Rae1 Is an Essential Mitotic Checkpoint Regulator That Cooperates with Bub3 to Prevent Chromosome Missegregation

The Journal of Cell Biology

JCB

Published January27,2003 T Rae1-null andBub3-nullmiceareembryonic lethal,although insufficiency and,surprisingly, Bub3haplo-insufficiency. show thatoverexpression ofRae1cancorrectforhaplo- checkpoint defectsandchromosome missegregation. We also Rae1 orBub3resultsinasimilarphenotypeinvolving mitotic combination. Hereweshow thathaplo-insufficiency ofeither out micethathave thesegenesdeletedindividually orin roles ofRae1andBub3inmammals,wegenerated knock- Bub3 inthemitoticcheckpoint. To determinetheinvivo that Rae1isinvolved inthemRNA exportpathway and and Jan M.van Deursen B.Jeganathan, Babu,J. Ramesh Karthik Darren J.Baker, Xiaosheng Wu, Ningling Kang-Decker, chromosome missegregation that cooperates withBub3toprevent Rae1 isanessentialmitoticcheckpoint regulator mitotic checkpoint,leadingtoinhibitionoftheanaphase- metaphase plateandattachedtomicrotubulesactivatethe Kinetochores ofchromosomesthatarenotyetalignedatthe 2001;Nigg,WassmannandBenezra,2001). Hieter, (Burke, 2000;ShahandCleveland,Kitagawa faithful segregationofduplicatedchromosomesduringmitosis partly resolvedintricatemolecularnetworkthathelpstoensure 1999; Gemmaetal.,2001).Themitoticcheckpointis a Bub1, BubR1,andMad2(Cahilletal.,1998;Imai in genesthatencodemitoticcheckpointproteins,such as with chromosomalinstability,mutationshavebeenidentified et al.,1998;Cahill1999b).Inasubsetofhumancancers of wholechromosomes(HartwellandKastan,1994;Lengauer the geneticeventsthatcaninitiateorpromotelossesgains vast majorityofhumancancers,butlittleisknownabout Abnormalities ofchromosomenumberareahallmarkthe ofPediatricDepartment andAdolescent Medicine, Mayo Clinic,Rochester, MN55905 Introduction http://www.jcb.org/cgi/doi/10.1083/jcb.200211048 The Journal ofCellBiology, Volume 160, Number 3,February 3,2003341–353  mosomal instability Rae1/Gle2;Bub3; mitoticcheckpoint;chro- Key words: mRNAexport; 0340. E-mail:[email protected] Street SW,Rochester,MN55905. Tel.:507-284-2524.Fax:507-266- Address correspondencetoJanvan Deursen,MayoClinic,200First

TheRockefeller University Press, 0021-9525/2003/02/341/13$8.00 Article similarity. However, previousstudieshave suggested sive sequencehomology, indicative offunctional he WD-repeat proteinsRae1andBub3show exten- cytosol, includingBubR1,Bub3,andMad2(Fangetal., is believedtoactivatemitoticcheckpointproteinsinthe 2001). Althoughthecompositionofthissignalisunclear, it Cleveland, 2000;Hoffmanetal.,2001;JallepalliandLengauer, act togenerateamolecular“anaphasewait”signal(Shahand chore sensors formicrotubule–kinetochoreattachmentandkineto- that checkpointproteinsassociatedwithkinetochoresact as condensedchromosomes.Currentmodelspropose chores of proteins arepositionedinthemitoticcytosolandatkineto- chromatids bycleavingcohesioncomplexes(Peters,2002). by activationoftheproteaseseparase,whichseparatessister point isreleased.TheAPCthenenablestheonsetofanaphase chromosomes arealignedatthemetaphaseplate,check- When allkinetochoresareattachedtomicrotubulesandthe (Morgan,1999;Peters,2002). promoting complex(APC)* mitotic checkpoint. with essential,overlapping, andcooperating rolesinthe for Rae1andcharacterize Rae1andBub3asrelatedproteins wild-type mice. Thus, ourdatademonstrate anovel function to dimethylbenzanthrene-induced tumorigenesisthan mice withmitoticcheckpoint defectsaremoresusceptible than singlehaplo-insufﬁcient cells.Finally, weshow that sister chromatid separation andchromosome missegregation cells fromthesemiceexhibitmuch greaterrates ofpremature haplo-insufﬁcient Rae1/Bub3miceareviable.However, nuclear exportofmRNA. Unlikenullmice,compound cells fromthesemicedidnothave adetectabledefectin complex; PMSCS,prematuresister chromatid separation. mouse embryonicfibroblast;NE,nuclear envelope;NPC,nuclearpore E, embryonicday;ES, stem;ICM,innercellmass;MEF, *Abbreviations usedinthispaper: APC, anaphase-promotingcomplex; During prophaseandprometaphase,mitoticcheckpoint tension. Intheabsenceofattachmentortension,they

