Published online 23 Oc obe 2017 Nucleic Acids Resea ch, 2017, Vol. 45, No. 21 12401–12412
doi: 10.1093/na /gkx974
Asymme ic cell di ision equi es speci ic
mechanisms o adjus ing global ansc ip ion
Ad iana Mena1,†, Daniel A. Medina1,†, Jos´
eGa c
´
ıa-Ma ´
ınez2,†, Vic o ia Begley3,
Abhyudai Singh4, Sebas i´
an Ch´
a ez3,Ma iC.Mu˜
noz-Cen eno3and Jos´
eE.P
´
e ez-O ´
ın1,*
1Depa amen o de Bioqu´
ımica y Biolog´
ıa Molecula and E.R.I. Bio ecmed, Uni e si a de Val`
encia, D . Moline , 50,
Bu jasso 46100, Valencia, Spain, 2Depa amen o de Gen´
e ica and E.R.I. Bio ecmed, Uni e si a de Val`
encia, D .
Moline , 50, Bu jasso 46100, Valencia, Spain, 3Depa amen o de Gen´
e ica, Uni e sidad de Se illa and Ins i u o de
Biomedicina de Se illa (IBiS), Hospi al Vi gen del Roc´
ıo-CSIC-Uni e sidad de Se illa, 41013 Se illa, Spain and
4Depa men o Elec ical and Compu e Enginee ing, Uni e si y o Delawa e, Newa k, DE 19716, USA
Recei ed Augus 29, 2017; Re ised Sep embe 22, 2017; Edi o ial Decision Oc obe 07, 2017; Accep ed Oc obe 10, 2017
ABSTRACT
Mos cells di ide symme ically in o wo app oxi-
ma ely iden ical cells. The e a e many examples,
howe e , o asymme ic cell di ision ha can gen-
e a e sibling cell size di e ences. Whe eas physical
asymme ic di ision mechanisms and cell a e con-
sequences ha e been in es iga ed, he speci ic p ob-
lem caused by asymme ic di ision a he ansc ip-
ion le el has no ye been add essed. In symme -
ically di iding cells he nascen ansc ip ion a e
inc eases in pa allel o cell olume o compensa e i
by keeping he ac ual mRNA syn hesis a e cons an .
This canno apply o he yeas
Saccha omyces ce e-
isiae
, whe e his mechanism would p o oke a ne e -
ending inc easing mRNA syn hesis a e in smalle
daugh e cells. We show he e ha , con a ily o o he
euka yo es wi h symme ic di ision, budding yeas
keeps he nascen ansc ip ion a es o i s RNA poly-
me ases cons an and inc eases mRNA s abili y. This
con ol on RNA pol II-dependen ansc ip ion a e is
ob ained by con olling he cellula concen a ion o
his enzyme.
INTRODUCTION
Du ing exponen ial g ow h, o al cell mass and olume in-
c ease exponen ially. This inc ease should be compensa ed
by an equi alen inc ease in he numbe o molecules (RNA
o p o eins) o main ain ibos asis and p o eos asis (1–3).
A he single cell le el, he simples consequence o ibos a-
sis o mRNA le els would be he mRNA concen a ion
([mRNA]) emaining cons an wi h cell olume. Howe e ,
compensa o y di e ences in mRNA syn hesis and deg a-
da ion a es [mRNA] be ween cells o di e en sizes could
exis . Recen ly, a s udy on mRNA le els and ansc ip ion
a es in di e en sized cells was done in mammalian ib ob-
las s (4). These au ho s ound ha hose cells adap ed RNA
polyme ase II (RNA pol II) nascen ansc ip ion a es
(nTRII) o cell olume in o de o keep [mRNA] ibos a-
sis a he single cell le el. A p e ious s udy conduc ed wi h
exponen ial g owing popula ions o Schizosaccha omyces
pombe yeas cell size mu an s (5) ob ained a simila esul :
nTRII inc eases linea ly wi h he popula ion a e age cell
olume. In bo h s udies, he measu ed pa ame e was nTR,
which concep ually di e s om he ac ual mRNA syn hesis
a e (SRII)(see(6) o a de ailed discussion). nTRII e alu-
a es he numbe o ansc ibed mRNA molecules, bu he
chemical equilib ium be ween mRNA syn hesis and deg a-
da ion uses [mRNA] ins ead o numbe o molecules pe
cell. Acco dingly, he SRII is he igh pa ame e o be con-
side ed when dealing wi h equilib ium and ibos asis:
SRII =kd[mRNA],
whe e kdis he mRNA i s -o de deg ada ion cons an ha
allows he mRNA hal -li e (HL) o be calcula ed. SRII can
be app oxima ed by di iding nTRII pe cell olume. Thus,
a ein e p e a ion o bo h s udies (4,5) can conclude ha
SRII is kep cons an because nTRII scales in pa allel wi h
cell olume. A co olla y o hose esul s is ha as SRII and
[mRNA] a e independen o cell olume, kd, and he e o e
he HL, a e also in a iable (Figu e 1A).
These wo biological sys ems di ide by cell ission o
(s ochas ically) p oduce simila olume cells (7). Howe e ,
o he cellula sys ems di ide asymme ically and p oduce
wo sibling cells o di e en sizes. The appea ance, a e
each di ision, o wo cells wi h di e en cell olumes im-
poses a new scena io o ansc ip ion a e con ol. Asym-
me ic cell di ision (ACD) is a mechanism ha gene a es
cell di e si y in single cells o in mul icellula o ganisms,
while main aining sel - enewing s em cell popula ions (8,9).
*To whom all co espondence should be add essed. Tel: +34 963543467; Fax: +34 963544365; Email: jose.e.pe ez@u .es
†These au ho s con ibu ed equally o his wo k as i s au ho s.
C
The Au ho (s) 2017. 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
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jou nals.pe [email protected]
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12402 Nucleic Acids Resea ch, 2017, Vol. 45, No. 21
Figu e 1. Asymme ical di ision in S. ce e isiae p o okes a concep ual
p oblem in ansc ip ion a e con ol along successi e cell gene a ions. (A)
In symme ical cell di ision inc eased cell size is pa alleled by nTR in such
a way ha SR is kep cons an , and bo h iden ical daugh e cells ha e ap-
p oxima ely he same olume, nTR and SR, as hei p e ious gene a ion.
