Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2

13 Pages·2016·6.39 MB·English

2846–2858 NucleicAcidsResearch,2016,Vol.44,No.6 Publishedonline9February2016
doi:10.1093/nar/gkw027
Foxo3 circular RNA retards cell cycle progression via
forming ternary complexes with p21 and CDK2
William W. Du1,2,†, Weining Yang1,†, Elizabeth Liu1,2, Zhenguo Yang1,2, Preet Dhaliwal1,2 and
Burton B. Yang1,2,*
1SunnybrookResearchInstitute,SunnybrookHealthSciencesCentre,Toronto,M4N3M5,Canadaand2Department
ofLaboratoryMedicineandPathobiology,UniversityofToronto,Toronto,M5S1A1,Canada
ReceivedJune7,2015;RevisedJanuary8,2016;AcceptedJanuary11,2016 Do
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ABSTRACT RNAspossessmicroRNA(miRNA)bindingsitesandfunc- e
d
Most RNAs generated by the human genome have tpiloen,tahsescpiorcnugleasrtRoaNrAresCtimRiSR-N7Acofnutnaicntisomnsa(n6y,7b)i.nFdoirngexsaimtes- from
noprotein-codingabilityandaretermednon-coding h
forthemicroRNAmiR-7,andcanfunctionasaspongeof ttp
RclNudAes.eAxmonoincgctirhceuslearinRcNluAdse(cciirrccuRlaNrAR),NmAasi,nwlyhifcohuinnd- mmaiRny-7b(in6,d7i)n.gAsintoesthfoerrmciirRcu-1la3r8RanNdAfucnacltlieodnsSaRsYa,mcoiRnt-a1i3n8s s://ac
a
in the cytoplasm, and intronic RNAs (ciRNA), pre- sponge (7,8). Due to their abundance and stability, circu- d
e
dominantly detected in the nucleus. The biological larRNAsarebelievedtobemoreeffectiverelativetonon- m
ic
functions of circular RNAs remain largely unknown, circularRNAsinspongingmiRNA(1,7,9).SincemiRNAs .o
u
although ciRNAs have been reported to promote areimportantinregulatingproteinexpressionandcellular p.c
physiology, circular RNAs may thus exert roles in modu- o
gene transcription, while circRNAs may function as m
latingcellularphysiologysuchascellproliferationanddif- /n
microRNA sponges. We demonstrate that the circu- a
lcaarncReNrAceclilrsc-Faonxdo3wewreasashsigohcliyateexdprweisthsecdelilncnyocnle- fdteiearsleinrgotnileaedtoiotfona.ecTxiprhcliousrlheaartshRinsNohtAybpceoiertnchuerlesaipsr.oFWrtoeexdoex3apninldorroeeugdrutslhateutidpnyogtwceeanls-l r/article
progression. Silencing endogenous circ-Foxo3 pro- cycleprogression. -ab
s
moted cell proliferation. Ectopic expression of circ- Both circular Foxo3 (circ-Foxo3) and linear Foxo3 tra
c
Foxo3 repressed cell cycle progression by binding (Foxo3mRNA)areencodedbytheFoxo3gene(10).Dereg- t/4
to the cell cycle proteins cyclin-dependent kinase 2 ulation of Foxo3 is associated with cancer development 4/6
(alsoknownascelldivisionproteinkinase2orCDK2) (11),whichappearstobetheconsequenceofincreasedAkt /2
8
and cyclin-dependent kinase inhibitor 1 (or p21), re- activityorPhosphataseandtensinhomolog(PTEN)inac- 46
sulting in the formation of a ternary complex. Nor- tivationandFoxo3isthusclassifiedasatumorsuppressor /24
9
gene(11,12).Ourpreviousstudyshowedthattheupregula- 9
mally, CDK2 interacts with cyclin A and cyclin E to 4
fthaceislietaitnetecrealclticoyncsleanendtrayr,rewshticleellpc2y1cwleorpkrsogtoreisnshioibnit. st(i1ioo3nn),.oCwfyhFciolcihxnosm3aniwgdahcstylcbinleiknae-ddsseotpoceinaddteeecdnretwaksiietnhdascceeesllll(uClcaDyrcKlseesn)pearsrocegetrnwecsoe- 52 by gu
The formation of this circ-Foxo3-p21-CDK2 ternary e
classesofregulatorsforcellcycleprogression.Asamember s
complex arrested the function of CDK2 and blocked ofthecyclin-dependentkinasefamily,CDK2isaSer/Thr t on
cellcycleprogression. protein kinase. Its activity is restricted to the G1-S phase 06
incellcycleprogression,andisessentialfortheG1/Stran- A
p
INTRODUCTION sition. In the G1 phase, CDK2 forms a complex with cy- ril 2
clinE.Thecyclincomplexphosphorylatesretinoblastoma 0
1
9
Non-codingRNAsrepresentthemajorityoftranscriptsin protein (Rb) and promotes gene expression leading to the
acell.CircularRNAsarealargeclassofnon-codingRNAs progression of cells from the G1 to S phase (14). The cy-
thatarecircularizedbyjoiningthe3(cid:2)endoftheRNAtothe clin E/CDK2 complex also phosphorylates p27 and pro-
5(cid:2)end,formingacircularstructure(1–7).Althoughcircular motesp27degradation,thusincreasingcyclinAexpression,
RNAsweredetecteddecadesago,theirfunctionsinmam- facilitatingG1toStransition.KnownCDKinhibitorsin-
maliancellsareonlyrecentlyemerging.Mostofthecircular clude p21 and p27 (15). p21 can bind CDK2 and inhibit
RNAs reported so far are exon-containing circular RNAs CDK2activity(16),thereforefunctioningasaregulatorof
and are detected in the cytoplasm. Some of these circular
*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Email:[email protected]
†Theseauthorscontributedequallytothepaperasfirstauthors.
(cid:3)C TheAuthor(s)2016.PublishedbyOxfordUniversityPressonbehalfofNucleicAcidsResearch.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by-nc/4.0/),which
permitsnon-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Forcommercialre-use,pleasecontact
[email protected]
NucleicAcidsResearch,2016,Vol.44,No.6 2847
cellcycleprogressionattheG1andSphase(17).Ourstudy were cultured in DMEM with different concentrations of
showed that circ-Foxo3 could interact with both p21 and FBSandharvesteddaily.Cellnumberwasdeterminedbya
CDK2 forming a ternary complex, resulting in the inhibi- coultercounterasdescribed(18,19).
tionofcellcycleprogression.
