Ato o orsodne([email protected]) correspondence for *Author USA. School, 01605, Medical MA Massachusetts Worcester, of USA. University 77030, Pharmacology, TX Molecular Houston, Center, Science Health A&M Texas Technology, a Lin Tao respectively genome synthesis, in ribosome functions and distinct protection exercise GNL3L and Nucleostemin ARTICLE RESEARCH ß 2302 2014 February 13 Accepted 2013; October 2 Received in only that 2 revealed separate studies 1 as Later exist 2007). GNL3L al., and al., et et nucleostemin (Du genes Yasumoto was nucleostemin-related GNL3L for 2006). 2006; screen al., et a Zhu in 2011; discovered Tsai, cells 2012; progenitor Bishop, or 2002). stem and of McKay, (Qu self-renewal at embryogenesis the and of early expressed maintenance (Tsai in the and role be cells indispensable cancer to an plays many found Nucleostemin in also levels was their preferentially elevated with and compared well-known cells progeny a stem as differentiated neural most embryonic discovered rat was in the family, expressed this Nucleostemin, and of motif member MMR-HSR1 GTP- localization. unique of their family nucleolar by recognized defined newly a binding constitute (also NGP1) GNL2 and as GNL3L known nucleostemin, proteins mammalian The INTRODUCTION Nucleolus, synthesis GNL3L, Ribosomal damage, Nucleostemin, DNA cycle, Cell WORDS: KEY vertebrates the as control cell-cycle a and evolved. for protection fine-tuned vertebrate was genome contradictory nucleolus versus in the role be invertebrate how of the suggestive to are of and seemed genes functions had the about what findings a for provide explanation results nucleostemin Our coherent paralogous function. genome-protective the during novel a whereas gene acquired that, biosynthesis, ancestral the indicate ribosome of role in results the retained These GNL3L evolution, inducing vertebrate damage. without DNA production ribosome appreciable impairs GNL3L markedly contrast, By depletion production. and ribosome total synthesis the (r)RNA affects ribosomal mildly of only step initial we cells the S-phase carcinoma perturbing Here, in breast damage without DNA renewal. human significant and a prompt cell triggers mammalian in line nucleostemin cell in of its implicated depletion nucleostemin that been has found mammalian instead, paralogous as has, The biosynthesis, GNL3L. an been ribosome has from descendent, GNL3 arose in Invertebrate that GNL3. implicated genes gene, paralogous GNL3-like invertebrate by and ancestral encoded nucleostemin are proteins (GNL3L) nucleolar mammalian The ABSTRACT rga nCl n eeomna yais eateto iceityand Biochemistry of Department Dynamics, Developmental and and Biosciences Cell of in Institute Program Biology, Cell Stem and Cancer for Center 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,20–32doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1 igu Meng Lingjun , 1 sn-hnLin Tsung-Chin , 1 ar .Wu J. Laura , 1 hr Pederson Thoru , r aal fbnigt T,ms fte ontpossess and not 2005) (Daigle do al., YjeQ et [i.e. them (Reynaud do of Lsg1 that 2002), most few al., the GTP, et For to activity. 2006), GTPase binding al., intrinsic of et family, (Meng capable MMR-HSR1 GNL2 are the and of GNL3L nucleostemin, members including many Although yeast from human. conserved highly to is that product gene single a represents in pombe Schizosaccharomyces Nug1 (K01C8.9), rmncelrrgosweerN rncito n processing and transcription rRNA where growth regions nucleolar the from rescue NST-1-deficient to in 2006). fails phenotype al., et nucleostemin (Du mammalian nucleostemin human Moreover, by by not restored be but can GNL3L, Rpl25a yeast human and Grn1-null processing of the phenotypes pre-rRNA study export in 35S a nucleolar impaired by action the contradicted that is nucleostemin showing processing mammalian pre-rRNA of of coherent in role target nucleostemin direct a a establish direct Indeed, studies to pathway. to a ribosomal-synthetic lead fail these eventually or they ribosomal might Although ribosomes, mechanism nucleostemin 28S of 2009a). of perturbation to loss the al., nucleostemin the pre-rRNA that et of 32S indicate of (Romanova knockdown processing (r)RNA prolonged a the by that suggested delayed was showing synthesis potential ribosomal a study in cells, mammalian nucleostemin In of 2009). al., role proteins et subunit (Rosby ribosomal L26 and large L11 the and of (Kudron accumulation rRNAs nucleolar in 26S and in and 18S 2008), of of Reinke, levels In the export 2006). al., decreases been et in nucleolar NST-1 (Du has complex and Grn1 (60S) on It Rpl25a processing the of NS1). performed (pre-r)RNA and deletion been preribosomal NST-1 that have Grn1, reported (i.e. a nucleostemin showing Tsai, GNL3 studies of the and invertebrate of most Pederson effect date, 1998; To ribosomal 2002). Pederson, al., et 2005; 2008; Scherl al., 2009; et Pederson, (Andersen that role and this a know such now Ma of serve we proteins function but nucleolar canonical all synthesis), not ribosome the (i.e. in in domain in involved nuclear involved of than are concentration that be higher presence nucleoplasm assumes at the to nucleolus hypothesis nucleolar the a thought in the such stationed proteins been course, of Of always biogenesis. Because ribosome has 2007). it nucleoplasm al., nucleostemin, the and et nucleolus the nucleolus-concentrated (Meng between many shuttle like also but, proteins, nucleolus the in localized relatively is activity GTPase detected the 2009)], weak. al., et (Rosby omnotoo fncesei n N3,termed in GNL3L, NS1 and named nucleostemin specifically contain melanogaster or of invertebrate available collectively The ortholog is GNL3 information 2009). common sequence Meng, a which and for (Tsai genomes species vertebrate ulotmn N3 n N2poen r conspicuously are proteins GNL2 and GNL3L Nucleostemin, C38) S- in NST-1 (CG3983), 2 n oetY .Tsai L. Y. Robert and Drosophila acaoye cerevisiae Saccharomyces N_0027) ycnrs,GNL2 contrast, (NM_001022573). By .elegans C. elto fN1poenresults protein NS1 of depletion .pombe S. anradtselegans Caenorhabditis n S- sabsent is NST-1 and , .elegans C. Drosophila 1, * andGrn1in etrs35S perturbs Drosophila osof loss , GNL3