This has been obse ed in S. pombe and human ib oblas s (4,5), whe e no
change in [mRNA] and, he e o e in he mRNA hal -li e (HL), has been
de ec ed. (B) Wi h asymme ical di ision ha p oduces a la ge mo he (M)
and a small daugh e (D) cells, a simila model o nTR con ol would p o-
duce daugh e cells wi h highe SRs han he p e ious gene a ion, which
would ende his model unsa is ac o y o explain ac ual beha io in S.
ce e isiae.(C) We p opose a model in which nTR emains cons an wi h
olume, which would p o oke a lowe SR in e sely o inc eased olume.
I [mRNA] ibos asis is s ic ly conse ed, an inc ease in HL will appea .
Numbe s inside cells ep esen SR alues. No e ha genome eplica ion oc-
cu s in be ween second and hi d g ow h s ages in he h ee models shown.
In he case o model C nTR should be duplica ed a he end o he eplica-
ion (2) o be hen di ided (1+1) be ween daugh e and mo he cells.
Di e en expe imen al sys ems ha e been widely s udied o
disco e he molecula mechanisms ha pa i ion RNA and
p o eins be ween sibling cells, which cause changes in cell
beha io and a e (10). No always do sibling cells ha e di -
e en olumes, bu i is a equen ou come o ACD (8),
e.g. he de elopmen o D osophila b ain depends on neu-
oblas s, a ype o s em cell ha p oduces ma kedly smalle
daugh e cells (11). Howe e , he consequences o di e en
sized sibling cells on gene al gene exp ession ha e no ye
been s udied.
The yeas Saccha omyces ce e isiae is a pa icula ly good
example o ACD ha in ol es ma ked di e ences in size.
Cell olume con ol in his yeas di e s o S. pombe gi en
ha i buds p oduce a smalle daugh e cell ha is pheno-
ypically di e en om he la ge mo he cell (12). ACD
condi ions many budding yeas li e ci cums ances. Fo in-
s ance, mo he cells expe ience aging and die a e a numbe
o gene a ions (13,14), a phenomenon ha does no happen
in symme ically di iding cells such as S. pombe (15).
No de ailed s udy has been done on he in luence o cell
olume on mRNA u no e in S. ce e isiae o da e, de-
spi e old s udies ha ing add essed he e olu ion o mRNA
u no e in he cell cycle (16–18), a p ocess du ing which
cell olume changes. This yeas is dis an ly ela ed (330 o
420 million yea s om i s common ances o ) o S. pombe
(19). In ac many genes, cell cycle pa ame e s and ansc ip-
ion egula ion a e qui e di e en be ween hese wo yeas
species (7,20–22).
Cell size in mic oo ganisms is in luenced by di e en
pa ame e s, including ploidy (5,23)andg ow h a es(24)
which, in u n, depend on cul u e condi ions (25–27). The
olume o indi idual cells also changes du ing hei cell
cycle (28). Changes in cell olume in all hese ins ances
ep esen di e en physiological si ua ions and can, he e-
o e, be a ec ed by addi ional pa ame e s, such as he
e men a i e/ espi a o y quo ien . Thus he selec ion o a
pa icula expe imen al s a egy o in es iga e he depen-
dence o S. ce e isiae mRNA u no e wi h cell olume may
be obscu ed by indi ec e ec s.
In his s udy we used di e en expe imen al s a egies and
e- isi ed p e iously published s udies o conduc a comp e-
hensi e s udy abou changes in mRNA u no e wi h cell
olume in an asymme ic di iding cell (S. ce e isiae)and
o ex ac obus conclusions om a ious esul s. We con-
clude ha in budding yeas mRNA u no e dec eases wi h
cell olume in bo h global syn hesis and deg ada ion a es,
and in such a way ha mRNA ibos asis is basically main-
ained simila ly o ha ound in o he euka yo es. Howe e ,
we ound ha S. ce e isiae keeps all i s RNA polyme ases
nTR cons an in spi e o olume changes. Wi h nTRII his
is achie ed by con olling he exp ession o RNA pol II i -
sel . We pos ula e new egula o y models o budding yeas
ha di e om ha ound in o he cellula sys ems wi h
symme ical cell di ision. This sugges s ha he quan i a-
i e cons ain s imposed by ACD ha e in luenced he e olu-
ion o di e en egula o y mechanisms o cells o possess
symme ic o asymme ic di ision.
MATERIALS AND METHODS
Yeas s ains, media and g ow h condi ions
The S. ce e isiae s ains used he ein a e lis ed in Supple-
men a y Figu e S4C. Yeas cells we e g own in liquid YPD
(2% glucose, 2% pep one, 1% yeas ex ac ). Expe imen al
assays we e pe o med wi h cells g own o a leas se en
gene a ions un il OD600 0.5 a 28◦C.
S anda d p ocedu es we e ollowed o synch oniza ion
a START and low cy ome y (29,30).
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Nucleic Acids Resea ch, 2017, Vol. 45, No. 21 12403
Cell olume and o he cellula de e mina ions
The median alues o cell olumes we e calcula ed by a
Coul e -Coun e Z se ies de ice (Beckman Coul e , USA).
Absolu e alues in em oli e s and ela i e alues a e shown
in Supplemen a y Figu e S4C.
We ob ained he g ow h a e (GR) by g owing 50 ml o
yeas cul u es in 250-ml lasks wi h shaking (190 pm) a
28◦C. 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.
RNA ex ac ion and poly(A) RNA measu emen s
To de e mine RNA amoun , cells we e g own in ich me-
dia un il he exponen ial phase o al RNA was ex ac ed by
phenol:chlo o o m ex ac ion as desc ibed in (31)in h ee
biological iplica es and was quan i ied by OD260 es ima-
ion in a Nanod op de ice (The mo-Fishe ). Se ial dilu ions
o o al RNA we e hen spo ed on a nylon memb ane (Ny-
an SPC, GE Heal hca e) and hyb idized wi h a speci ic
oligo d(T)40 p obe, e minally labeled wi h Polynucleo ide
Kinase (Roche) and ␥-32P-ATP. Memb anes we e exposed
o an Imaging pla e (BAS-MP, Fuji ilm) and scanned by a
Fuji ilm FLA3000 Phospho image . The signal in ensi y o
he spo s was quan i ied wi h he A ay Vision so wa e.