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342 (embryonic day[E]8.5)yielded 9Rae1 1, AandC).Dissectionofembryos at8.5dpost-coitum development andgenotyped them byaPCRmethod(Fig. embryos fromheterozygousintercrosses atvariousstagesof mouse embryogenesis. offspring,demonstrating thatRae1isessentialfor liveborn quency (7 no Rae1 Results erozygous micebutfoundnoRae1 guishable fromwild-typelittermates.Weintercrossedhet- line (Fig.1B).TheseRae1heterozygousmicewereindistin- mice thatpassedthedisruptedRae1genethroughgerm- targeted embryonicstem(ES)cellclonesyieldedchimeric a lacZ-neoselectioncassette(Fig.1A).Threeindependently gene byinterruptingitscodingregionataminoacid50with targeting approachinthemouse.WeinactivatedRae1 We addressedthephysiologicalroleofRae1throughagene- Rae1 isessentialforearlyembryogenesis some missegregation. Rae1 allelecausesamitoticcheckpointdefectandchromo- ogous recombination.Weshowthatthelossofasingle pothesis, wehavedisruptedthemouseRae1genebyhomol- might actasamitoticcheckpointprotein.Totestthishy- al., 2001).ThisfindingledustohypothesizethatRae1 Rae1 bindsnotonlytoNup98butalsoBub1(Wanget GLEBS sequencesofmitoticcheckpointproteins.However, for Bub3(Wangetal.,2001).exclusivelybindsto proteins Bub1andBubR1,wheretheyserveasbindingsites GLEBS motifsarealsopresentinthemitoticcheckpoint Rae1 (Pritchardetal.,1999).Wehaverecentlyshownthat contains amotifnamedGLEBSthatdirectsbindingto 2000; Zenklusenetal.,2001).ThenucleoporinNup98 1999; Pritchardetal.,Bachi2000;Yoon McKeon, 1997;Baileretal.,1998;Martinez-Exposito Murphy etal.,1996;KraemerandBlobel,1997;Taylor but whosepreciseroleremainsunclear(Brownetal.,1995; is involvedinthepathwayformRNAexportinterphase, mrnp41) isahighlyconservednucleartransportfactorthat arates WDrepeats3and4.Rae1(alsocalledGle2or protein lengthandisespeciallyhighinthesegmentthatsep- each ofthefourWDrepeatmotifs;itextendsoverentire Exposito etal.,1999).Theirhomologyisnotconfinedto quence homologywithRae1(Tayloretal.,1998;Martinez- vate theAPC. anaphase waitsignals,allowingCdc20tobindandacti- of allchromosomesatthemetaphaseplatequenches and itsemissionofanaphasewaitsignalsstops.Alignment kinetochore-associated checkpointproteinspartiallydetach bule–kinetochore attachmentandkinetochoretension,its vating theAPC.Whenachromosomehaspropermicrotu- teins thenbindtoCdc20,therebypreventingitfromacti- 2001; Fang,2002;Yu,2002).Activatedcheckpointpro- 1998; GillettandSorger,2001;Hoyt,Sudakinetal., Rae1 fied among26 blastocysts).TheseRae1