Journal of Cell Science i.2,tepretg of percentage the 2A, Fig. isnhssfnto fivrert N3 ti osbethat GNL3. invertebrate possible and from is GNL3L diverged ribosome- functionally mammalian it have the GNL3, might retains nucleostemin invertebrate GNL3L mammalian of only are function that of evidence biosynthesis Because that GNL3L, the 2005). and given al., nucleostemin regions and mammalian et of (Politz subnucleolar descent rRNA paralogous mammalian the 28S in Furthermore, nascent in localized 2008). deficient is Reinke, and nucleostemin (Kudron occur ARTICLE RESEARCH oass hte atclrsaeo h elccewas cycle of cell percentage the the of in increase stage the particular quantified a also we and whether effect impacted, damage assess DNA the of to immediacy the after determine event To early an is depletion cells nucleostemin S-phase in damage DNA response. damage 23.5%, of to foci 3.3% 1B, with 30.2%, (Fig. cells to (ATR) 5.3% of rad3-related from and percentage telangiectasia the ataxia in protein the increase an immunostaining cells revealed Parallel also of 1A). (Fig. percentage immunofluorescence increased using by an and blots, with by western ( shown as of H2AX damage), analysis DNA phosphorylated pronounced with of a associated the modification was amount in histone there and the confirmed Notably, in 2008), blotting). is elevation western cells al., (by MDA-MB-231 1A et has Fig. in siNS (Meng efficiency of specificity previously knockdown target demonstrated The transfection. been control after analysis hours with for 48 collected were at or were and duplexes line, (siNS) siRNA (siScr) cell scrambled nucleostemin-specific carcinoma with breast human transfected response a damage cells, DNA prompt MDA-MB-231 a elicits nucleostemin of Loss in genome RESULTS vertebrate function in conserved during role a that, unique retained synthesis. ribosome GNL3L a indicate whereas DNA acquired thus any, protection, if nucleostemin results little, perturbs evolution, evoking Our GNL3L while of pre-rRNA depletion damage. of contrast, processing striking up the By for structure hours. nucleolar 48 or a to synthesis hours incurring rRNA 12 without on as depletion, effect early nucleostemin significant following as of arises initiation damage event the DNA MDA-MB- after that initial cancer found we breast the cells, human 231 actually Using depletion. impaired is nucleostemin than consider 2013; for rather to production, al., damage, began et DNA role we ribosome Lin of 2013), 2012; accumulation al., a et the al., Yamashita whether et of 2013; integrity (Hsu al., al., cells genome et discovery Meng progenitor et the and recent and Romanova stem telomeres 2009a; of the protecting al., in by et nucleostemin (Romanova Motivated a days over 2009b). 5 knockdown of nucleostemin following RNAi-mediated period observed of been have rounds noteworthy is synthesis depletion two it ribosome respect, nucleostemin on this effects In effect. upon that direct a observed represent not synthesis might ribosome in elto eutdi nices ntepretg fS-phase of percentage nucleostemin the of in analysis that increase FACS an hours. in revealed and 48 resulted knockdown first depletion cells the nucleostemin within of propidium-iodide-stained rise initiation to the continued after hours 12 H2AX hs paetdsrpnisrieteiseta perturbations that issue the raise discrepancies apparent These c HA oi(rm23 o12.2%, to 2.3% (from foci -H2AX + el vr1–8husb sn AS ssonin shown As FACS. using by hours 12–48 over cells P 5 .2,bt fwihaeidctv faDNA a of indicative are which of both 0.02), P , .1 rwt 3P oi(i.1,from 1C, (Fig. foci 53BP1 with or 0.01) c -H2AX + el nrae seryas early as increased cells P , .1 a revealed was 0.01) c HA,a -H2AX, c - 1pplto smaue yFC fclslbldwith labeled cells of FACS G0/ by the in measured that with as non-BrdU-labeled compared population the bar) gray G1 in an 2E, damage showed (Fig. DNA population cells cell with 2E, cells non-S-phase (Fig. of foci the percentage 53BP1 of of 11% number increased only whereas cell), iStetdcls(i.2) The 2C). (Fig. cells siNS-treated n ee el nG/0cmae ihthe with compared G1/G0 in cells cells S-phase fewer more significantly and contained population depleted obelblFC a efre omauetecell-cycle the measure to the performed in distributions was possibility, time-point this FACS 48-hour address double-label the To triggers damage. depletion at nucleostemin DNA only that in replication-dependent suggested decrease cells This a 2B1,2). G1/G0 was (Fig. there of the that at percentage and cells time-points, G2/M-phase the 48-hour of and percentage the 24 in 12, decrease a and cells on ht2.%(2hus o4.%(8hus fteS-phase the of hours) (48 40.2% showed to hours) We cells (12 knockdown. nucleostemin 24.5% of that with duration the found cells of function S-phase shows a versus as foci 2D G1-phase Fig. of percentages 2C3,4). the (Fig. population nucleostemin-depleted paeo h rdn nlg5ehnluiie(-U,adits and (5-EU), 5-ethynyl-uridine ribosomal-synthetic analog the uridine the the if on based of in expected was design uptake rRNA be experimental The inhibited. processed would was pathway of as nucleus, accumulation the the affected knockdown was synthesis panel). rRNA right Materials of 3A, (see impairment (Fig. days significant observed 6 a of RNAi- total Methods), 47S of a and rounds the for of two lasting of 3A, to knockdown, (Fig. hours subjected mediated levels 48 when intermediate combined However, pre-rRNA the panel). At 45S the of center the level on precursors. and the transcript or on 45S primary transcript effect primary and no was 47S 47S there the depletion, the spans nucleostemin 5 PS-2 both the and for region detects pair 18S the primer 47S between the the junction the only whereas reflects spans product transcript, 3 (PS-1) PCR (left primary its the 1 therefore and 3A and Site Fig. region (ETS), spacer Processing 28S in the for shown between pair junction As primer 45S RT-PCR. the and 47S panel), real-time of levels by the the on pre-rRNAs measured knockdown next nucleostemin