FACSanalysis
MATERIALSANDMETHODS
Cells were washed and resuspended in cold phosphate
Materials bufferedsaline(PBS)andincubatedinice-cold70%ethanol
for3h.Thecellswerethencentrifugedat1500rpmfor10
The monoclonal or polyclonal antibodies against cy-
minandresuspendedinpropidiumiodide(PI)mastermix
clin A, cyclin B, cyclin C, cyclin D, cyclin E, CDK2,
(40 mg/ml PI and 100 mg/ml RNase in PBS) at a density
CDK4, CDK6, p16, p18 and p27 were purchased from
of5×105cells/mlandincubatedat37◦Cfor30minbefore
Santa Cruz Biotechnology. The monoclonal antibodies
against p21 and p57 were obtained from BD Biosciences. analysiswithflowcytometryasdescribed(20,21). D
o
w
Horseradish peroxidase-conjugated goat anti-mouse IgG n
and horseradish peroxidase-conjugated goat anti-rabbit Westernblotanalysis loa
d
IgGwereobtainedfromBio-Rad.RNAandDNAextract e
kwietrs,eRobNtAainRedTfarnodmpQoilaygmeenr.aNseocrhthaeinrnrMeaActXionkit(PwCaRs)frkoimts CmeMllsKwCerl,e2lymseMd inMlgyCsils2baunfdferPI()2,0amndMthHeeppreost,epinHsa7m.2p,l1e0s d from
weresubjecttowesternblotasdescribed(22,23). h
Ambion. Immunoblotting was performed using the En- ttp
hkaitn(cAedmcehrsehmaimlumBiinoescscieennccees)(.EBCiLot)inweCshterronmbolgoetnidcetDecettieocn- Nativegelanalysisofthep21/CDK2complex s://ac
a
tion kit was from Thermo Scientific. Protein A-Sepharose d
4B Conjugate and Dynabeads MyOne Streptavidin C1 About 7–15% gradient polyacrylamide gels containing no em
sodium dodecyl sulphate (SDS) were prepared and used ic
magnetic beads were obtained from Invitrogen. The cell .o
with a non-SDS running buffer containing 25 mM Tris- u
lines used in this study were from American Type Culture p
Cl, 190 mM glycine and 1 mM DTT adjusted to pH 8.0. .c
Collection(ATCC). o
m
Therunningbuffer,thenativegels,andgelrunningequip-
ment were cooled to 4◦C. The gels were subject to pre- /na
CWoengsternuecrtastaedndapcroimnestrrsuct expressing mouse circular RNA ewleercetrloypshedo,reasnisdaotn1e00pVarftoorf1thhebepfroorteesinamsapmleplloeawdiansgm.Cixeellds r/article
Foxo3(circ-Foxo3).Briefly,theplasmidscontainedaBlue- withonepartofthenativesampleloadingbuffer(62.5mM -ab
script backbone, a CMV promoter driving mouse circ- Tris-Cl, pH 6.8, 40% glycerol, 0.01% Bromophenol Blue). stra
Fgroexeon3fleuxoprreesscseinotnporortaeinno(Gn-FrePla)teexdprceosnsitoronlusneiqtuweansceli.nTkhede Ttrhoephdoilruetseisdwsaasmcpalrersiewdeoreutlouasidnegd8o0nVtoatth4e◦Cgeulsn,tailntdheeldeyce- ct/44
to the cir-Foxo3 but contained an internal ribosome entry migratedtothebottomofthegels.Thegelswerethensub- /6/2
site(IRES)allowingtheGFPtobeexpressedseparately. jecttowesternblotting. 84
6
Togeneratecirc-Foxo3,abasicsequencecontainingcirc- /2
4
Foxo3wassynthesized,whichcontainedthe3(cid:2)-half-intron- RT-PCRandreal-timePCR 99
4
exon-IIfragment(spliceacceptororSA)ofbacteriophage 5
2
T4 td gene, a small space sequence (SSS), a exon-I splice Thiswasperformedasdescribedpreviously(24).Inbrief,2 b
donor(SD)-5(cid:2)-half-intronsegment.TheSSScontainedtwo ×106 cellswereharvested,andtotalRNAswereextracted y g
u
restriction enzyme sites HindIII and SalI for insertion of withtheQiagenRNeasyminikit.Twomicrogramsoftotal e
s
circular RNA fragment (circ-Fox). In addition, the basic RNAs were used to synthesize cDNA, a portion of which t o
sequencealsocontainedanIRES.Thebasicsequencewas (1(cid:2)l,equalto0.2(cid:2)gcDNA)wasusedinaPCRwithtwo n 0
6
clonedintothemultiplecloningsitesofpEGFP-N1,which appropriate primers. Real-time PCR was performed with A
aSlSloSwweidthgtehneecrairtciounlaorfFcoixrocu3l,aprroRdNuAcinagncdirGc-FFPoxb-yGrFePpl(aScuinpg- masiStecmripptlaStYesB.RThGerpereinmPeCrsRuKseidt(fQoriargeeanl-)tiumsienPgC1R(cid:2)linctDerNnaAl pril 20
1
plementaryFigureS1a).Togeneratethecontrolvectorcirc- controlsweremouse-U6RNAfandmouse-U6RNAr. 9
RS,arandomsequencewassynthesizedandclonedintothe
HindIII-SalIsitesofcirc-Fox-GFP.
Immunoprecipitationassay
All primer sequences used are listed in Supplementary
Figure S1b. The anti-circ-Foxo3 siRNA and DNA oligo 107cellswerewashedinice-coldphosphate-bufferedsaline,
probes against endogenous or ectopic expression of circ- andlysedin500(cid:2)lco-IPbuffer(20mMTris-CL,pH7.5,
Foxo3,labeledwithbiotinorCy5,wereobtainedfromIn- 150mMNaCl,1mMethylenediaminetetraaceticacid,0.5%
tegratedDNATechnologies(SupplementaryFigureS1c). NP-40,and5(cid:2)g/mlaprotinin).Equalamountsofproteins
were incubated with 5 (cid:2)g of primary antibody and 50 (cid:2)l
of 50% slurry of protein A-Sepharose at 4◦C for 4 h. The
Cellproliferationassay
pelletwaswashed3×withPBSandwasresuspendedin2×
Cells(4×104)wereseededonto6-welldishesin10%Fetal Laemmlibuffer(0.125MTris–HCl,4%SDS,20%glycerol,
BovineSerum(FBS)/DMEM(Dulbecco’smodifiedEagle’s 10%2-mercaptoethanol,0.004%bromphenolblue,pH6.8),
medium)mediumandmaintainedat37◦Covernight.Cells followedbywesternblotanalysis.
