed by so ing (Supplemen a y Figu e S6). We
isola ed he 10% op and bo om subpopula ions o mic o-
colonies acco ding o hei size (Supplemen a y Figu e S6).
These wo subpopula ions exhibi ed an a e age cell num-
be o 32 and 2519, espec i ely, which in ol es appa en
doubling imes o app oxima ely 162 and 76 min acco d-
ing o he incuba ion ime unde g owing condi ions (Figu e
4C). This longe duplica ion ime o he small mic ocolony
cells ag ees wi h hei longe G1 phase and e lec s hei
di e en ial cell-cycle egula ion (Delgado–Ramos, Mu˜
noz–
Cen eno and Ch´
a ez, o be published elsewhe e). The a e -
age cell numbe and appa en doubling ime o non-so ed
mic ocolonies ga e in e media e alues. Howe e , when all
he analyzed mic ocolonies we e conside ed as a single pop-
ula ion, he esul ing popula ion doubling ime was compa-
able o he s anda d liquid cul u es (Figu e 4C).
This simple p ocedu e allowed us o isola e mRNA
p epa a ions om he wo ypes o so ed colonies, and o
Nucleic Acids Resea ch, 2016, Vol. 44, No. 8 3651
Figu e 3. Ribosome biogenesis, mi ochond ia- ela ed and s ess-induced genes use di e en mechanisms o adjus mRNA le els o he g ow h a e. Using
he da a om he Figu e 1A da a se , we ex ac ed da a o he RiBi egulon (GO: 0042254, panels Aand B), o he mi ochond ial ibosome (GO: 0005761,
panels Cand D), and o he induced pa o he ESR (51)(panels Eand F). The genes in he Mi ochond ial ibosome GO ca ego y (and simila ly in he
Pu 3 egulon, see Figu e 5B) seem o ha e lowe mRNA le els (RA) wi h he g ow h a e (GR) o he cul u e, mainly by mRNA des abiliza ion (inc eased
deg ada ion cons an , kd). The mRNA le els o he RiBi genes (and he ibosomal p o ein genes, see Supplemen a y Figu e S4) end o ise wi h he GR
due o inc easing ansc ip ion (nTR). The induced pa o he ESR con e sely lowe s, and RA educes wi h dec easing ansc ip ion. Pea son’s co ela ion
coe icien s ( ) and associa ed P- alues a e shown.
3658 Nucleic Acids Resea ch, 2016, Vol. 44, No. 8
54. Lesage,P., Yang,X. and Ca lson,M. (1996) Yeas SNF1 p o ein kinase
in e ac s wi h SIP4, a C6 zinc clus e ansc ip ional ac i a o : a new
ole o SNF1 in he glucose esponse. Mol. Cell. Biol.,16, 1921–1928.
55. Vincen ,O. and Ca lson,M. (1998) Sip4, a Sn 1 kinase-dependen
ansc ip ional ac i a o , binds o he ca bon sou ce- esponsi e
elemen o gluconeogenic genes. EMBO J.,17, 7002–7008.
56. Gasmi,N., Jacques,P.E., Klimo a,N., Guo,X., Riccia di,A., Robe ,F.
and Tu co e,B. (2014) The swi ch om e men a ion o espi a ion in
Saccha omyces ce e isiae is egula ed by he E 1 ansc ip ional
ac i a o / ep esso . Gene ics,198, 547–560.
57. Mille ,M.A., Russo,J., Fische ,A.D., Lopez Leban,F.A. and
Oli as,W.M. (2014) Ca bon sou ce-dependen al e a ion o Pu 3p
ac i i y media es apid changes in he s abili ies o mRNAs in ol ed
in mi ochond ial unc ion. Nucleic Acids Res.,42, 3954–3970.
58. Lee,C.D. and Tu,B.P. (2015) Glucose- egula ed phospho yla ion o
he PUF p o ein Pu 3 egula es he ansla ional a e o i s bound
mRNAs and associa ion wi h RNA g anules. Cell Rep.,11,
1638–1650.