we of Introduction), effects depletion (see nucleostemin synthesis whether rRNA on affects reports conflicting the Given damage the DNA with of coincident activation structure rRNA nucleolar perturb or significantly biogenesis not does depletion Nucleostemin iueBd us nsN-rae el,floe by followed cells, 30- siNS-treated BrdU. a and 53BP1 on performed against we antibodies pulse with cells, immunofluorescence BrdU S-phase damage in DNA minute commences, preferentially the nucleostemin-knockdown-triggered depletion substantiate occurs as To that nucleostemin arrest. cells 3.5% after cell-cycle result S-phase intra-S-phase to hours in an 12 induced triggering hours) is as (12 2D, damage early (Fig. 1.8% DNA cells that G1/G0 only demonstrate the of with hours) (48 compared depletion, ouain tmgtas edet h ifrn estvte of sensitivities different non-BrdU-labeled the to the due in FACS. be versus labeling immunofluorescence also BrdU might of It already time were population. of that the inclusion cells the S-phase at to some due arrested as be well might as bar) cells gray G2/M-phase 2D, (Fig. PI and H2AX ( foci 53BP1 (BrdU-labeled) of S-phase number the increased of an 27% showed time-point, cells 48-hour the At ete ogtt eemn hte nucleostemin whether determine to sought then We ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal c -H2AX c -H2AX + inl nrsos onucleostemin to response in signals + versus c -H2AX c -H2AX P 9 , 9 P T,adteeoeit therefore and ETS, .1.Teeresults These 0.01). 2 xenltranscribed external , .1.Tehigher The 0.01). uppltosof subpopulations + nucleostemin- § oiper foci 5 c -H2AX c -H2AX 2303 c 2 -

Journal of Cell Science EERHARTICLE RESEARCH 2304 These 3D2). previous the (Fig. the a to in described days secondarily response might but 6 section. damage processing DNA depletion the 3D1), and for of (Fig. nucleostemin induction transcription siNS prolonged days rRNA 2 that perturb with of indicate for some treated results in siNS cells structure structure with the nucleolar nucleolar nucleolin-labeled treated nucleolin-labeled disorganized the cells we support, in In in 3C2). changes (Fig. no knockdown apparent 6-day was found a for structure DFC after DFC down cells disorganized fibrillarin-labeled knocked some a in but was 3C1), the nucleostemin (Fig. which days in 2 in changes cells of in we discernible (DFC) structure fibrillarin, protein component no the fibrillar by found defined dense is depletion the which nucleostemin structure, of nucleolar report that dispersion previous showing a a 2009b) causes to al., contrast et in compared (Romanova Finally, signal reduction cells. differential substantial siScr-treated 5-EU a with nucleoplasmic displayed their days in 6 (75%) for down had nucleostemin knocked which been in cells However, cells. compared siScr-treated knockdown nucleostemin small with of a days showed 2 after rRNAs) (20%) mature decrease representing thus of cells, that the from untreated as subtracted cells (calculated actinomycin-treated differential in level 5-EU signal nucleoplasmic dye the nuclear 3B, shown the As Fig. time 1966). (Penman, in which state cells by steady attained hours, The has inhibitor. 2 labeling rRNA the for of 5-EU in absence with incubated label or presence were dye knockdown the of versus on control amount based in the cells, transcripts in assess non-rRNA to inhibited versus possible rRNA selectively be (Perry, actinomycin would be of it concentration 1962), low can a reasoned with synthesis cells we mammalian rRNA Moreover, newly 2008). because the Salic, that, and of (Jao detection reaction dye- click-chemistry-mediated a coupling by by cells fixed followed in RNA synthesized RNA, into incorporation western panel, left Upper (A) nM. 100 at hours 48 proteins. for DNA-damage-response duplexes multiple siRNA for (siNS) staining by nucleostemin-specific visualized or and as (siScr) foci nucleostemin scrambled damage of DNA with blots of treated number were the cells increases nucleostemin MB-231 of Loss 1. Fig. ht ros xmlso ATR of examples arrows, White iha anti- an with ) 20 C), B ecnaeo ATR of Percentage (B) m B.Brgah ersn mean represent graphs Bar (B). m c HA nioy pe-ih ae,qatttv esrmn ftepretg of percentage the of measurement quantitative panel, Upper-right antibody. -H2AX + el lf)a eemndb sn muoloecne(ih) aes1ad2so anfe iw ftergosotie nwhite. in outlined regions the of views magnified show 2 and 1 Panels (right). immunofluorescence using by determined as (left) cells c HA.Poenlaigwscnrle yteaon of amount the by controlled was loading Protein -H2AX. + el.()Tepretg f53BP1 of percentage The (C) cells. 6 s.e.m. + el rgt sdtrie yuigimnfursec lf) cl as 50 bars: Scale (left). immunofluorescence using by determined as (right) cells hcpitadtu fe nepaaino h seemingly the on of knockdown nucleostemin cycle. explanation of cell an effect the the offer on findings thus conflicting G2/ and the until checkpoint phase S M through progress a nucleostemin with of cells loss that indicate partial results G2/M- These the 4B2). S-phase in (Fig. decrease population 84%, phase concomitant a was depletion with depletion nucleostemin apparent, nucleostemin became least arrest the the When with 4B1). samples (Fig. G2/ of in percentage the cells in increase M-phase an was there Notably, 4A3). (Fig. A14 rhmnGL A16.Aayi fwsenblots western of Analysis (Ab136). GNL3L human GNL2 either human analyzed antibodies recognize or proteins, cell-cycle specifically and GNL2 (Ab134) to and duplexes and GNL3L generated detect were siRNA To damage later. hours (siGNL2) their 48 DNA how GNL2-specific (siG3L) determine GNL3L-specific on or with to transfected interest were impact Cells of progression. might was it homologs, depletion nucleostemin GNL2, other and two GNL3L contain cells human Because with depletion contrasts nucleostemin knockdown GNL2 or GNL3L time-point 48-hour the of percentage at the 92% that confirmed and analysis nucleostemin FACS 84% to we 4A1,2). led (Fig. 54%, possibility, that concentrations this of siRNA investigate depletions of the To range a checkpoint. Because tested 50– G2/M speculated 2006). of the we transit study, range S-phase until al., present for permissive the the is depletion of et in nucleostemin results partial that the was Zhu on cases based 2008; and both 60%, in al., G2/M-phase depletion et a nucleostemin display (Meng shRN both arrest cells and osteosarcoma human depleted fibroblasts mouse nucleostemin-haploinsufficient embryonic that found arrest G2/M previously a We induces depletion nucleostemin Partial H2AX a tbln(u) S ulotmn oe aes immunofluorescence panels, Lower nucleostemin. NS, (Tub). -tubulin + ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal el nrae npooto oncesei depletion nucleostemin to proportion in increased cells c -H2AX + el nsSr n iStetdsamples. siNS-treated and siScr- in cells mrmdae nucleostemin- Amir-mediated m Aand (A m MDA- c -