These da a we e used o poly(A) cell concen a ion cal-
cula ions, as desc ibed in (32). Al e na i ely, he poly(A)
quan i ica ion in indi idual cells was done essen ially as
desc ibed (doi.o g/10.1101/044735), bu an oligo d(T)30V
labeled wi h Cy3 was used. These samples we e analyzed
in an LSR Fo essa cy ome e (Bec on Dikinson). Poly(A)
amoun was aken as es ima o o o al mRNA and hen di-
ided by cell olume o ob ain o al mRNA concen a ion
([mRNA]).
Global mRNA hal -li e de e mina ion by hiolu in shu o
To de e mine he global mRNA hal -li e, we used a simila
do -blo s a egy o ha desc ibed in he p e ious sec ion,
excep o he ac ha RNA samples we e collec ed a 0, 5,
12 and 25, min a e ansc ip ional shu -o , ollowing he
addi ion o hiolu in o 5 ␮g/ml. All he samples we e lash-
ozen in liquid ni ogen and RNA ex ac ion was hen pe -
o med. The de ailed p o ocol is desc ibed in (32).
De e mina ion o nascen ansc ip ion a es and syn hesis
a es by di e en me hods
Genomic Run-On (GRO) analyses we e done as in (33) wi h
he modi ica ions desc ibed (34). All he da a se s we e ob-
ained om he exponen ially g owing yeas popula ions
g own a 28◦C in YPD. The nascen ansc ip ion a e al-
ues o he indi idual genes we e measu ed as a ios wi h
ega d o a wild- ype haploid (BY4741) s ain. Indi idual
alues we e summed o ob ain he global RNA pol II an-
sc ip ion a e es ima es (nTRII).
Some da a we e ob ained om o he published da ase s
(35,36) (see below) by a di e en echnique (cDTA, (35)),
which calcula es ma u e mRNA ansc ip ion a es. These
a es, howe e , a e no co ec ed by cell olume and a e,
hus, chemically equi alen o nTRII.nTRandSR ep e-
sen di e en aspec s o he same phenomenon (see (6) o a
u he explana ion). In his pape , o he sake o simplici y,
we used he ac onym SR o e e o he molecula p ocess
o RNA syn hesis by any RNA polyme ase and o di e -
en ia e be ween RNA polyme ases using he subindex (e.g.
SRII,SR
I). The SR can be in e ed om expe imen al nTR
da a (GRO) by assuming ha a s able pe cen age o nascen
mRNA molecules eaches he cy oplasm and om cDTA
da a by di iding hem by ela i e cell olumes, as desc ibed
(37). The me a-analysis o hese da a se s is desc ibed below.
Fo he o al SR (SRT: RNA pol I + II + III), he o-
al adioac i i y inco po a ed du ing a un-on expe imen
(nTRT) was de e mined by TCA p ecipi a ion on a glass-
ibe il e and hen di ided by cell olume. Fo hese expe -
imen s, he S. ce e isiae cells g own unde he same con-
di ions as abo e we e collec ed in 3-ml aliquo s by cen-
i uga ion a 4000 pm o 2 min. Fo he con ol sample
(blank), he cell pelle was esuspended in 5 ml o dis illed
wa e and was ecollec ed. Then he pelle was esuspended
in 1 ml o dis illed wa e and ans e ed o an eppendo
ube. Fo he expe imen al samples he cell pelle was esus-
pended in 5 ml o 0.5% Sa kosyl and was ecollec ed. Then
he pelle was esuspended in 1 ml o Sa kosyl and ans-
e ed o an eppendo ube. The cells in eppendo ubes
we e cen i uged a 6000 pm o 1 min. The supe na an
ha con ained Sa kosyl (samples) o wa e (blank) was e-
mo ed and he pelle was esuspended in 7.2 ␮l o dis illed
wa e . The un-on pulse was pe o med by adding, pe sam-
ple, 9.87 ␮l o he ansc ip ion mix (7.5 ␮lo 2.5×T an-
sc ip ion bu e : 50 mM T is-HCl pH 7.7, 500 mM KCl, 80
mM MgCl2,1␮lo 10mMATP,CTPandGTP,0.375␮l
o 0.1 M DTT, 0.66 ␮lo 3␮M UTP and 0.34 ␮lo 3␮M
[␣-33P] UTP (Pe kin Elme , 3000 Ci /mmol, 10 ␮Ci/␮l o
a inal olume o 18 ␮l). To allow ansc ip ion elonga ion,
he mix was incuba ed by agi a ion (650 pm) o 5 min a 30
C. The pulse was s opped by adding 82 ␮l o cold dis illed
wa e o he mix and being s o ed on ice. To measu e he o-
al amoun o adioac i i y p esen in he mix (‘To al’), 10
␮l o he eac ion we e di ec ly spo ed on o pape and d ied
in an ae a ed hea e a 65◦C. To measu e he pe cen age o
adioac i i y ha had been inco po a ed in o he nascen
RNA chain, ano he 10-␮l olume o he mix was spo -
ed on o glass ibe pape and d ied in he ae a ed hea e ,
ollowed by nucleic acid p ecipi a ion pe o med in echni-
cal duplica es. Fo nucleic acid p ecipi a ion, glass ibe pa-
pe was soaked in 4 ml o 10% ( / ) o ichlo oace ic acid
(TCA) and incuba ed a 4◦C o 20 min. TCA was emo ed
by decan ing and 4 ml o cold TCA (10% / ) was added
again, ollowed by incuba ion a 4◦C o 10 min. TCA was
emo ed and glass ibe pape was washed wi h 3 ml o cold
70% ( / ) E OH, ollowed by washing wi h 3 ml o cold
96% ( / ) E OH. Glass ibe pape was d ied in a hea e
a 65◦C. Once d ied, 5 ml o scin illa ion liquid was added
o each ial o adioac i e coun ing. Fo each indi idual
sample, he pe cen age o inco po a ion was calcula ed as:
(p ecipi a ed/ o al) x 100] – blank, whe e ‘blank’ was calcu-
la ed as [(p ecipi a ed/ o al) ×100] in he con ol sample.