59. Gup a,I., Claude -Muns e ,S., Klaus,B., Ja elin,A.I., Aiya ,R.S.,
Benes,V., Wilkening,S., Hube ,W., Pelechano,V. and S einme z,L.M.
(2014) Al e na i e polyadenyla ion di e si ies pos - ansc ip ional
egula ion by selec i e RNA-p o ein in e ac ions. Mol. Sys . Biol.,10,
719.
60. Rio dan,D.P., He schlag,D. and B own,P.O. (2011) Iden i ica ion o
RNA ecogni ion elemen s in he Saccha omyces ce e isiae
ansc ip ome. Nucleic Acids Res.,39, 1501–1509.
61. Sain -Geo ges,Y., Ga cia,M., Dela eau,T., Jou d en,L., Le C om,S.,
Lemoine,S., Tan y,V., De aux,F. and Jacq,C. (2008) Yeas
mi ochond ial biogenesis: a ole o he PUF RNA-binding p o ein
Pu 3p in mRNA localiza ion. PloS One,3, e2293.
62. Po ua,O. and Lib i,D. (2015) T ansc ip ion e mina ion and he
con ol o he ansc ip ome: why, whe e and how o s op. Na . Re .
Mol. Cell Biol.,16, 190–202.
63. Pelechano,V., Cha ez,S. and Pe ez-O in,J.E. (2010) A comple e se o
nascen ansc ip ion a es o yeas genes. PloS One,5, e15442.
64. Ke en,L., an Dijk,D., Weinga en-Gabbay,S., Da idi,D., Jona,G.,
Weinbe ge ,A., Milo,R. and Segal,E. (2015) Noise in gene exp ession
is coupled o g ow h a e. Genome Res.,25, 1893-1902
65. Shalem,O., G oisman,B., Chode ,M., Dahan,O. and Pilpel,Y. (2011)
T ansc ip ome kine ics is go e ned by a genome-wide coupling o
mRNA p oduc ion and deg ada ion: a ole o RNA Pol II. PLoS
Gene .,7, e1002273.
66. Molin,C., Jauhiainen,A., Wa inge ,J., Ne man,O. and
Sunne hagen,P. (2009) mRNA s abili y changes p ecede changes in
s eady-s a e mRNA amoun s du ing hype osmo ic s ess. RNA,15,
600–614.
67. Pe ez-O in,J.E., Alepuz,P.M. and Mo eno,J. (2007) Genomics and
gene ansc ip ion kine ics in yeas . T ends Gene .,23, 250–257.
68. Ge be ,A.P., He schlag,D. and B own,P.O. (2004) Ex ensi e
associa ion o unc ionally and cy o opically ela ed mRNAs wi h
Pu amily RNA-binding p o eins in yeas . PLoS Biol.,2, E79.
69. Diaz-Ruiz,R., Rigoule ,M. and De in,A. (2011) The Wa bu g and
C ab ee e ec s: on he o igin o cance cell ene gy me abolism and
o yeas glucose ep ession. Biochim. Biophys. Ac a,1807, 568–576.
70. Lagunas,R. (1986) Misconcep ions abou he ene gy me abolism o
Saccha omyces ce e isiae. Yeas ,2, 221–228.
71. Dashko,S., Zhou,N., Compagno,C. and Pisku ,J. (2014) Why, when,
and how did yeas e ol e alcoholic e men a ion? FEMS Yeas Res.,
14, 826–832.
72. Wang,J., A olia,E., Hua,B., Sa i ,Y., Escalan e-Chong,R. and
Sp inge ,M. (2015) Na u al a ia ion in p epa a ion o nu ien
deple ion e eals a cos -bene i adeo . PLoS Biol.,13, e1002041.
73. Ven u elli,O.S., Zule a,I., Mu ay,R.M. and El-Samad,H. (2015)
Popula ion di e si ica ion in a yeas me abolic p og am p omo es
an icipa ion o en i onmen al shi s. PLoS Biol.,13, e1002042.
74. Abe na hy,E., Gilbe son,S., Alla,R. and Glaunsinge ,B. (2015) Vi al
Nucleases induce an mRNA deg ada ion- ansc ip ion eedback loop
in mammalian cells. Cell Hos Mic obe,18, 243–253.