Journal of Cell Science oe hnte1.%of 19.3% the than lower EERHARTICLE RESEARCH rti Fg A.FC nlsssoe htol .%of 3.9% only that showed analysis FACS were cells GNL3L-depleted 5A). GNL3L (Fig. of amount protein the by reduced treatment protein siG3L that confirmed nucleostemin. of knockdown during event early an is damage DNA S-phase 2. Fig. N2dpee el were in change cells the no GNL2-depleted elicited in GNL2 of amount decrease the c showed in slight blotting reduction Western 62% a 5C1,2). a (Fig. that and cells the G1/G0 in cells of increase percentage G2/M-phase an of showing percentage in cells nucleostemin-depleted differ from cells GNL3L-depleted addition, In 5B). (Fig. sample depleted h .%of 1.5% the elppltos cl a:50 bar: Scale populations. cell uniaieyaaye yuigFC ihanti- with FACS using by analyzed quantitatively el ihmr hn2 oiso hoooe.()Cl-yl rflso the of profiles Cell-cycle (C) . of copies 2N than more with cells E muoloecn mgn rgt sn ni5B1adat-rUatbde nMAM-3 el rae ihsN 10n)fr4 or.Quanti hours. 48 for nM) mean (100 the siNS show with Data treated time-points. cells 48-hour MDA-MB-231 and in 24 antibodies 12, anti-BrdU mean the the and at represent anti-53BP1 (left) compared using data were (right) sample imaging nucleostemin-knockdown Immunofluorescent the (E) in G1/G0-phase versus S-phase D-B21cl ape rae ihsSro iS(0 M o 2 4o 8hus aaso h mean the show Data hours. 48 or 24 12, for nM) (100 siNS or ( siScr analyses with quantitative treated samples cell MDA-MB-231 HA rti Fg D.FC nlssrvae ht54 of 5.4% that revealed analysis FACS 5D). (Fig. protein -H2AX . c -H2AX 2 u i o nraeteaon of amount the increase not did but 82% n + 5 el ntesSrtetdsml u distinctly but sample siScr-treated the in cells ,B)o ic-adsN-rae D-B21clsa ifrn ncdw iepit.Tefte rflssae nga represent gray in shaded profiles fitted The time-points. knockdown different at cells MDA-MB-231 siNS-treated and siScr- of B2) 4, 6 ...o h ecnaeo 53BP1 of percentage the of s.e.m. m c c c m. -H2AX -H2AX -H2AX + + + hc ssihl ihrthan higher slightly is which , hc ssilsignificantly still is which , el ntenucleostemin- the in cells c HA n rpdu oie(I.()Bsdo h nlsssoni ,tepretgsof percentages the C, in shown analyses the on Based (D) (PI). iodide propidium and -H2AX + c el ntennBd-aee nnSpae seik essBd-aee Spae arrows) (S-phase, BrdU-labeled versus asterisk) (non-S-phase, non-BrdU-labeled the in cells -H2AX c -H2AX hs retse pnncesei depletion. S- nucleostemin results the upon trigger not seen These does proteins arrest these phase 5F). of either (Fig. of depletion depletion, that nucleostemin arrest as does and elicits as response GNL2 damage G2/M nor DNA a GNL3L extensive neither a S-phase of of depletion percentage that of demonstrate the in indicative decrease slight cells, a and G2/ of cells percentage the M-phase in increase an to led depletion GNL2 addition, ( cells nucleostemin-depleted for than lower slkl ob h ebro hspoenpi hthsrtie the retained has that pair protein this elicits of member but GNL3L the that be nucleostemin reasoned to to we likely depleted, is paralogous when damage is DNA minimal GNL3L that processing pre-rRNA Given perturbs GNL3L of Loss + versus A ASbsdqatfcto ftepretg of percentage the of quantification FACS-based (A) ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal c -H2AX 2 el nncesei-ncdw ape were samples nucleostemin-knockdown in cells 6 ...()Cl-yl rfls(1 and (B1) profiles Cell-cycle (B) s.e.m. P , .0)(i.5) In 5E). (Fig. 0.001) c c -H2AX -H2AX 6 + + s.e.m. el in cells el in cells 2305 tative

Journal of Cell Science n ulotmnlbldncelrsrcue nsRAtetdclsa h -a n -a iepit.Wiedse ie hwncesoutlines nucleus show lines dashed White time-points. 6-day immunofluorescen and Confocal 2-day (D) the time-points. at 6-day cells and siRNA-treated 2-day 10 in the structures at nucleolar cells nucleostemin-labeled siRNA-treated in and structure nucleostemin-labeled and component fibrillar EERHARTICLE RESEARCH 2306 al., et to (Dousset known perturbed that 2013). is Pederson, as rDNA and such Ma the structure 2000; of nucleolar transcription in when not change occur does a processing 47S-to-45S as on manifest knockdown effect GNL3L the 2-day that the indicating of 6C,D), fibrillari (Fig. n the structures subnucleolar alter labeled (2-day) not did short-term depletion However, with 6B). transcript, (Fig. primary knockdown as a GNL3L 47S after to rRNA the nucleoplasmic reveal assay led of 5-EU differential level the depletion 47S-to the of the inhibition GNL3L an indicating in 6A). increase (Fig. substantial cells t determined GNL3L-depleted we hypothesis, the this of function synthesis ribosome o siScr with were treated transcripts cells target MDA-MB-231 differe of The in arbitrarily levels mean nucleoplasm. quantified were the the expression were in samples The show nucleoplasm present siScr Data (6d). rRNA the corresponding mature days. days in the synthesized 6 6 samples] newly in nucleost or or (ActD)-treated of transcript of amount actinomycin days (2d) each assays the minus 2 days qRT-PCR of measure (Ctrl) 2 to panels, levels [control for designed left expression signals was siNS and The assay 5-EU or Middle click-chemistry HMG-14. siScr 5-EU 5 circles. of modified with 18S, gray expression A represent treated by the (B) boxes cells grey indicated to and MDA-MB-231 are normalizing black in sites by White, time-point. levels recognition quantified PS-2). 