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12404 Nucleic Acids Resea ch, 2017, Vol. 45, No. 21
RT-qPCR analysis o he RPB1 mRNA le els in s ains wi h
di e en olumes
The quan i ica ion o he exp ession o Rpb1, he la ges
subuni o RNA polyme ase II, was measu ed by RT-
qPCR and no malized agains he ACT1 mRNA le -
els. Speci ic p ime s we e designed o his aim: Rpb1-F:
5-CCAGAAGTGGTCACACCATATAA-3and Rpb1-R:
5-GGTCTCCGCTATCACGAATG-3. Re e se ansc ip-
ion o mRNA was ca ied ou using an oligo d(T)15VN
wi h The mo-Scien i ic Maxima Re e se T ansc ip ase
(The mo Scien i ic). cDNA was labeled wi h SYBR P e-
mix Ex Taq (Tli RNase H Plus) om Taka a and he Cq
alues we e ob ained om he CFX96 Touch™Real-Time
PCR De ec ion Sys em (BioRad).
Wes e n blo analysis o RNA polyme ase II quan i ica ion
Wes e n blo s we e pe o med as desc ibed p e iously (32)
using h ee di e en an ibodies o RNA pol II de ec-
ion: an i-POLR2C an ibody (Abcam) agains Rpb3 sub-
uni , an i –Rpb1 N- e minal (␥-80, San a C uz Bio echnol-
ogy) and an i-phospho-S2 (Abcam). The i s wo de ec all
RNA pol II molecules and he hi d one de ec s elonga ing
Se 2-phospho yla ed molecules. To al p o ein was quan i-
ied in he same samples by he B ad o d me hod (38).
Me a-analysis o he expe imen al da a se s
To e alua e he co ela ion be ween cell olume and mRNA
u no e in asynch onous exponen ial g ow h phase cells,
a se o 38 yeas mu an s was used (35). In ha s udy, he
global SRII, [mRNA] and he a e age hal -li e (HL, in e se
o kd) we e de e mined acco ding o he cDTA p o ocol.
They we e all ep esen ed as being ela i e o hei wild
ype. Those da a we e p ocessed as p e iously desc ibed (37)
and plo ed agains he cell olumes ob ained om a ious
sou ces (23,39). The obse ed Pea son’s co ela ion coe i-
cien , , 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. Wi h he R package BayesVa Sel (40), a
Bayesian analysis o he inclusion p obabili ies among GR,
SRII and olume con i med he SRII dependence on cell ol-
ume (see Supplemen a y Figu e S3).
To e alua e he co ela ion be ween cell olume and
mRNA u no e du ing indi idual cell g ow h h oughou
he cell cycle, da a om he s udy o (36) we e used. In ha
s udy, SRII, RA and HL (kd) we e also de e mined acco d-
ing o he cDTA p o ocol.
En ichmen analyses o gene ca ego ies in he se o poly-
ploid and cell size mu an s ains
The gene exp ession alues ob ained om he GRO expe -
imen s on he s ains lis ed in Supplemen a y Figu e S4C
we e done in iplica e and he acqui ed aw da a we e no -
malized using he A ayS a s a is ics so wa e (Imaging Re-
sea ch Inc.) by he median absolu e de ia ion (MAD) ap-
p oach. Volume-gene exp ession co a ia ion was quan i-
ied using a modi ied e sion o Pea son’s co ela ion (see
(41) o de ails) be ween bo h alues. A gene- o-gene anal-
ysis o he di e en ial beha io , as ega ds o he global a -
e age, was pe o med by he Signi icance Analysis o Mi-
c oa ays (SAM) me hod (41) 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 gene se s wi h di e en beha io s. 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 (GO) g oups. Analyses
we e un wi h R packages (40). The whole R code used in
his pape is ound as Supplemen a y Ma e ial.
RESULTS
Global mRNA syn hesis and deg ada ion a es lowe wi h he
indi idual cell olume du ing he cell cycle
As a i s app oach o s udy he in luence o cell olume on
gene exp ession in S. ce e isiae, we analyzed global mRNA
syn hesis and deg ada ion acco ding o cell olume ac oss
he cell cycle. We en isioned ha he di ec ansposi ion o
he es ablished model o symme ically di iding cells o he
budding yeas would be un iable (compa e Figu e 1Aand
B). By assuming ha bo h copies o he genome should ha e
iden ical nTR, and ha he cell olume was ∼50% highe in
mo he cells (28), SRs would di e a e cy okinesis. In ac
i nTR inc eased in pa allel o cell olume du ing he cell
cycle, as in symme ically di iding cells, he daugh e cell
would ha e an inc eased SR as ega ds he o iginal alue a
he beginning o cycle. This would p o oke a ne e -ending
inc ease in he SR o newbo n daugh e cells, unless he
budding yeas had a mechanism o compensa e (in ad ance)
changes in SR (Figu e 1B). Mo eo e , i HL emained con-
s an , which seems o happen in cells ha unde go symme -
ic di ision (4,5), [mRNA] would also inc ease in daugh e
cells and mRNA ibos asis would be comp omised. Al e -
na i ely, we easoned ha S. ce e isiae should ha e a ype o
con ol o i s ansc ip ion a e as ega ds cell olume ha
di e s om he linea inc ease in nTR seen in S. pombe (5)
and human cells (4). We hypo hesized an al e na i e model
whe e nTR would emain cons an and SR would dec ease
wi h olume. I he e was a compensa o y change in SR and
HL, [mRNA] ibos asis would be p ese ed (Figu e 1C).