2848 NucleicAcidsResearch,2016,Vol.44,No.6
RNAbindingproteinimmunoprecipitationassay(RIP) thedetectionofcirc-Foxo3inthecells(SupplementaryFig-
ure S2b). These results suggested that expression of circ-
Infunctionalassays,cellswereharvestedwhentheyreached
Foxo3wasassociatedwithcellcycleprogressionandprolif-
70–80% confluence. In brief, 107 cells were washed in ice-
eration.WeculturedthecelllinesMEF,NIH3T3,4T07and
coldPBS,lysedin500(cid:2)lco-IPbufferandincubatedwith5
(cid:2)gofprimaryantibodyat4◦Cfor2h.Atotalof40(cid:2)lof 4T1totheconfluencyof50,80,100%andoverconfluence,
thenmeasuredcellcycledistributionandcirc-Foxo3levels.
50%slurryofproteinA-Sepharosewasaddedtoeachsam-
ple,andthemixtureswereincubatedat4◦Cfor4h.Thepel- We found that circ-Foxo3 levels were significantly upregu-
lated (Figure 1B) when cell cycle was arrested (Figure 1C,
letswerewashed3×withPBSandresuspendedin0.5mlTri
SupplementaryFigureS2candd).Thelevelsofcirc-Foxo3
Reagent(Sigma-Aldrich).Theelutedco-precipitatedRNA
decreasedwhenthecellsweretreatedwithcellproliferating
in the aqueous solution was subject to qRT-PCR analysis
factorEpidermalgrowthfactor(EGF)butincreasedwhen
todemonstratethepresenceofthebindingproductsusing
thecellsweretreatedwithEGFinhibitorAG1478(Figure
respectiveprimers.
1D). D
o
To validate the essential roles of circ-Foxo3 in cell cycle w
n
RNApull-downassays progression,wedesignedsiRNAspecificallytargetingcirc- lo
a
Foxo3toknockdowncirc-Foxo3function(Figure1E,left). d
pR(2hN5o,A2sp6h).pauteIln-l-bduobfwfreineref,dass1as0ali7ynsec,elwyllseseredwinepre5er0fo0wr(cid:2)malsehcdoed-IaPsinbudfeifcsecerr-,icabonelddd Tlervaenlssf(eFctigiounrew1itEh,thriigshstiR, SNuAppelfefmecetinvtealryysiFleingcuerdecSi2rce-)F.oFxuor3- ed from
thermore,mousecardiacfibroblaststhatexpresshighlevels h
ietnenmcduopbgeaertanetoduurwseitofhorr3e2(cid:2)cthgo.bpAiiocattilonlytyalelaxtoepfdre5Ds0sNe(cid:2)dAlcwoiralicsg-hoFeopdxrooS3btr,eesapattagrvaoiiodnmisnt ogeftcinirgc-cFirocx-oF3oxwoe3reotrrtawnosfoeclitgeodswwitithhsriaRnNdAomspseecqiufiecnaclleys.taSri-- ttps://ac
lencingcirc-Foxo3wasconfirmedbyreal-timePCRinthe a
C1magneticbeads(Invitrogen)wereaddedtoeachbinding d
siRNA-transfected cells relative compared with wild-type em
reactionandfurtherincubatedatroomtemperatureforan-
cellsorthecellstransfectedwiththeoligos(Supplementary ic
otherhour.Thebeadswerewashedbrieflywithco-IPbuffer .o
Figure S2f). Cell proliferation assay showed that the cells u
forfivetimes.Theboundproteinsinthepull-downmateri- transfected with circ-Foxo3 siRNA had an increased pro- p.c
alswereanalyzedbywesternblotting. om
liferativecapacityrelativetocellstransfectedwiththecon-
/n
trololigosandtheuntransfectedwild-typecells,suggesting a
NRoNrAthsernwebrloetisolated with RNA extract kit. Northern a1erFrao)t.lieoInnofBceo1nm6dpcoeaglrelesn,dowuwesitachlisrtcoh-Fdeeoctxoeonc3tteriodnlsacen(lFlinipgcruroerleaifse1erGaint)i.ocenll(Fpirgoulirfe- r/article-a
blot analysis was performed with northern blot kit (Am- b
bion) as described (27). Briefly, the preparation of to- CellcycleanalysisrevealedthatfewersiRNA-transfected stra
MCF cells were detected in the G1 phase, but more were c
tal RNAs (30 (cid:2)g) was denatured in formaldehyde and detectedinSandG2phasesascomparedwiththecontrols t/44
tThheenReNlecAtrsowpehroertehseendtirnanasf1er%redagoanrtoosea–Hfoyrmboanldde-hNy+dneygloenl. (Figure 1H, Supplementary Figure S2g), which suggested /6/2
anincreaseincellcycleprogression. 84
membrane(Amersham)andhybridizedwithbiotin-labeled 6
DNA probes. Biotin Chromogenic Detection kit (Thermo We further examined the relationship between cell cycle /24
progressionandcirc-Foxo3expression.NIH3T3cellswere 9
Scientific)wasusedtodeveloptheboundRNAs. 9
culturedinserum-freemediumfor48h,thenchangedwith 45
2
medium containing 10% FBS. Cells were collected hourly b
y
Statisticalanalysis forupto24h,followedbycellcycleanalysis(Figure2a)and g
u
circ-Foxo3expression(Figure2b).Plottingofbothrevealed e
Allexperimentswereperformedintriplicateandnumerical s
data were subject to independent sample t-test. The levels astrongcorrelationbetweencellcycleprogressionandcirc- t on
ofsignificanceweresetat*P<0.05and**P<0.01. Foxo3expression(Figure2B,inset). 06
Weexaminedtheroleofcirc-Foxo3inmediatingcellac- A
RESULTS tFiovixtoy3beyxsptraebslsyiotnracnosnfescttriuncgt,NaIHco3nTtr3ofilbvreoctbolarsotsrwoitthhercierxc-- pril 2
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pressionplasmids.Expressionofcirc-Foxo3wasconfirmed 19
Expressionofcirc-Foxo3repressedcellcycleprogression
by RT-PCR (Supplementary Figure S3a), real-time PCR
It has been proposed that circular RNAs are conserved (SupplementaryFigureS3b)andnorthernblotting(Figure
across species (28,29). We analyzed the sequence of hu- 2C, Supplementary Figure S3c). The RNA samples were
mancirc-Foxo3andmousecirc-Foxo3andfoundthatthey treatedwithRNAse-Rpriortoreal-timePCR.OnlyRNA
were91%homologous(SupplementaryFigureS1d).Wean- isolatedfromthecirc-Foxo3-transfectedcellsdisplayedre-
alyzed circ-Foxo3 and Foxo3 levels in mouse cancer cells sistancetoRNAse-Rcleavage,confirmingcircularizationof
and non-cancer cell lines by RT-PCR and real-time PCR. circ-Foxo3(SupplementaryFigureS3d).