48-hour PS-2 (PS-1, primer the 2 and PS-2 at and PS-1 and integrity 1 sites (NS), nucleolar PS-1 processing or The the synthesis respectively. flanking rRNA rRNAs, positions on primer 28S effects the and minimal step has pre-rRNA-processing nucleostemin the of Knockdown 3. Fig. m m. cesei ncdw,GNL3L knockdown, ucleostemin dasgiiat(7)rdcinin reduction (57%) significant a ed elvl frN rncit in transcripts rRNA of levels he 6 neta rti,GL.T test To GNL3. protein, ancestral 4Sprocessing -45S - uloi-o nucleostemin- or nucleolin- n-, ... * s.e.m.; P , .1 ** 0.01, tp naddition, In step. P , .0,*** 0.001, P , .01 C ofclimnfursec ftefbilrn(i)lblddense (Fib)-labeled fibrillarin the of immunofluorescence Confocal (C) 0.0001. efre eceeprmns ulotmnGPexpression Nucleostemin–GFP experiments. we damage, rescue DNA replication-linked performed in nucleostemin of function estvt fMAM-3 el oH-nue replication HU-induced the to H increased cells ( depletion MDA-MB-231 thymine stalling and of Nucleostemin species sensitivity oxygen respectively. reactive dimers, stalling, fork replication estvte fcnrladncesei-eltdclsto cells nucleostemin-depleted and H the measured To control the we hydoxyurea, hydrolysis. involved, of of damage base stalling DNA DNA sensitivities of by or type caused the stress determine be oxidative fork, can replication damage DNA damage DNA GNL2, Spontaneous or replication-induced GNL3L against of protection not confers but nucleostemin, of Overexpression 2 O 2 idcdDAdmg Fg A.T ute netgt the investigate further To 7A). (Fig. damage DNA -induced ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal P , .0,rpae esrsAOA u o oU-or UV- to not but ANOVA) measures repeated 0.001, 2 O 2 rU etrain htintroduce that perturbations UV or A et iga showing diagram Left, (A) eo nucleolin- of ce cl bars: Scale . iSfor siNS r e s1. as set 8 and .8S emin ntial

Journal of Cell Science EERHARTICLE RESEARCH aaoospoen N3,ta a eandterl in role the retained the is has it protecting that a that by GNL3L, has and stability protein, nucleostemin damage, genomic DNA that paralogous maintaining one replication-induced show least in against at nucleolar investigation the role or together, this require effect, Taken unique sites. direct conferred of not replication a to results be also proximity does to in occurs likely Thus, – nucleostemin that is panel). and of upper nucleoplasm localization 7B, property (Fig. the and damage this – DNA in that against (NSdB–GFP) nucleostemin protection accumulated signal of version nucleolus-localization a thus of the expression However, mechanism the lacked nucleostemin. indirect that of found some localization we through of nucleolar the the occur replication-linked concentration requiring might against in high nucleostemin whether damage of investigated DNA the cells we effect compare nucleolus, Given protective the the (GFP-positive) was panels). in 7B, protein effect lower (Fig. nucleostemin transfected rescue and This samples upper panel). in upper was 7B, nucleostemin–GFP-transfected only (Fig. GNL2–GFP so DNA nor seen do GNL3L–GFP replication-linked to neither able against whereas cells damage, protected significantly R time-point. 48-hour the at collected were blots. Samples the siNS. below and shown are siScr samples of siNS cells. and (conc.) nucleostemin-depleted siScr of concentrations paired arrest different between cell-cycle (%) with intensities the treated nucleostemin of cells nature MDA-MB-231 the dictates in efficiency efficiency knockdown The 4. Fig. ( elmrhlg r hw yDP tiigaddfeeta nefrnecnrs DC mgn,rsetvl.Saebr:10 bars: Scale respectively. imaging, (DIC) contrast interference differential and of percentage staining the DAPI of by quantification shown are and morphology anti-nucleostemin cell using performed Immunofluorescence (A2) n 5 )o D-B21clstetdwt 25n B)o 0n B)sRA o 8hus aaso h mean the show Data hours. 48 for siRNAs (B2) nM 50 or (B1) nM 12.5 with treated cells MDA-MB-231 of 4) c -H2AX + el nsSr n iStetdsmls ** samples. siNS-treated and siScr- in cells c HA nioisi ic-adsN-rae D-B21cls ula tutr and structure Nuclear cells. MDA-MB-231 siNS-treated and siScr- in antibodies -H2AX Rmnv ta. 09) twsaantti akrudta we that background this against was biosynthesis It report ribosome 2009a). another al., in et and (Romanova nucleostemin 2009), al., mammalian et implicated (Rosby biosynthesis ribosome iooesnhssetbihdi h neta pre-vertebrate ancestral the in protein. established synthesis ribosome ncl rlfrto,atog h ietrlso h proteins the appeared of evidence roles time, Zhu same 2011a; direct proteins 2009; the in At both al., the al., proven. of rigorously et although et not involvement Zhu were the proliferation, Meng 2008; to (Lin cell al., attention 2007; in proteins. drew et GNL3L Pederson, 2006) Meng al., and 2011b; GTP-binding and et al., Ma nucleostemin et nucleolar 2010; Meng both al., two on mammalian et possible work these of the landscape Subsequent of GNL3L, the broadened origin. of functions evolutionary nucleostemin, its of identification not cells, paralog on the course, subsequent in of was, determinant The focus a the as 2002), role neural McKay, its embryonic and rat (Tsai in cells discovered stem first was nucleostemin When hypothesis The DISCUSSION P Drosophila , .0,*** 0.001, ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal a tbln(u)i hw salaigcnrl S nucleostemin. NS, control. loading a as shown is (Tub) -tubulin P , ikn h netbaepoen N3 to GNL3, protein, invertebrate the linking .01 B elccepoie n uniaieanalyses quantitative and profiles Cell-cycle (B) 0.0001. 6 ...o he needn experiments. independent three of s.e.m. A)Wsenbost hwteknockdown the show to blots Western (A1) m .(3 FACS-based (A3) m. elative 2307