To es his hypo hesis we i s an a me a-analysis o he
published RNA pol II da a om (36). The exhaus i e anal-
ysis done in ha pape con ained many da a poin s du ing
h ee cell cycles using S. ce e isiae cells synch onized wi h
␣- ac o . We analyzed only he da a om 0 o 40 min, which
co esponded o he G1 phase o he i s cell cycle be o e
budding. Ou me a-analysis esul s indica ed no inc ease in
nTRII, bu a educed mRNA u no e (dec ease in nTRII
and inc ease in HL) associa ed wi h cell cycle p og ession
in S. ce e isiae (Figu e 2). As we do no ha e expe imen al
cell olumes o (36) da a poin s, i was impossible o de e -
mine ac ual SRII a ia ion. Howe e , as HL is independen
on concen a ion and, he e o e, on cell olumes, he e was
an ac ual inc ease in global mRNA s abili y wi h cell ol-
ume, which ag eed wi h ou p edic ed model.
In o de o comple e he in es iga ion o ibos asis du -
ing he cell cycle we pe o med a cell synch oniza ion expe -
imen wi h ␣- ac o and elease, in which we measu ed he
a e age cell olume by Coul e Coun e , o al RNA by phe-
nol ex ac ion, and mRNA by do -blo and low cy ome y.
We ocused on he i s 25 min a e ␣- ac o elease which,
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Nucleic Acids Resea ch, 2017, Vol. 45, No. 21 12405
Figu e 2. Me a-analysis o he published da a o mRNA u no e depen-
dence on cell olume in synch onized cells. Me a-analysis o he esul s
om Ese e al. (36). Al hough he o iginal au ho s use he cDTA me hod,
which measu es newbo n ma u e mRNAs, and hey desc ibed hei da a
as syn hesis a es (SRII) gi en ha hey do no ake in o accoun cell ol-
umes hei ansc ip ion da a should be conside ed o mally equi alen o
nascen ansc ip ion a es (nTRII). The cDTA s udy also p o ided alues
o mRNA deg ada ion a es ha can be con e ed in o mRNA hal -li es
(HL).Weshowhe e ha nTR
II lowe s and he HL inc eases wi h ime a e
␣- ac o elease. The e is no cell olume measu emen in his expe imen ,
al hough i is concei able ha i inc eases wi h ime.
in ou hands, co esponded o he G1 pe iod (Supplemen-
a y Figu e S1) be o e budding (28,36). Figu e 3Ashows
how cell olume inc eases by ∼25% du ing a simila ime
cou se o ha depic ed in he expe imen o (36). In ou ex-
pe imen , o al [RNA] emained cons an (Supplemen a y
Figu e S2) and [mRNA] has a sligh inc ease (Figu e 3B)
as ega ds cell olume du ing he cell cycle. Thus i seems
ha mRNA ibos asis was main ained by a compensa o y
change in SRII and mRNA s abili y du ing he cell olume
changes h oughou he cell cycle.
P o ein concen a ion emains cons an , bu ac i e RNA pol
II dec eases du ing G1 phase
In o de o know he eason o he d op in SRII wi h cell
olume, we analyzed he ac i e RNA pol II concen a ion
in he same samples by wes e n blo using an an ibody ha
quan i ies he molecules engaged in elonga ion (an i Se 2P).
Figu e 3C depic s ha whe eas he o al p o ein concen-
a ion is cons an (Supplemen a y Figu e S2B), elonga -
ing RNA pol II dec eases in concen a ion by up o 25%
in pa allel o he inc eased cell olume h ough G1 a e he
alpha ac o elease. This esul sugges s ha S. ce e isiae
dec eases SRII by keeping he numbe o elonga ing RNA
pol II molecules on o ch oma in (cons an nTRII) cons an
in spi e o an inc easing cell olume.
Global mRNA syn hesis a e (SRII) lowe s and mRNA s a-
bili y inc eases wi h he a e age cell olume in asynch onous
cul u es
Cell olumes a y in a yeas cell acco ding o se e al ci -
cums ances. In he i s pa o his s udy we saw ha a i-
a ions in cell olume du ing g ow h du ing he G1 pe iod
did no beha e as hey do in o he euka yo es. Gi en ha
he expe imen al se up using cell synch onized cul u es can
Figu e 3. Ribos asis and p o eos asis analysis in synch onized cells. We
pe o med a simila expe imen o ha o Ese e al. (36)(Figu e2) by mak-
ing a ␣- ac o synch oniza ion o yeas cells (see ex o de ails) and we
measu ed cell olume (A) and [mRNA] (B) a di e en imes a e ␣- ac o
elease. [mRNA] was calcula ed as he poly(A) amoun pe cell (di ided
by cell olume) by an assay based on luo escen oligo-d(T) hyb idiza ion
and cy ome y quan i ica ion. Equal p o ein amoun s we e used in Wes -
e n blo analyses o RNA pol II quan i ica ion wi h he phospho-S2 an i-
body, which measu es elonga ing RNA pol II (C). Fou independen bio-
logical eplica es we e used o calcula e he a e age and s anda d de ia ion
(SD) alues. To al [RNA] and [p o ein] om he same samples a e seen in
Supplemen a y Figu e S2A and B. A ep esen a i e Wes e n blo is shown
in Supplemen a y Figu e S2C. The le els o he o al and elonga ing RNA
pol II we e no malized agains he in e nal glucose 6-phospha e dehyd o-
genase (G-6-PDH) con ol.
impose some cons ain s o biases o s udying he egu-
la ion mRNA syn hesis a e wi h cell olume, we decided
o do s udies ha used di e en yeas s ains wi h a iable
cell olumes caused ei he by di e en geno ypes o di e -
en ploidies. In ac i ely g owing yeas , as cell cul u es a e
composed o cells in di e en cell cycle s ages and o a i-
ous eplica i e ages, he alues ob ained in his s udy ep e-
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12406 Nucleic Acids Resea ch, 2017, Vol. 45, No. 21
sen he a e age (median) cell olumes o he whole pop-
ula ion. P e ious expe imen s done in he yeas S. pombe
ha e shown ha he o al mRNA amoun pe yeas cell can
inc ease wi h cell olume o main ain [mRNA], which oc-
cu s h ough an inc ease in he global SRII (5), bu wi h
no changes in mRNA s abili ies. In S. ce e isiae i has
been p e iously shown ha global [mRNA] emains wi hin
ce ain limi s (±50%) in di e en physiological si ua ions
(32,35,37). In e es ingly enough, his mRNA ibos asis is
main ained by a coo dina ed pa allel change in i s syn he-
sis (SRII) and deg ada ion a es (in e se o HL) (37,42).