The levels of circ-Foxo3 and Foxo3 were inversely corre- Circ-Foxo3 and vector-transfected NIH3T3 cells were
latedinnon-cancerandcancercelllines,althoughtwocan- cultured in DMEM 5% FBS. Cell proliferation assays
cercellsline4T1andB16expressedlowlevelsofbothtran- showed that cells expressing circ-Foxo3 proliferated less
scripts(Figure1A,SupplementaryFigureS2a).RNAsiso- rapidly when compared with cells transfected without or
latedfromtheabovecelllinesweresubjecttonorthernblot- withthevectororGFPplasmid(Figure2D,Supplementary
ting with a probe specific for circ-Foxo3, which confirmed Figure S3e). Expression of circ-Foxo3 was confirmed by
NucleicAcidsResearch,2016,Vol.44,No.6 2849
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Figure1. Theeffectofcirc-Foxo3oncellproliferation.(A)Left,Real-timePCRshowedthatcirc-Foxo3washighlyexpressedinthenon-cancercelllines 1
9
ofmouseembryofibroblast(MEF),mousecardiacfibroblast(MCF)andNIH3T3,ascomparedwiththecancercelllines67NR,66C14,4T07,4T1and
B16.Right,ThelevelsofFoxo3linearmRNAwerenotcorrelatedwithcirc-Foxo3.Asterisksindicatesignificantdifferences.**P<0.001,Errorbars,SD
(n=4).(BandC)Differentcelllinesasindicated(B)orNIH3T3fibroblasts(C)wereseededatthecelldensityof1×105cells/wellon6-welldishesin10%
FBS/DMEMmediumuntil50,80,100%orover-confluencebasedonthecoverageofthesurfaceofthetissuecultureplates,followedbydetermination
ofcirc-Foxo3levelsandcellcycledistribution.Increasedcelldensitiesexpressedhigherlevelsofcirc-Foxo3(B)andmorecellsweredetectedintheG1
phase(C).(D)NIH3T3fibroblastswereincubatedinbasalmediumwithEGF(0,2,10and50ng/ml,left)orAG1478(0,0.5,1.5and5(cid:2)M,right)for
24h.Real-timePCRshowedcirc-Foxo3expressiondecreasedafterEGFtreatmentbutincreasedafterAG1478treatment.**P<0.001,Errorbars,SD
(n=4).(E)Left,AsiRNAwasdesignedtospecificallytargetcirc-Foxo3.Right,CellsweretransfectedwithsiRNAtargetingcirc-Foxo3oracontrol
oligo.RNAsisolatedweresubjecttoreal-timePCRtoconfirmdownregulationofcirc-Foxo3inthesiRNA-transfectedcells.(F)MCFcellstransfected
without(wild-type)orwithcirc-Foxo3siRNAor2oligoswithrandomsequenceswereculturedinDMEMsupplementedwith5%FBSforupto5days.
ThesiRNA-transfectedcellsgrewfastcomparedtothecontrols.**P<0.001,Errorbars,SD(n=4).(G)B16cellstransfectedwithoutorwithcirc-Foxo3
siRNAorthecontrololigoswereculturedinDMEMwith2.5%FBSforupto5days.Cellproliferationassaysshowedthatcirc-Foxo3siRNA-transfected
cellsgrewfastcomparedtothecontrols.**P<0.001,Errorbars,SD(n=4).(H)Silencingcirc-Foxo3decreasedthenumberofcellsinG1phase,but
increasedthenumberofcellsinSandG2phase.**P<0.01.Errorbars,SD(n=4).
2850 NucleicAcidsResearch,2016,Vol.44,No.6
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Figure2. circ-Foxo3repressedcellcycleentry.(A)NIH3T3cellswereculturedinserum-freemediumfor48h,thenchangedwithmediumcontaining10%
FBSforcellcyclesynchronization.Cellswerecollectedeachhourforup-to24handsubjecttoflowcytometryforcellcycleanalysis.Errorbars,SD(n
=3).(B)Circ-Foxo3levelsweredeterminedbyreal-timePCR.Inset,thecorrelationbetweencirc-Foxo3levelsandpercentageofcellsinG1phasewas
analyzedbyGraphPrism4.Itshowedthatcirc-Foxo3levelswerehighlycorrelatedwiththepercentagesofcellsinG1phase.Rsquares,0.778;P<0.001,
n=25.(C)Levelsofcirc-Foxo3weredeterminedbyreal-timePCRinNIH3T3cellstransfectedwithGFPvector,Foxo3,mockcontrol,circ-Amotl1and
circ-Foxo,orinoverconfluence(OC)cells.**P<0.01.Errorbars,SD(n=4).(D)NIH3T3fibroblaststransfectedwithoutorwithcirc-Foxo3,thecontrol
vector,oraGFPplasmidwereculturedinDMEMwith5%FBS.Cellproliferationassaysshowedthatcirc-Foxo3expressingcellsgrewslowlycompared
tothecontrols.**P<0.01.Errorbars,SD(n=4).(E)ThecellswerealsoculturedinDMEMsupplementedwith5%FBSfor2days,andthenprocessed
toflowcytometry.Expressionofcirc-Foxo3significantlyincreasedthenumberofcellsintheG1phase,anddecreasedthenumberofcellsintheSandG2
phases.**P<0.01.Errorbars,SD(n=4).(F)B16cellsweretransfectedasaboveandculturedinDMEMwith5%FBS.Cellproliferationassaysshowed
thatcirc-Foxo3expressingcellsgrewslowlycomparedtothecontrols.**P<0.01.Errorbars,SD(n=4).(G)Thecellswerealsosubjecttoflowcytometry.