To determinewhenRae1 The mitoticcheckpointproteinBub3sharesextensivese-

The JournalofCellBiology

embryoswerepresentatanormal Mendelianfre-

embryos.At3.5dofgestation, however,

, 13

, and6

Volume 160,Number3,2003

embryosdie,wecollected

embryoswere identi-

/ miceamong203

, 22Rae1

embryoswere

/ , but technique usingFITC-labeled oligo(dT) cellular distributionofpoly(A) chard etal.,1999).Surprisingly, boththelevelandsub- (Fig. 2C).DisruptionoftheRae1homologue our gene-targetingstrategyhadindeedgeneratedanullallele pronounced NElocalizationinRae1 with Rae1attheNPC(Pritchardetal.,1999),exhibited a lished data).Nup98,anucleoporinthatformscomplex G). Similarresults wereobtainedwhenearlier embryonic cells ofRae1-deficient embryonicoutgrowths (Fig.2,Fand mained viableuptoandbeyondE8.5(Fig.1D). tocysts developednormallyintoaflattenedlayerandre- The abilitytocultureRae1 E8.5, culturedRae1 ing thefirst2dofculture(Fig.1D).FromE6.5through cida andattachedtothesurfaceoftissueculturedishdur- type, mostculturedblastocystshatchedfromthezonapellu- properties (vanDeursenetal.,1996).Regardlessofthegeno- intercrosses atE3.5andthenanalyzedtheirinvitrogrowth Rae1 depletion,weharvestedblastocystsfromheterozygous heterozygous counterparts. morphologically indistinguishablefromtheirwild-typeand Rae1 isnotessentialfornuclearexportofmRNA were stainedforpoly(A) E8.5 embryonicoutgrowths from heterozygousintercrosses cells lackingthisproteinaccumulate mRNAintheirnuclei. Bachi etal.,2000;SabriandVisa,2000),weaskedwhether al., 1996;KraemerandBlobel,1997;Pritchardet1999; for nuclearexportofmRNA(Brownetal.,1995;Murphy et of Nup98toNPCs. and E),demonstratingthatRae1isnotneededforbinding charomyces cerevisiae shortly afterattachmentofRae1 post-mitotic trophoblastcells(Fig.1D).WeobservedICMs panded theirinnercellmass(ICM)onanunderlyingsheetof that NPCsealingdoesnotoccurinRae1 growths bytransmissionelectronmicroscopydemonstrated phy andWente,1996).Examinationofembryonicout- tion ofmembranousstructuresthatsealnuclearpores(Mur- Another featureofGle2p-deficientyeastcellsistheforma- ing thatknockoutcellshaveanormaldistributionofNPCs. mAb414, similartothatofcontrolcells(Fig.2C strong anduninterruptedrim-likelabelingoftheNEwith (Murphy andWente,1996).Rae1-depletedcellsshoweda detected incellsfromRae1 nucleus andthecytoplasm(Fig.2B).NoRae1stainingwas though significantamountsofRae1werealsofoundinthe prominently localizedtothenuclearenvelope(NE),al- 2001). Incellsfromcontrolembryonicoutgrowths,Rae1 a markerofthenuclearporecomplex(NPC)(Wuetal., mouse Rae1(188–368)andmonoclonalantibodymAb414, bryonic outgrowthswithapolyclonalantibodyagainst port (Fig.2A).First,wedoublestainedE7.5–E8.5em- ther investigatetheroleofRae1innucleocytoplasmictrans- On theotherhand,trophoblastcellsfromRae1 instead, degenerated(34outof144embryosdegenerated). these massesfailedtoexpandfromE6.5throughE8.5and, To furtherinvestigatethedevelopmentaldefectscausedby Because previousstudieshavelinkedRae1tothepathway causessevereclusteringofnuclearpores / andRae1 RNAbyaninsituhybridization / / blastocystsallowedustofur- outgrowths,confirmingthat RNAappearednormalin / / blastocystsatE5.5,but blastocystsrapidlyex- / embryos(Fig.2,D 50 -mer probe(Prit- / cells(unpub- GLE2 ), indicat- in /

blas-

Sac-

Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 (D) InvitrogrowthofrepresentativeRae1 to thecytoplasm whenRae1islacking. bulk ofmRNAs synthesizedinthenucleus can beexported outgrowths (E5.5–E7.5)werestainedforpoly(A) and hybridizedtoa3 identification ofwild-type(WT)andknockout(KO)Rae1allelesareshown.(B)SouthernblotgenomicEScellDNA,digestedwith BamH1 vector (middle),andthedisruptedRae1allele(bottom).BamHIrestrictionsites3 Figure 1. T, trophectodermcells. multi-well slidesinDME/15%FCS.Theywereinspecteddailyand photographedat48(E5.5),72(E6.5),and96h(E7.5)afterseeding. I,ICM; Rae1 normal subcellularlocalizations foralltheseproteinsin splicing-dependent mRNA-binding proteinY14showed hnRNP proteinsAandC2, the SRproteinSC35,and the mRNAexportfactorsTap (Fig.2,HandI)Aly,the Rae1. Immunolabelingexperiments withantibodiesagainst mRNA bindingproteinswouldbealteredintheabsenceof asked ifthesubcellulardistributionofvariouspre-mRNA/ not coincidewithnuclearaccumulationofmRNA.Wealso (unpublished data),revealingthatICMdegenerationdoes embryos fromaRae1 / cells(unpublisheddata).Thus, itappearsthatthe Targeted disruptionofthemouseRae1gene. / externalprobe,revealingtheexpected10.2-kbwild-typeand8.5-kb mutantfragments.(C)PCRgenotypingofE3.5 intercrossshowingwild-typeandknockoutallele-specificamplification productsofrespectively516and650bp. / andRae1 /

embryos.EmbryosfromheterozygouscrosseswererecoveredatE3.5 andgrownon (A)Indicatedarepartoftheendogenousmouse(m)Rae1gene(top),targeting

RNA

and Benezra,2001). Thisinformationprompted ustoana- ical forcellswith adefectivemitoticcheckpoint (Wassmann drug nocodazole(Micheletal., 2001),aresponsethatistyp- cultured inthepresenceof microtubule-depolymerizing in prometaphaseandexitmitosis withoutcytokinesiswhen one copyofthemitoticcheckpoint geneMad2failtoarrest sis. Othershaverecentlyshown thatHCT116cellswithonly terest todeterminewhetherRae1 wouldberequiredinmito- tinez-Exposito etal.,1999;Wang2001),itwasofin- Bub3 andcaninteractwithBub1(Tayloretal.,1998;Mar- Because Rae1hasahighdegreeofsequencesimilarityto checkpoint dysfunction Haplo-insufficiency attheRae1locuscausesmitotic Novel roleforRae1inthemitoticcheckpoint| DNAprobe(solidbar)usedforSouthernblot Babuetal.