We i s made a me a-analysis o he da a published by
(35), whe e 38 yeas mu an s o di e en cell olumes we e
analyzed o [mRNA], SRII and HL by he cDTA me hod.
To ob ain SRII alues om he o iginal nTRII ones, we
used cell olumes o his s ain lis , which we e aken om
(23,39). We ound a dec easing endency o SRII and an
inc easing one in HL wi h inc eased olume (Figu e 4A
and B). [mRNA], howe e , displayed a la endency (Figu e
4C). Thus i seems ha S. ce e isiae ends o keep ibos asis
in spi e o a dec easing mRNA u no e .
The gene al endency o dec ease SRII and o inc ease
mRNAs HL wi h cell olume seen in he me a-analysis o
he mu an s was clea , bu he e is a po en ial p oblem. This
da a se showed an in e se co ela ion be ween GR and cell
olume (37). We p e iously desc ibed he di ec dependence
o SRII wi h GR (37). Thus he in e se co ela ion be ween
cell olume and SRII can be an indi ec esul o his. In o -
de o es his possibili y, we used a Bayesian model. The
esul s o his s a is ical analysis con i med ha he e was a
ue dependence o SRII on cell olume, apa om i s de-
pendence on GR (Supplemen a y Figu e S3).
On his gene al endency, he pa icula physiology o
each mu an p o okes noise and some pa icula mu an s
especially beha e disc epan ly o he gene al ule (e.g. some
he ou lie s in Figu e 4A). To sol e his p oblem and o ex-
pe imen ally con i m he p e ious me a-analysis, we used a
se ies o polyploid s ains wi h an iden ical geno ype con-
s uc ed by he D. Pellman labo a o y (43) and wo hap-
loid mu an s (whi5 and cln3) known o ha e e y di e -
en cell olumes, bu wi h simila g ow h a es o hei wild
ype (BY4741, see Supplemen a y Figu e S4). We measu ed
he a e age cell olume and DNA con en (see Supplemen-
a y Figu e S4A–C). We also measu ed acuole size in each
s ain o ule ou ha o al cell olumes we e no good
e alua o s o cy oplasm olumes. By acuole-speci ic s ain-
ing, we de e mined acuole and cy oplasmic olumes, and
showed a linea 1:1 co ela ion be ween o al cell olumes
and cy oplasmic olumes (Supplemen a y Figu e S5).
In his s udy we used a di e en me hod o quan i y
mRNA concen a ions and syn hesis a es. Ou Genomic
Run-On me hod (GRO (33)) allows he quan i ica ion o
global nTRII which, by knowing cell olume, can be used as
a p oxy o SRII (6). Figu e 5A shows a signi ican dec ease
in global SRII ( he sum o all he syn hesis a es o all he
genes) wi h cell olume, whe eas he global [mRNA] plo
shows no signi ican slope. The calcula ed global mRNA
s abili y (HL =[mRNA]/SRII) inc eased (i.e. kddec eased)
wi h cell olume.
We ound ha he SRII o mos genes ollowed he gen-
e al endency wi hin he s a is ical limi s, which is consis-
Figu e 4. Me a-analysis o published da a on di e en cell size mu an
s ains. We used he da a o he a e age o al mRNA syn hesis a e (SR),
[mRNA] and mRNA s abili y (HL) om 38 mu an s ains desc ibed in
Sun e al. (35). The o iginal nTR and RA da a we e co ec ed by cell ol-
ume o exponen ially g owing cell cul u es o he same mu an s aken om
e e ences (23,39). Pea son’s co ela ion coe icien ( ) and he associa ed
P- alue o he non- la plo s a e shown. (A)SR;(B)HL,(C)[mRNA].
en wi h p e ious s udies in o budding yeas (43,44)and
S. pombe (5). Only some gene unc ional ca ego ies showed
a pa icula di e gen endency. Fo ins ance, he genes e-
la ed wi h ansposons and mi ochond ia and espi a ion
lowe SRII mo e slowly wi h olume as ega ds he a e -
age popula ion, and hose ela ed wi h plasma memb ane
lowe ed mo e quickly (Supplemen a y Figu e S6). The h ee
RNA pol II la ges subuni s genes (RBP1,RPB2 &RPB3)
also displayed beha io ha esembled he gene al endency
(see below).
Finally, in o de o e i y he inc ease in he global
mRNA s abili ies calcula ed om SRII and [mRNA], we
decided o use an independen app oach o e alua e he
mRNA s abili ies in he se o polyploid s ains. Fo his
pu pose, we used a me hod based on oligo dT hyb idiza-
ion o he iden ical o al RNA amoun s in a do -blo (see
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Nucleic Acids Resea ch, 2017, Vol. 45, No. 21 12407
Figu e 5. Examina ion o mRNA u no e in polyploid and cell size mu an s ains. (A) Global ansc ip ional s udy. Genomic Run-On (GRO) in he
wild ype haploid BY4741 and isogenic polyploid s ains was pe o med o ob ain a nascen ansc ip ion a e (nTRII). nTRII was di ided by he median
cell olume as a p oxy o SRII. [mRNA] was calcula ed as desc ibed in Figu e 3legend. Essen ially he same esul was ob ained by hyb idiza ion wi h
adioac i e oligo d(T) in a do -blo p o ocol (no shown). The whole cell popula ion mRNA hal -li e (HL) was de e mined by di iding [mRNA] by SRII.
(B) RNA pol II p o ein egula ion. Iden ical p o ein amoun s we e used o pe o m Wes e n blo analyses o RNA pol II quan i ica ion by he an iSe 2-
phospho yla ed an ibody ha measu es elonga ing RNA pol II, and he An i-N- e minal-Rpb1 an ibody ha ecognizes all he RNA pol II molecules.
The shown quan i ica ion co esponds o he a e ages and s anda d de ia ions (SD) o ou independen biological eplica es. To al p o ein concen a ion
does no a y wi h cell olume (see Supplemen a y Figu e S8). The le els o o al and elonga ing RNA pol II we e no malized agains he in e nal G-6-
PDH con ol. Examples o ep esen a i e Wes e n blo s a e shown in panel (C). The same s udy on aliquo s wi h he same samples and wi h An i-Rbp3
an ibody is shown in Supplemen a y Figu e S8. (C) The RT-qPCR analysis o he Rpb1 mRNA le els in he same cells no malized agains ACT1 mRNA.