Expressionofcirc-Foxo3significantlyincreasedthenumberofcellsinG1phaseanddecreasedthenumberofcellsinSandG2phases.**P<0.01.Error
bars,SD(n=4).
NucleicAcidsResearch,2016,Vol.44,No.6 2851
real-timePCR(SupplementaryFigureS3f).Cellcycleanal- transfectedwithanumberofexpressionconstructs(Foxo3,
ysisindicatedthatthereweremorecirc-Foxo3cellsdetected circ-Amotl1 and circFoxo3) and control vectors. The cells
in the G1 phase but fewer in S and G2 phases compared werealsoovergrowntomaintainhighlevelsofcirc-Foxo3.
tothecontrols(Figure2E,SupplementaryFigureS4a).We Western blot analysis showed that expression of p21 and
alsotestedtheroleofcirc-Foxo3inthecancercelllineB16. CDK2 was not affected by transfection of the constructs
Expression of circ-Foxo3 was confirmed by RT-PCR and nor affected by cell over growth (Figure 4D). However, in
real-time PCR (Supplementary Figure S4b). Similarly, the immunoprecipitationassays,anti-p21antibodyonlypulled-
circ-Foxo3-transfected cells grew slower than the controls down high levels of CDK2 in the circ-Foxo3-transfected
(Figure2f,SupplementaryFigureS4c)andmorecellswere cells or when NIH3T3 fibroblasts were overgrown (Figure
found in the G1 phase but fewer cells in S and G2 phases 4D).Similarly,anti-CDK2antibodyonlypulled-downp21
(Figure2G). inthesameconditionswhilecirc-Foxo3levelswerehigh.In
thecellstransfectedwithcirc-Foxo3orovergrown,wecon-
firmedthatwhileexpressionofp21andCDK2wasnotaf- D
Formation of ternary complexes by circ-Foxo3, CDK2 and o
fected,thecirc-Foxo3probepulled-downhighlevelsofp21 w
P21 n
andCDK2relativetothecontrololigo(Figure4E). lo
a
Wetestedpotentialinteractionsofcirc-Foxo3withcellcycle Since circ-Foxo3 was found to stay mainly in G1 phase d
e
awsesroecsiautbejdecptrtooteiimnsm.Cuneoll-lpyrseicsippirteaptaiorned(IfPro)mwiNthIHan3tTi-3racbebllist (mFeidguiarteed3Db)y,cwirect-eFsotexdo3ifmthaeininlyteorcaccutirornedofinp2G11apnhdaCseD. WK2e d from
IgG,mouseIgG,cyclinA,cyclinB,cyclinC,cyclinD,cy- confirmedthattheanti-p21antibodywasabletopull-down h
clinE,CDK2,CDK4,CDK6,p16,p18,p21,p27andp57 highlevelsofCDK2,andthatanti-CDK2antibodycould ttps
antibodies, followed by real-time PCR with primers spe- pull-downhighlevelsofp21inG1phase(Figure4F). ://a
c
cificforthelinearFoxo3mRNAorcirc-Foxo3.Theexperi- Theseresultssuggestedthatcirc-Foxo3,CDK2andp21 a
d
mentshowedthatcirc-Foxo3waspulled-downbyimmuno- formed ternary complexes. To validate this, we resolved em
precipitation experiments with antibodies against CDK2, protein lysates prepared from the vector and circ-Foxo3- ic
.o
CDK6, p16, p21and p27, which did not pull-down linear transfected cells in native gradient gels, followed by west- u
p
Foxo3mRNA(Figure3A).Celllysatespreparedfromthe ernblottingprobedwithantibodiesagainstp21andCDK2. .c
o
m
vector- and circ-Foxo3-transfected cells were subject to IP In the circ-Foxo3-transfected cells, p21 mainly migrated
/n
assaywiththeantibodiesindicatedabove.Wevalidatedthe as a single band with a size slightly larger than the sum a
faunnticbtoiodnieosfaaglaliannsttibCoDdiKes2,(FCigDuKre63,Bp)1,6b,upt2f1oaunnddpth2a7twonerlye ovefctCoDr-Ktra2nsafnedctepd2c1el(l∼s,7a0lakrDgeap,oFrtigiounreof4Gp2,1lemfti)g.raItnedthaes r/article
able to pull-down significantly higher levels of circ-Foxo3 a monomer, and a small quantity of p21 migrated with a -a
b
fromthecirc-Foxo3cellsthanthosefromthecontrolcells massof70kDa.Similarly,antibodyagainstCDK2detected stra
(Figure 3c). It was noted that the CDK2 and p21 showed CDK2 monomer and hetero-dimer with p21 (Figure 4G, c
thegreatestdifferenceinourexperiments,suggestingacrit- right).Theaboveresultsaresummarizedinthediagramin t/44
icalroleforCDK2andp21incirc-Foxo3-mediatedcellcy- Figure4H. /6/2
cleprogression.Cellsweresortedbyflowcytometrytoob- 84
6
tainG1-,S-andG2-phasecellsforlysatepreparation.Anti- /2
Disruptionoftheternarycomplexincreasedcellcycleentry 4
CDK2 and anti-p21 antibodies pulling-down circ-Foxo3 9
9
mainlyoccurredmainlyinG1phase(Figure3D).Thiswas We validated the effect of circ-Foxo3 in mediating the in- 45
2
consistentwithourresultsthatectopicexpressedcirc-Foxo3 teraction of CDK2 and p21. Asynchronized NIH3T3 fi- b
y
arrestedcellsinG1phase. broblaststransfectedwithsiRNAtargetingcirc-Foxo3were g
u
Toanalyzethespecificityoftheinteraction,weexamined confirmed to retain their capacity in expression of CDK2 e
s
the levels of other circular RNAs including circ-DNSJA1, andp21(Figure5A).IPassayshowedthattheassociation t o
n
circ-MRPL47, circ-NDUF53, circ-RPS5 and circ-PRL5. of p21 with CDK2 was reduced in the siRNA-transfected 0
6
We found that antibodies against p21 and Cdk2 did not cells(Figure5B).Similarly,CDK2associationwithp21de- A
pciufilcl-idnotwernactthieosnebciertcwuelaenrRciNrcA-Fso(xFoi3guarned3tEh)e,sseutgwgoesptirnogtesipnes-. cCrDeaKse2dainntitbhoedsyi,RbNuAt t-htreancasfpeaccteitdycoefllCsDafKte2r IbPinwdiinthgawnittih- pril 2
0
1
We tested the interactions between circ-Foxo3 with cyclinAandcyclinEwasrecovered(Figure5C).Further- 9
CDK2 and p21. Western blot analysis indicated that anti- more,whiletheexpressionofcyclinAandcyclinEwerenot
CDK2antibodyimmuno-precipitatedhigherlevelsofp21, affectedbysiRNAtransfection(Figure5D),bothantibod-
butlowerlevelsofcyclinAandcyclinE,inthecirc-Foxo3- iesagainstcyclinAandcyclinEpulled-downmoreCDK2
transfectedcells(Figure4A).However,inthecontrolgroup, in the cells transfected with circ-Foxo3 siRNA compared
anti-CDK2antibodydidinfactpull-downcyclinAandcy- withthecontrololigo(Figure5E).