Journal of Cell Science h mean the EERHARTICLE RESEARCH 2308 diverged functionally nucleostemin that distinct strongly genome hypothesis and in the reveal respectively, GNL3L support biosynthesis, findings and ribosome and Our nucleostemin protection mammalian functions. a of acquired or activities nucleostemin function ribosome paralogous in different protein the ancestral the whereas of mammalian role biosynthesis, that the was retained hypothesis has Our GNL3L study. present the launched and GNL2 GNL3L, of blots Western damage. (A,D) DNA hours. minimal of 48 with for nM arrest 100 G2/M of at amount causes duplexes GNL2 siRNA or D–F) (siGNL2, GNL3L GNL2-specific of Loss 5. Fig. c -H2AX 6 + a s.e.m. tbln(u) h eaieaon ftesRAtree rti sidctdbnahtebos BE ASbsdqatfcto ftepercentag the of quantification FACS-based (B,E) blots. the beneath indicated is protein siRNA-targeted the of amount relative The (Tub). -tubulin el nsRAtetdsmls CF elccepoie n uniaieaaye ( analyses quantitative and profiles Cell-cycle (C,F) samples. siRNA-treated in cells rmisvrert aao,GLL n h invertebrate the and GNL3L, paralog, GNL3. vertebrate ortholog, its from iooeboeei a o encer otpeiu studies previous Most clear. in been role not direct a has plays biogenesis nucleostemin ribosome whether above, discussed depletion As an nucleostemin is following biosynthesis, event ribosome early of impairment not damage, DNA D-B21clswr rae ihGLLseii sGL –)or A–C) (siG3L, GNL3L-specific with treated were cells MDA-MB-231 ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal n 5 )o iN-rae D-B21cls aarepresent Data cells. MDA-MB-231 siRNA-treated of 4) ic rsGLfr2dy.Wiedse ie show um. lines 10 dashed bars: White Scale days. outlines. 2 nucleus for siG3L with or treated siScr cells and in nucleolin- days. structures the 2 nucleostemin-labeled of for siG3L immunofluorescence or Confocal siScr (D) with treated cells (NS)-labeled in nucleostemin structures and (Fib)- fibrillarin the mean show the show Data with ActD. *** treatment of (Ctrl) dose without low or a (ActD) GNL3L-knockdown with and cells (siScr) (siG3L) of control immunofluorescence in Right, signals (left). 5-EU days siScr 2 with for treated siG3L cells or MDA-MB-231 in quantified nucleoplasm were the minus in (Ctrl) samples] 1. [control (ActD)-treated as signals actinomycin set 5-EU arbitrarily differential were The samples (B) each siScr of the levels in expression transcript The the HMG-14. target to of of normalizing expression levels by expression quantified The were or (2d). transcripts siScr days with 2 treated for cells siG3L MDA-MB-231 in levels of PS-2 processing the perturbs GNL3L pre-rRNAs. of Loss 6. Fig. P , .01 C ofclimnfursec to immunofluorescence Confocal (C) 0.0001. c HA.Poenlaigwscnrle ythe by controlled was loading Protein -H2AX. A R-C saso N3,P- and PS-1 GNL3L, of assays qRT-PCR (A) 6 s.e.m. e

Journal of Cell Science EERHARTICLE RESEARCH rnucdctpti vns hsitrrtto ftheir of to interpretation succumb This cells events. 5-day-knockdown 1966), cytopathic Penman, these (e.g. kinetics pronounced studies than that slower labeling other much nucleostemin numerous rRNA were suggesting al. in of the 5-day et reported Romanova their that by those days to study 6 noted the comparable in is also reported after that We time inhibited of knockdown. rRNAs period of severely a maturation 2-day knockdown, and both a transcription al. the after et are that Romanova nucleoplasm found the we the to study, relevant in Most species steady- knockdown. the nucleostemin rRNA in reduction labeled minor within a state only differential perturbed reveals the assay appreciably Moreover, labeling knockdown. not 5-EU nucleostemin is of hours precursors 48 rRNA of initiation 45S the and Our after shortly knockdown. triggers ( arrest were of depletion knockdown cell-cycle cells rounds nucleostemin and which two damage that DNA after in show day cells, analyses 5th HeLa al. timecourse the et in on Romanova 2009a) whole- analyzed of al., both temporal study 2009) et to al., nucleostemin-knockdown (Romanova et the applies the gene Rosby to 2008; issue Reinke, resolving nucleostemin and This and (Kudron events. of without studies organism the results knockdown, of terminal relationship or the knockout only examined , 2hus.B otat h isnhsso 47S of biosynthesis the contrast, By hours). 12 hsas lre st h osblt htncesei is nucleostemin that possibility synthesis. rRNA the not to and it protection us genome where in alerted 2013). involved sites, al., et also molecular damage (Meng RAD51 the direct protein This DNA repair a DNA to is the as with there interacts nucleostemin under that well was of suggested investigation study recruitment as present parallel a the depletion, way, while cell and Furthermore, the nucleostemin function cycle. nucleolar that long- between of confounded versus relationship believe interconnecting immediate be effects the might we surrounding al. term that issues Thus, et an unsuspected Romanova showing to synthesis. by by leads literature reached rRNA cells the conclusion mammalian of in transformed arrest. inhibition of evidence cell-cycle arrest 5-day- abundant a cell-cycle the elicit is would that There indicates fact literature the substantial given a display traction cells knockdown further gains findings ta. 09 raG/ ret(ege l,20;Nkore al., et Nikpour 2008; al., et Nikpour (Meng 2007; arrest Pederson, G2/M and a Ma or 2008; 2009) al., al., et et include (Dai shown arrest Effects studies. G1/S of different a progression among vary the cycle on cell depletion the nucleostemin of effects exact The cell-cycle on depletion progression nucleostemin of effect The A D-B21clstetdwt ic rsN eetse o their for tested H were HU, with siNS treatment or following siScr survival with clonogenic treated damage. cells or DNA MDA-MB-231 GNL3L (HU)-induced of (A) hydroxyurea not against but protects nucleostemin, GNL2, of Overexpression 7. Fig. ae,tepretg of percentage the panel, 4hu Utetet oe ae,tepretg of percentage the a panel, without Lower or treatment. with HU GNL2–GFP, 24-hour or GNL3L–GFP mutant), nucleoplasmic nucleostemin (a overexpressing NSdB–GFP (NS)–GFP, transiently nucleostemin cells GFP, MDA-MB-231 of populations positive) nlzdb eetdmaue NV.Dt ersn the represent Data ANOVA. mean measures repeated were by Differences analyzed populations. (GFP-negative) non-transfected the ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal 6 s.e.m. c -H2AX , 0 eraei itn B which 2B, histone in decrease 50% + el ntetasetd(GFP- transfected the in cells 2 O 2 rU.()Upper (B) UV. or c -H2AX + el in cells 2309