(D) Regula ion o he RNA pol II subuni s ha encode mRNAs. The qRT-PCR analysis o RPB1 mRNA ( ed do s) was done on he samples o he same
yeas s ains se . Da a ep esen he a e age and SD o h ee biological epea s. The a e age SR and SEM o he h ee genes RPB1,RPB2 and RPB3,
which encode he h ee la ges RNA pol II subuni s in he same s ains, is also shown (blue iangles). These indi idual SR gene da a co espond o he
GRO da a used o he o al SRII used in panel (A).
Ma e ials and Me hods). In his way we ob ained a decay
cu e ha ep esen ed he a e age s abili y o he global
poly(A) mRNA popula ion. Supplemen a y Figu e S7 dis-
plays a clea inc ease in he global mRNA s abili y wi h cell
olume. This esul a i ied he p e ious esul (Figu e 5A),
in which he a e age global s abili y o mRNAs was ma he-
ma ically in e ed, and con i med ha mRNA u no e de-
c eased wi h a e age cell olume in exponen ially g owing
yeas cell cul u es.
RNA pol II concen a ion lowe s wi h cell size
In ou p e ious expe imen done wi h synch onized cells,
we showed ha he numbe o ac i e elonga ing RNA pol
II molecules on o ch oma in emained cons an despi e he
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12408 Nucleic Acids Resea ch, 2017, Vol. 45, No. 21
inc ease in cell olume (Figu e 3C), which led SRII o de-
c ease. We hypo hesized ha his si ua ion would also hap-
pen in expe imen s wi h exponen ial unsynch onized cul-
u es, and would cause SRII o lowe (as seen in Figu e 5A).
We checked his hypo hesis by a Wes e n blo wi h h ee
di e en an ibodies agains o al (an i-Rpb3 and an i-N-
e minal Rpb1) and elonga ing RNA pol II (an i Se 2P).
Figu e 5B and C and Supplemen a y S8 indica e ha all
h ee an ibodies led o a 3- old change in he RNA pol II
concen a ion (when compa ing he whi5 mu an and he
4n polyploid), which was almos an ipa allel o he cell ol-
ume inc ease (3.2- old, see Supplemen a y Figu e S4). This
esul con i ms ha S. ce e isiae speci ically con ols RNA
pol II le els as ega ds cell olume, bu di e en ly o human
cells, which keep he RNA pol II concen a ion cons an in
pa allel o he o al p o ein concen a ion (4).
We wonde ed i RNA pol II concen a ion egula ion in
esponse o cell olume ook place a he mRNA le el. In
o de o answe his ques ion, we pe o med an RT-qPCR
analysis o he RPB1 (which encodes he la ges subuni o
RNA pol II) mRNA le els. We ound ha he le el o his
mRNA lowe ed wi h cell olume as ega ds bo h o al RNA
(mos ly RNA- RNA, no shown) and ACT1 con ol (Fig-
u e 5D). Since he SRII o his gene and he o he wo la ge
RNA pol II subuni s (measu ed in he GRO expe imen s
desc ibed in Figu e 5A) beha ed iden ically (Figu e 5D), we
deduced ha RPB1 exp ession dec eased wi h cell olume
gi en i s d op in SRII. As his dec ease was iden ical o ha
o he global SRII d op (see Figu e 5A), we concluded ha ,
in ac , he ac ual eason o he lowe RPB1 (and possibly
o he o he speci ic RNA pol II subuni s) exp ession was
ha i did no unde go he gene al s abiliza ion obse ed o
he global ansc ip ome.
The di e en beha io o he o he wo RNA polyme ases
In e es ingly, he e alua ion o o al nTR (nTRT), he sum
o all h ee RNA polyme ases, showed an inc easing p o-
ile wi h olume (Figu e 6A), which caused an app oxima e
cons an SRT(Figu e 6B). Al hough ou me hod did no
dis inguish SRII om he SR o he o he wo RNA poly-
me ases as SRI + III was ∼75% o he o al SR (45), we con-
cluded ha he beha io o RNA pol II lowe ing SRII wi h
cell olume was speci ic o his polyme ase, and ha RNA
pol I+III, which ansc ibe ncRNAs, kep SRI + III cons an
wi h cell olume in he popula ion a e age cell olume ex-
pe imen .
As we used cells wi h di e en ploidies (n–4n), we won-
de ed how he genome copy numbe would in luence an-
sc ip ion a es. When dealing wi h genome copy changes,
we dis inguished be ween nTR/cell omanTR/genome
copy, which was mo e con enien because i would e lec
he ac ual densi y o ansc ibing RNA polyme ases on o
hei empla es (6). When we ep esen ed he nTR/genome
o he se ies o yeas s ains om ou s udy we saw ha
whe eas nTRT/genome ac i i y sligh ly dec eased (25%
om n o 4n, see Figu e 6C), he nTRII/genome dec eased
almos p opo ionally o ploidy (70% om n o 4n, Figu e
6C). In ac he 25% dec ease in nTRT, which included 25%
o RNA pol II ac i i y app oxima ely, can be explained al-
mos en i ely by he d op in he RNA pol II componen .
Figu e 6. To al ansc ip ion in he polyploid and cell size mu an s ains.
(A) Changes in he o e all nTRT(RNA pol I+ II + II) in ela ion o he
wild- ype s ain (BY4741) o he di e en yeas s ains, calcula ed by a
un-on p o ocol as desc ibed in he Me hods sec ion. (B)nTR
Twas hen
di ided by he ela i e cell olume o ob ain SRT.(C) The esul s om
bo h nTRT(panel A) and nTRII (Figu e 5A blue do s) a e ep esen ed he e
as pe genome copy by di iding he ploidy o each s ain (Supplemen a y
Figu e S4C). All he da a ha e been ela i ized agains he alues o wild
s ain BY4741, and co espond o he a e age and SD o h ee expe imen s.