clinE,butnotp21,sinceCDK2isknowntointeractwith We also validated the effect of circ-Foxo3 on the for-
cyclinAandcyclinE(30–32).Inthecirc-Foxo3transfected mation of the ternary complexes. Protein lysates prepared
cells, anti-cyclin A and anti-cyclin E antibodies pulled- from siRNA- and control oligo-transfected cells were re-
down less CDK2 compared with the control cells (Figure solvedinnativegradientgels,followedbyWesternblotting
4B).Nevertheless,expressionofcyclinAandcyclinEwas probedwithantibodiesagainstp21andCDK2.Inthecirc-
notaffectedbycirc-Foxo3transfection(Figure4C). Foxo3 siRNA-transfected cells, p21 mainly migrated as a
We tested the specificity of circ-Foxo3 in mediating the singlebandofp21monomer(∼20kDa,Figure5F,left).In
interaction of p21 and CDK2. NIH3T3 fibroblasts were the control, some endogenous circ-Foxo3 appeared to fa-
2852 NucleicAcidsResearch,2016,Vol.44,No.6
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Figure3. circ-Foxo3interactedwithCDK2andP21.(A)CelllysispreparedfromNIH3T3cellsweresubjecttoimmuno-precipitation(IP)withantibodies g
againstrabbitIgG,mouseIgG,cyclinA,cyclinB,cyclinC,cyclinD1,cyclinE,CDK2,CDK4,CDK6,p16,p18,p21,p27andp57,followedbyreal-time ue
PFoCxRo3wmithRpNriAm.eIrtswspaesceifispcefocirallilnyeoabrvFiooxuos3thmaRtpNrAeciopritcairticn-FgoCxDo3K.A2notri-PC2D1Kpu2,llCedD-dKo6w,np1c6ir,cp-2F1oaxnod3.p*2*7Pan<tib0o.0d1i.esEprruollredb-adros,wSnDci(rnc-=Fo4x)o.3(,Bb)uCtneloltlylisnaetaesr st on
preparedfromNIH3T3cellstransfectedwithcirc-Foxo3ormockcontrolweresubjecttoimmunoprecipitationwithanti-rabbitIgG,mouseIgG,cyclinA, 0
6
cyclinB,cyclinC,cyclinD,cyclinE,CDK2,CDK4,CDK6,p16,p18,p21,p27andp57antibodies.Westernblotshowedthatimmunoprecipitationpulled A
dPoCwRnwsiimthilparrimamerosusnptecoifficprfootrecinirsc-iFnobxoot3h.Aconnttir-oClDaKnd2,cCircD-FKo6x,op31-6tr,apn2s1feacntdedp2ce7llasn.t(iCb)odTiheespimulmleudn-doo-pwrnecmipoitraetecdircm-Fixotxuor3esfrwoemreNaIlHso3sTu3bjceecltlsttorarneasfle-tcitmede pril 2
withcirc-Foxo3thanfrommockcontrol.*P<0.01.Errorbars,SD(n=4).(D)NIH3T3cellsweresubjecttoflowcytometrytosortcellsinG1,Sand 01
G2phases,followedbyimmunoprecipitationwithanti-rabbitIgG,mouseIgG,p21andCdk2antibodiesandreal-timePCRwithprimersspecificforcirc- 9
Foxo3.Antibodiesagainstp21andCdk2precipitatedsignificantlymorecirc-Foxo3inG1phasethaninG2anSphases.**P<0.01.Errorbars,SD(n=4).
(E)Celllysatespreparedweresubjecttoimmunoprecipitationwithanti-rabbitIgG,mouseIgG,p21andCdk2antibodies,followedbyreal-timePCRwith
primersspecificforcirc-Foxo3,circ-DNAJA1,circ-MRPL47,circ-NDUF53,circ-RPS5andcirc-RPF5.Antibodiesagainstp21andCdk2pulled-down
circ-Foxo3,butnottheothercircularRNAs.**P<0.01.Errorbars,SD(n=4).
cilitate the interaction of p21 and CDK2. Moreover, anti- fected with the control oligo (Figure 5G), while the total
CDK2antibodydetectedaCDK2monomerinthesiRNA- levelsofcirc-Foxo3appearedunaffectedasshownbycirc-
transfected cells, and it also detected the hetero-dimmer Foxo3pull-downassayusingcirc-Foxo3probe(Figure5H).
withp21(Figure5F,right). The anti-p21 antibody pulled-down trace amounts of p21
We further examined whether silencing p21 and CDK2 andCDK2(Figure5I).
affectedtheinteractionofcirc-Foxo3withtheseproteins.In Similarly, silencing CDK2 did not affect circ-Foxo3
cellstransfectedwithp21siRNA,anti-p21antibodypulled- probe to pull-down circFoxo3 (Figure 5j), but resulted
down decreased levels of circ-Foxo3 relative to cells trans- in pulling down decreased levels of circ-Foxo3 using an
NucleicAcidsResearch,2016,Vol.44,No.6 2853
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Figure4. circ-Foxo3enhancedtheinteractionbetweenp21andCDK2.(A)LysatespreparedfromNIH3T3cellstransfectedwithcirc-Foxo3ormock ril 2
controlweresubjecttoIPwithanti-CDK2antibody,followedbyWesternblotting.CDK2precipitationpulleddownmorep21,andlesscyclinAand 01
cyclinEinthecirc-Foxo3-transfectedcellsthanthecontrol.(B)Left,thelysatesweresubjecttoIPwithanti-cyclinAantibody,followedbyWestern 9
blotting.CyclinAprecipitationpulleddownlessCDK2inthecirc-Foxo3-transfectedcellsthaninthecontrol.Right,thelysatesweresubjecttoIPwith
anti-cyclinEantibody,followedbywesternblotting.CyclinEprecipitationpulled-downlessCDK2inthecirc-Foxo3-transfectedcellsthaninthecontrol.