Journal of Cell Science nSpaecls oprdwt oto el,nucleostemin- to cells, not but control damage HU-induced H to with sensitive DNA more Compared are that cells depleted cells. show S-phase data in occurs preferentially Our knockdown nucleostemin by damage. induced damage UV-induced intra- increased to and an show bases sensitivity NER dysfunctional depurinated with Cells removes crosslinks. strand machinery (NER) repair h ulols ne odtoso uloa teso mitotic or stress nucleolar 2008), of conditions transit. al., under to nucleolus et nucleoplasm the from the mobilized (Meng is protein the nucleostemin one when primarily place of takes function previous -regulatory activity its whereas genome-protective a constitutively, occurs the and that al., study, emphasize et This (Beekman of embryos function 2006). -null obligatory the in with documented synchrony nucleostemin in MDM2–p53 more the is of and independently pathway act of to activity genome-protective expected MDA-MB-231 observed is nucleostemin the because mutated, Finally, p53 takes are replication. that cells damage DNA DNA of during repair place the promoting by cells actively dividing of integrity genomic the suggest safeguard findings might nucleostemin These that damage. DNA HU-induced rescue to able is hc ercmlt rprilncesei ncdw might knockdown in nucleostemin the previously, partial to or reported resolution near-complete arrest a potential G2/M- cell-cycle which a on a provides a results This produces that contradicting 4). and (Fig. nucleostemin arrest arrest cell of S-phase phase the an in knockdown is p16 depletion less-efficient or cell-cycle nucleostemin primary Rb the of p53, that reveals event of study present status The the used. discrepancy models by This 2006). explained al., be et Zhu cannot 2009a; al., et Romanova 2009; ARTICLE RESEARCH N3 htpasamjrrl nrN isnhss hs the Thus, 2310 biosynthesis. fact rRNA in in is role it major a that only plays and occurs that knockdown, GNL3L biosynthesis nucleostemin ribosome prolonged of the after that impairment as show significant we nucleostemin Here, a regard GNL3. invertebrate to of equivalent investigators 2009), mammalian al., some et knockdown led Rosby nucleostemin mammalian 2008; naturally of Reinke, effects and similar biosynthesis of Kudron ribosome reports 2006; impair al., invertebrate to et been the observed (Du of ribosome have was deletion GNL3, which nucleostemin genetic on ortholog, in because mammalian studies Thus, knockdown present earlier and investigated. of The its interpretation GNL3 consequence. the invertebrate in the secondary of genome-protective aid Instead, a can effect a findings be case. be might the the Our might biosynthesis be protein. and nucleostemin not multitasking of a function, might cell is role this this in primary that that nucleostemin notion suggest of the results to biosynthesis role rise ribosome the and gave regulation on MDM2–p53 reports proliferation, proteins conflicting family nucleostemin The of view evolving An H to sensitivity to machinery leads, experimentally, impairment forks (BER) BER thus and replication deamination, repair or alkylation oxygen collapsed species, reactive excision by or caused lesions base base stalled non-helix-distorting repairs The of 2008). repair (Helleday, and restart cells the for hydrolysis, responsible stalling, mechanism primary base replication the homologous is machinery The or recombination of mechanisms. repair species distinct result mounting oxygen by a respond reactive as of occurs formation protection damage damage DNA DNA in When nucleostemin of role arrest. The G2/M and S intra-S-phase late an arrest, a G1/S) or (apparent arrest S-phase early an in result 2 O 2 rU-nue aae n vrxrsino nucleostemin of overexpression and damage, UV-induced or - 2 O 2 h uloieexcision nucleotide The . 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Journal of Cell Science CCTT-3 rae ihbooexuiie(rU 10 (BrdU, with were incubated bromodeoxyuridine cultures cells, and with S-phase X-100 detect treated To Triton temperature, antibodies. 0.3% secondary room and at with primary minutes permeabilized ice- 15 or were for staining) cells staining) anti-nucleolin (nucleolin for except methanol cases cold in all fixation After (in were FITC. formaldehyde or 4% Rhodamine-X Nucleoli with Secondary conjugated CA). respectively. were Diagnostics, FL) antibodies were Research groups, Gainesville, (4E2, EnCor, cells antibody siNS (38F3, anti-nucleolin siNS 1615 antibody and and and anti-fibrillarin and using siScr siScr by 1572 the labeled the for staining, for counted 53BP1 were counted For For cells respectively. respectively. 592 groups, siNS and groups, and 580 Cell siScr staining, the (#4937, for ATR 53BP1 counted were and For cells TX) MA). 1113 Dallas, Danvers, Cruz, Technology, Santa Signaling (N-19, ATR H2AX, ncdw xeiet ihtoqC repeats. qPCR two with experiments knockdown TCTC-3 ACTCTGGACAATGG-3 TGCCCTTCGTCCTGGGAAAC-3 CCGTCGGCATGTATTAGCTCT-3 N aaefc eedtce ysann ihatbde against antibodies with staining by detected were foci damage DNA Immunofluorescence ARTICLE RESEARCH ytei frNswsdtce yuigteClick-iT the using by detected was rRNAs chemistry click of by Synthesis synthesis rRNA of Imaging quantitative For transcriptase. murine Moloney reverse and the (Mo-MLV) primers (q)PCR, virus hexanucleotide random leukemia using cDNAs (5 samples RNA cell transcripts Total rRNA of analysis qRT-PCR experiments. data independent All four software. 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Helleday, 8hus fe eoeigfr2 or,clswr eltda low at for replated were duplexes cells nM) (100 hours, 24 siRNA for desired cells/cm (55 recovering the density After with hours. treated 48 were Cells assay survival Clonogenic ses .B,Prbo,T . os .J,Prde,J n ains .W. A. Macinnes, and J. Paridaen, J., Y. Goos, C., T. Pereboom, B., P. Essers, D. Balasundaram, and S. Mahalingam, W., Wu, Q., X. Chen, R., M. Rao, and X., Du, D. Hernandez-Verdun, D., Chen, C., Verheggen, C., Wang, T., Dousset, Brown, and V. E. Koonin, L., Aravind, M., A. Berghuis, L., Rossi, M., D. Daigle, H. Lu, and X. X. Sun, S., M. Dai, W., Wurst, T., Floss, M., Maetens, S., Clercq, I. De A. M., Lamond, E., Nichane, C. Lyon, C., E., Beekman, S. Ong, K., A. Leung, W., Y. Lam, S., J. Andersen, References [grant T.P. Foundation to Science Council National MCB-1051398] and Research number L. R.O.C.) Cancer T.