Thus we concluded ha he e was no dose compensa ion
in RNA pol I nTR in S. ce e isiae. On he con a y, RNA
pol II showed p onounced dose compensa ion by spli ing
nTRII among he ac ual genome copies. Gi en he inc ease
in cell olume wi h ploidy (Supplemen a y Figu e S4C),
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Nucleic Acids Resea ch, 2017, Vol. 45, No. 21 12409
Figu e 7. Model o ansc ip ion a e con ol du ing he cell cycle in eu-
ka yo es wi h symme ic and asymme ic cell di ision. In symme ically
di iding cells, such as human ib oblas s and S. pombe, RNA pol II is
much less concen a ed han hei DNA a ge s. As he e is a s ong bias
o he equilib ium owa d he bound s a e (kon >> ko ), all he RNA pol
II molecules a e bound o ch oma in and nTR is s ic ly dependen on
he RNA pol II numbe o molecules (Scena io #1). In asymme ically di-
iding cells, such as S. ce e isiae, nTR does no depend on he RNA pol
numbe o molecules, ei he because he e is a as excess o hem o e
hei a ge s (e.g. o RNA pol I+III) in which i becomes dependen on he
numbe o DNA a ge s numbe (Scena io #2), o because he RNA pol
concen a ion is egula ed in such a way ha i a ies wi h cell olume ( ep-
esen ed as an supe sc ip in he igu e). This scena io (#3) co esponds
o RNA pol II. In i nTR is cons an and p opo ional o he ac ual num-
be o ac i e RNA pol II molecules. No e ha scena ios #1 and #3 allow
he egula ion o he nascen ansc ip ion a e by con olling he concen-
a ion o RNA pol II, whe eas scena io #2 does no allow egula ion a
he RNA polyme ase le el because i is in as excess.
his dose compensa ion led he ac ual SRII o dec ease om
haploid o e aploid (Figu e 5A).
Model o he S. ce e isiae con ol o mRNA u no e
A e conside ing all he p e ious esul s and a gumen s, we
p opose a gene al model o cell con ol o e RNA an-
sc ip ion (Figu e 7). This model is an ex ension o he model
p oposed in (4) o ib oblas s and S. pombe, and o ha
discussed in (46). Euka yo ic cells wi h a symme ical di i-
sion ha e a limi ing amoun o RNA pol compa ed o i s
a ge s. By assuming a e y high associa ion cons an o
RNA pol binding and a limi ing concen a ion o his en-
zyme, he nTR becomes dependen on cell olume (scena io
#1). This si ua ion has been shown o RNA pol II in mam-
malian cells (4). Howe e , i DNA a ge s a e limi ing in e-
la ion o RNA pol, which is he case o budding yeas RNA
pol I, nTR becomes independen on cell olume (scena io
#2). Finally i he limi ing RNA pol concen a ion lowe s
wi h cell olume, nTR also becomes cons an and indepen-
den on cell size (scena io #3). This scena io ma ches he ex-
pe imen al esul s ha we ound o RNA pol II-dependen
genes exp ession in S. ce e isiae.
DISCUSSION
Syn hesis and deg ada ion a es (mRNA u no e ) de e -
mine concen a ions o mRNAs. We ound in he yeas S.
ce e isiae ha he global mRNA u no e a e lowe s wi h
cell olume, while he global mRNA concen a ion emains
app oxima ely cons an . In ou s udy we ound no only a
dec eases in global syn hesis a es wi h cell olume when we
used da a om wo di e en echniques (GRO and cDTA),
bu also inc eases in mRNA s abili y when we used da a
om h ee di e en ones (GRO, cDTA ad ansc ip ional
shu o ). We also ound a simila beha io in he asyn-
ch onous cell cul u es o di e en well-known cell-size mu-
an s ains, cul u e condi ions o ploidy, and in synch o-
nized cells h oughou he G1 phase. In all hese cell olume
change cases, we obse ed an in e se change in mRNA syn-
hesis and deg ada ion a es, while he global mRNA con-
cen a ion emained cons an . Thus he conclusion ha we
ob ained is e y obus . I is no ewo hy ha ou esul s e-
e o he global mRNA popula ion, bu do no apply o all
indi idual mRNAs. Some examples o di e en ial exp es-
sion and asymme ic dis ibu ion o mRNA species ha e
been desc ibed in mo he and daugh e cells in budding
yeas (47).
Some pas pape s ha e add essed he beha io o o-
al RNA and mRNA syn hesis a es du ing he cell cycle
in budding yeas (16,18). Al hough hey ha e no speci i-
cally s udied mRNA u no e dependence on cell olume,
i is possible o e- isi hose esul s by compa ing cell cy-
cle changes o measu ed nTR, SR and cell olumes. In
(18) ound ha nTRI emained cons an h oughou he G1
phase in spi e o an inc easing olume, indica ing a d op
in SRI). Thei s udy also ound ha nTRII was cons an
(SRII lowe ed) wi h cell olume inc eases, wha well ma ches
ou esul s. Ou cu en esul s e ealed ha , simila ly o
ha obse ed in o he euka yo es, o al [RNA], [mRNA]
and [p o ein] emained app oxima ely cons an in ela ion
o cell olume in budding yeas . I seems ha p o eos a-
sis emained by keeping he p o ein ansla ion (and deg a-
da ion) a es cons an o all cells (16). This could co e-
la e wi h o al [RNA] ibos asis gi en ha o al RNA is
composed mainly o ansla ion- ela ed ncRNA ( RNA +
RNA), as obse ed by (18). Addi ionally, we ound ha
budding yeas p ese es [mRNA] ibos asis by a pa allel
change in i s syn hesis and deg ada ion a es. To do so, S.
ce e isiae keeps o al nTRII cons an and p o okes a change
in SRII in e sely o he change in cell olume. This esul
con as s wi h p e iously published esul s in S. pombe and
mammalian cells, in which nTRII inc eased as ega ds cell
olume by keeping SRII cons an . Thus in hose o ganisms,
and unlike budding yeas , [mRNA] homeos asis was p e-
se ed because no change in SR and HL ook place.
We p opose ha he special beha io o RNA pol II in
budding yeas is ela ed o ACD (Figu e 1). Unlike o he
euka yo es, S. ce e isiae gene a es wo cells wi h a la ge di -
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