(C)Circ-Foxo3transfectiondidnotchangeexpressionofcyclinAandcyclinE.(D)CelllysatespreparedfromNIH3T3cellstransfectedwithGFPvector,
Foxo3,circularRNAvector,circ-Amotl1andcirc-Foxo3,orover-confluencecultureweresubjecttowesternblotprobedwithantibodiesagainstCDK2,
p21and(cid:3)-action.Thelysateswerealsosubjecttoimmunoprecipitationwithantibodyagainstp21orCDK2.Anti-p21antibodypulled-downmoreCdk2
andanti-CDK2antibodypulled-downmorep21inthecellstransfectedwithcirc-Foxo3orovergrown.(E)LysatespreparedfromNIH3T3cellstransfected
withcirc-Foxo3andavector,orover-growncellsweremixedwithbiotinylatedprobesagainstcirc-Foxo3oranoligo.Westernblottingshowedthatlevels
ofCDK2andp21werenotaffected(upper).However,thecirc-Foxo3probepulleddownmoreCDK2andp21inthecellstransfectedwithcirc-Foxo3
orover-grownrelativetothecontrols(lower).(F)LysatespreparedfromNIH3T3cellssortedintoG1,SorG2phaseweresubjecttoWesternblotting.
Thelevelsofp21andCDK2weresimilarincellsofdifferentphases(upper).Anti-p21andanti-CDK2antibodiespulleddownmoreCDK2andp21,
respectively,intheG1phasecells.(G)Thelysatespreparedfromcirc-Foxo3-andmock-transfectedcellsweresubjecttonativegradientgelelectrophoresis
followedbywesternblottingprobedwithantibodiesagainstp21orCDK2.(H)Diagramofourhypothesisshowingtheeffectofcirc-Foxo3oncellcycle
progression.Circ-Foxo3retardscellcycleentryviaenhancinginteractionbetweenp21andCDK2,whichrepressedCDK2–cyclincomplexformation.
2854 NucleicAcidsResearch,2016,Vol.44,No.6
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Figure5. Silencingcirc-Foxo3enhancedcellcycleentryanddecreasedtheinteractionbetweenp21andCDK2.(A)Lysatespreparedfromsubconfluence
NIH3T3cellstransfectedwithcirc-Foxo3siRNAoracontrololigoweresubjecttowesternblotting.Silencingcirc-Foxo3didnotchangeexpressionof
p21andCDK2.(B)ThelysatesweresubjecttoIPwithanti-p21antibodyfollowedbywesternblotting.p21precipitationpulled-downlessCDK2inthe
circ-Foxo3siRNA-transfectedcellsrelativetothecontrol.(C)ThelysatesweresubjecttoIPwithanti-CDK2antibody,followedbywesternblotting.CDK2
precipitationpulled-downlessp21,butmorecyclinAandcyclinEinthesiRNA-transfectedcellscomparedtothecontrol.(D)Silencingcirc-Foxo3did
notchangeexpressionofcyclinAandcyclinE.(E)CyclinAandcyclinEprecipitationspulleddownmoreCDK2inthecirc-Foxo3siRNA-transfected
cells.(F)Thelysatesweresubjecttonativegradientgelelectrophoresisfollowedbywesternblottingprobedwithantibodiesagainstp21orCDK2.(G)
Celllysatespreparedfromp21siRNA-andacontrololigo-transfectedNIH3T3cellsweresubjecttoimmunoprecipitationwithanti-rabbitIgG,mouse
IgG,p21andCdk2antibodies,followedbyreal-timePCR.Anti-p21antibodypulled-downlesscirc-Foxo3inthep21siRNA-transfectedcells.**P<0.01.
Errorbars,SD(n=4).(H)Thelysateswerehybridizedwithcirc-Foxo3probeoracontrololigoforRNApull-downassays.Real-timePCRshowedthat
thecirc-Foxo3probepulleddownsamelevelsofcirc-Foxo3inthecellstransfectedwithp21siRNA.Errorbars,SD(n=4).(I)Silencingp21didnotaffect
CDK2expression.However,anti-p21antibodypulled-downlessCdk2andp21inthep21siRNA-transfectedcells.Anti-CDK2antibodypulled-downless
p21,butthesameamountofCdk2inthep21siRNA-transfectedcells.(J)SilencingCDK2didnotaffectedcirc-Foxo3levels.(K)Anti-CDK2antibody
pulled-downlesscirc-Foxo3intheCdk2siRNA-transfectedcells.**P<0.01.Errorbars,SD(n=4).(L)SilencingCDK2didnotaffectp21expression.
However,anti-CDK2antibodypulled-downlessCdk2andp21intheCDk2siRNA-transfectedcells.Anti-p21antibodypulled-downlessCDK2,butthe
sameamountofp21intheCDK2silencingcells.