-C. (Taiwan to M Council Award & Fellowship Science A Postdoctoral National Texas R.Y.L.T., by to part Award in Incentive supported was work This Funding wrote R.Y.L.T. and T.P. L.J.W paper. data; and the analyzed R.Y.L.T. T.-C.L. and T.P. L.M., T.L., T.L., research; research; performed designed R.Y.L.T. and T.P. L.M., T.L., contributions Author interests. competing no declare authors assay. The 5-EU the on interests advice Competing for laboratory T.P. the in Ma Hanhui thank We Acknowledgements xoe o24n ih tdsso –0J/m were 0–20 cells of and doses removed at was light supernatant 254-nm PBS, to with exposed washed were cells h iueaonso r-RAi h uloiaeol rsn at present only are nucleoli the in pre-rRNA the levels. and of period, mass, background amounts labeling RNA reflecting this minute equilibrium, after to that, the labeled fact been was the has label to rRNA the little owing nuclear hours, by nucleoli, 2 the of actinomycin-treated determined times in label seen the was 5-EU With between rRNA groups. per non-treated intensity nuclear the and cells nucleoplasmic using of 27 in condition level from each difference The for measured program. repeats was independent ImageJ nucleoplasm three and the experiment in intensity signal 5mnts emaiie ih05 rtnX10i B for PBS in Click-iT X-100 the Triton with for incubated PBS 0.5% then in 5- and formaldehyde with with 4% minutes of 15 permeabilized with hours absence or 2 fixed presence minutes, were for continued 15 the cells incubated in The mM then 1 actinomycin. at and (5-EU) uridine minutes ethynyl 20 for actinomycin ifrn ocnrtoso yrxue rH or hydroxyurea of concentrations different o or h el eete ahdwt is ufradDAwas DNA and buffer rinse with TO-PRO washed then with were cells counterstained The hour. 1 for o 5mntsa omtmeaueadsann ih00%Crystal 0.05% with staining colony and allow temperature to room formaldehyde 3.7% weeks at 2 with minutes fixing 15 for by for visualized medium were growth Colonies formation. normal in maintained one.Fnldt ersn h vrg ftreindependent three of were cells average ANOVA. more the measures or repeated by represent 50 analyzed are containing data and experiments Colonies Final minutes. 30 counted. for Violet niios-anweai agtdcne therapy. cancer targeted in era new a - inhibitors 21) oprtv td fncesei aiymmesi zebrafish in members family nucleostemin biogenesis. ribosome of in roles study specific reveals comparative A (2014). nucleolar of the processing in and role yeast a play fission and growth from pre-rRNA. for Grn1p required are GTPases GNL3L putative human homologous The (2006). transcription. I polymerase S. and Huang, G-motifs permuted circularly with GTPase kinetics. unusual burst marked an is coli, Escherichia MDM2. D. of E. inhibition via arrest cycle cell induces Biol. early and during p53 activates cells stem/progenitor of C. development. proliferation J. vertebrate Marine, controlling and nucleostemin: E. Bellefroid, M. Mann, and 28 20) jQ nesnil osre,ucaatrzdpoenfrom protein uncharacterized conserved, essential, an YjeQ, (2002). ora fCl cec 21)17 3221 doi:10.1242/jcs.143842 2302–2312 127, (2014) Science Cell of Journal 4365-4376. , 20) mlfigtmu-pcfcrpiainlsosb N repair DNA by lesions replication tumour-specific Amplifying (2008). o.Bo.Cell Biol. Mol. 20) ntaino uloa sebyi needn fRNA of independent is assembly nucleolar of Initiation (2000). 20) uloa rtoedynamics. proteome Nucleolar (2005). 2 .A 4hus el eetetdfr2 or with hours 24 for treated were cells hours, 24 At ). Biochemistry o.Cl.Biol. Cell. Mol. 17 o.Bo.Cell Biol. Mol. 460-474. , H 3fr3 iue.Teaeae5-EU average The minutes. 30 for -3 20) bratepeso fnucleostemin of expression Aberrant (2008). 41 20) vltoaiycnevdrl of role conserved Evolutionarily (2006). 11109-11117., 26 11 9291-9301. , 2705-2717. , e.Biol. Dev. u.J Cancer J. Eur. 2 2 O l utrswr then were cultures All . 2 o Virradiation, UV For . Nature TM 385 ecincocktail reaction 304-315. , 433 44 77-83. , 921-927. , o.Cell. Mol. 2311

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SUMOylated to recruitment PML-IV 197 promoting by damage ppoi ntebadrcne ellns53 n SW1710. and and 5637 arrest lines bladder on the suppression in Nucleostemin apoptosis of effects Differential the (2009). maintains that cells. mechanism progenitor and essential the 11415-11420. stem an of with stability reveals genomic association deletion dynamic Nucleostemin its shortens and dimerization telomere. TRF1 inhibits cell arrest. and G2/M p53-dependent progression induces and G2-M promotes and MDM2 survival. stabilizes nucleostemin mechanisms. protein-specific versus 27 common proteins: family nucleoplasm. and 5124-5136. nucleolus between partitioning nucleostemin arrest. 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Kikyo, and protein cell stem the by revealed nucleolus the nucleostemin. in landscape nonribosomal USA Sci. Acad. Natl. tblzsteTF ope n rmtsmttctransition. mitotic promotes and 827-839. complex TRF1 the stabilizes stability. coactivator protein TRF1 for 9279-9290. regulates negatively competing and senescence by gamma receptor estrogen-related binding. of activity .Bohm elBiol. Cell Biochem. nucleostemin. J. of cycling dynamic 184. the controlling cells. cancer and cells stem in proliferation proteins. J. J. cell nucleolus. Diaz, precursor human and D. midgut Hochstrasser, A., and growth, cell biogenesis, maintenance. subunit ribosomal large J. P. of integrity and architecture complex. telomerase nucleolar the 284 of and ribonucleoproteins maintenance nucleolar the small in nucleostemin of to compartmentalization. of evolution cells the with correlates somatic that of R. 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