NucleicAcidsResearch,2016,Vol.44,No.6 2855
anti-CDK2 antibody (Figure 5K). In the CDK2 siRNA- of RNAse A, the enzyme was too potent which caused it
transfected cells, anti-CDK2 antibody precipitated de- to destroy the complex, resulting in the presence of only
creased levels of CDK2 and p21, while anti-p21 antibody the monomer. Similarly, antibody against CDK2 detected
onlyprecipitateddecreasedlevelsofCDK2(Figure5L). CDK2 monomer and the heterodimer with p21 when the
lysatesofcirc-Foxo3-transfectedcellsweretreatedwithlow
concentrationsofRNAseA(Figure7B).Athighconcentra-
Pullingdowncirc-Foxo3boundbothp21andCDK2
tionsofRNAseA,theanti-CDK2antibodyagainonlyde-
WetestedwhethertheprobeusedforNorthernblotcould tectedmonomer.Nevertheless,anti-p21antibodynotonly
pull-down p21 and CDK2. Cell lysate prepared from pulleddownp21,butalsopulleddownhighlevelsofCDK2
NIH3T3 fibroblasts transfected with circ-Foxo3 or the inthecirc-Foxo3-transfectedcellswithorwithoutRNAse
mock control was subject to the pull-down assay. While A treatment (Figure 7C), suggesting protection of the p21
we confirmed that the levels of circ-Foxo3 were similar in andCDK2bindingsitesincirc-Foxo3formingtheternary
thelysatesmixedwiththeprobeorthecontrololigo(Sup- complexes. As well, anti-CDK2 antibody not only pulled D
o
plementary Figure S4d, left), we found that the probe sig- down CDK2, but also pulled down high levels of p21 in w
n
nificantly pulled-down more circ-Foxo3 than the control thecirc-Foxo3-transfectedcells withorwithout RNAse A lo
a
oligo (Figure 6A). In the protein precipitation assay, af- treatment. d
e
tmerixctuorneficromnitnagineinqguatlheamprooubnetsorofthpe2c1onatnrdolCoDligKo2(Finiguthree whWeneathlseocperllespwareerdeloyvsaertegsrforwomn acenldlsiantc5u0b%atecdontflhueelnycseatoesr d from
6B, left), we found that both p21 and CDK2 were pulled- withRNAseAatdifferentconcentrations.Afterbeingsep- h
downbytheprobebutnotbythecontrololigointhecirc- arated in native gradient gels, protein bands were probed ttps
Foxo3-transfectedcells,butthisdidnotoccurinthevector- with a mix of p21 and CDK2 antibodies. We detected the ://a
c
transfectedcells(Figure6B,right). largerbandsonlytheovergrowthcellswhenthelysateswere a
d
We also tested the effect that circ-Foxo3 knock-down treatedwithlowconcentrationsofRNAseA(Figure7D). em
wouldhaveonthepulling-downp21andCDK2.Whilewe ic
.o
confirmed that the siRNA targeting circ-Foxo3 knocked- u
down significant levels of circ-Foxo3 (Supplementary Fig- DISCUSSION p.c
o
m
ureS4d,right),wefoundthattheprobepulled-downsignif- Inthisstudy,wefoundthatexpressionofthecircularRNA
/n
icantlylowerlevelsofcirc-Foxo3inthesiRNA-transfected circ-Foxo3 repressed cell proliferation and cell cycle pro- a
wceiltlhstchoimsrpeasureltd,lwowitehrtphreotceoinntlerovells(Foifgpu2re1a6nCd).CCDoKns2iswteenret gorfeesxspioenri.mTehnistsc.oWnchleunsicoenllswwaseroebgtaroinwendbbeaysoedndoncoannfluumenbceyr, r/article
pulled-downbytheprobecomparedwiththecontrololigo levelsofcirc-Foxo3increasedandmorecellswerearrested -a
b
(Figure6D). atG1phase,withapositivecorrelationbetweenbothcirc- stra
Inthep21siRNA-transfectedcells,thecirc-Foxo3probe Foxo3andcellsinG1phase.CelltreatedwithmitogenEGF c
couldonlypulldowntraceamountofp21butpulleddown haddecreasedlevelsofcirc-Foxo3,whilecellstreatedwith t/44
equal levels of CDK2 in the p21 siRNA-transfected cells EGFinhibitorproducedincreasedlevelsofcirc-Foxo3.Si- /6/2
relative to the oligo-transfected cells (Figure 6E). Reduc- lencingendogenouscirc-Foxo3promotedcellproliferation 84
6
tion of p21 did not disturb the interaction of circ-Foxo3 anddecreasedthenumberofcellsinG1phase.Itshouldbe /2
4
with CDK2, suggesting the exiting of circ-Foxo3–CDK2 notedthatthesiRNAtargetsiteincirc-Foxo3canonlybe 9
9
complexinadditiontotheternarycomplex.IntheCDK2 locatedinthejunctionofcirc-Foxo3,becausesiRNAstar- 45
2
siRNA-transfected cells, the circ-Foxo3 probe could pull getingotherareaswouldalsosilencelinearFoxo3mRNA. b
y
downequallevelsofp21butpulleddowndecreasedlevelsof To ensure that the silencing siRNA produced the desired g
u
CDK2(Figure6F),suggestingpresenceofthecirc-Foxo3– effects, it was necessary to include more that one negative e
s
p21complex. control. We included two control oligos with random se- t o
n
quences,aswellasnon-transfectedcellculturesinthefunc- 0
6
PWreotfeucrttihonerofcothnefiprm21edantdhCeDfoKrm2abtiinodninogfsitthee ternary com- ttpiororenssasailonandsseaaxypsn.eoSrniimm-terialnantrssl.yfe,Tcwatkeedeanlcsetoollignecctulhultedur,erdewtiewnbotehulienevreeecltatohtpeaditcvteehxcis-- April 2
0
1
plex.Celllysatespreparedfromthecirc-Foxo3-andvector- demonstratestheectopicexpressionofcirc-Foxo3couldin- 9
transfectedcellswereincubatedwithRNAseAatdifferent hibitcellproliferationandcellcycleprogression.
concentrations.Thelysateswereseparatedinnativegradi- To understand how circ-Foxo3 functioned in regulat-
entgels,followedbywesternblottingprobedwithantibod- ing cell proliferation, we explored the possibility that circ-
ies against p21 and CDK2. In the vector-transfected cells, Foxo3mightinteractwithcellcycleassociatedproteinsand
p21 migrated as a single band (Figure 7A). However, in found that CDK2 and p21 could bind to circ-Foxo3. For-
the circ-Foxo3-transfected cells, two bands, one monomer mation of the circ-Foxo3–p21–CDK2 ternary complex hi-
and one with size slightly larger than the sum of CDK2 jackedCDK2togetherwithp21avoidingtheformationof
and p21, were detected when the lysates were treated with cyclinE/CDK2complex,thusblockingthetransitionfrom
lower concentrations of RNAse A. This suggests that the G1toSphase.Italsoabolishedtheinhibitoryeffectofp21
binding site for p21 and CDK2 on the circ-Foxo3 was on cyclin A/CDK2 complex, therefore blocking the pro-
protected from being degraded by RNAse A. It also con- gression of the cell cycle in S phase. As a result, cell cy-
firmedtheformationofaternarycomplexformedbycirc- cleprogressionwasarrestedintheG1phaseandunableto
Foxo3, p21 and CDK2. However, at high concentrations transittoSphase.