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Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 344 Figure 2. of Rae1 (A) Overviewofthe experimentaldesign.Blastocysts fromintercrosses The JournalofCellBiology / micewerecultured for Rae1 isnotessentialfornuclear export ofmRNA.

Volume 160,Number3,2003 4–5 dandthenanalyzed by

into mediumwithhighserumandnocodazole,collected MEFs thatweresynchronizedinG1bylowserum,released we measuredthecyclinB–associatedCdc2kinaseactivityof as withthe4-hrecovery(unpublisheddata). extended to12h,essentiallythesameprofilewasobtained contained 4NDNAcontent.Whentherecoveryphasewas prematurely withoutdividingtoproduceacellinG1that sis inthepresenceofnocodazolehadexitedfrommitosis Rae1 that 15%ofRae1 each timepointusingfluorescencemicroscopy.Wefound mitotic index(definedasthepercentageofcells)at antibody toidentifymitoticcells,andthenmeasuredthe for 0,2,6,12,or24h,stainedwithphospho-histoneH3 spindle damage.Wetreatedcellswith200ng/mlnocodazole sured theresponseofRae1haplo-insufficientcellsto on therateofcellproliferation(Fig.3B).Wethenmea- that thereductionofRae1proteinhadnosignificantimpact Bub3 andMad2proteinlevelsweresimilarinRae1 presence ofnocodazolethanRae1 Rae1 and threeRae1 Rae1 zole. Wefirstintercrossedheterozygousmicetoderive lyze theresponseofRae1haplo-insufficientcellstonocoda- (F andG)Localizationofpoly(A) Shown arehigh-resolutionimages oftrophectodermcells. polyclonal antibodyagainstNup98(151–224) (Wuetal.,2001). (D andE)ImmunostainingofE8.5 embryonicoutgrowthswitha are representativehigh-resolution imagesoftrophectodermcells. antibody mAb414,amarkeroftheNPC(Wuetal.,2001).Shown mouse Rae1(188–368)(Pritchardetal.,1999)andmonoclonal E8.5 embryonicoutgrowthswithapolyclonalantibodyagainst immunostaining orinsituhybridization.(B–C’)Doublestainingof profile ofRae1 with 2NDNAcontent(Fig.3E,3).Incontrast,the cells wereabletocompletetheirmitosisandenterG1phase MEF cellsquicklyreturnedtonormalasmitotically-arrested without nocodazolefor4h,theDNAprofileofRae1 E, 2and5).Whencellswereallowedtorecoverinmedium with 4NDNAcontentirrespectiveofthegenotype(Fig.3 sure tonocodazolefor12hresultedinaccumulationofcells MEFs hadsimilarDNAprofiles(Fig.3E,1and4).Expo- protein inRae1 (Fig. 3E,6),indicatingthatRae1 human Tap(Braun etal.,1999). (H andI)Trophoblast cellsstainedwithapolyclonal antibodyagainst was usedforvisualizationofpoly(A) by flowcytometry.UnsynchronizedRae1 Rae1 point wasdefective.Consistentwiththisobservation, rested atthattimepoint,indicatingtheirmitoticcheck- In contrast,only2.5%ofhaplo-insufficientcellswerear- or 5.Forthestudiesdescribedbelow,atleastthreeRae1 at passages2and3usedforexperimentationpassage4 from individual13.5-d-oldfetuses.TheseMEFswerefrozen E8.5 Rae1

To furtherverifythelossofmitoticcheckpointactivity,

Western blotanalysisrevealedthattheamountofRae1 To confirmandextendtheseanalyses,weexaminedcells

/ / / /

andRae1

cells(Fig.3A).Growthratemeasurementsshowed celllines(Fig.3A);additionaltestsshowedthat culturesaccumulatedfarfewerroundedcellsinthe

andRae1

/ /

cloneswereexamined. / MEFcellsshowedonlylimitedrecovery

cellswasconsistentlylowerthanin

cellswerearrestedby12h(Fig.3C).

mouseembryonicfibroblasts(MEFs) /

outgrowths.AFITC–oligo(dT)

RNAintrophectodermcellsfrom

byinsituhybridization.

cellsthatenteredmito- cultures(Fig.3D). / andRae1 50 probe / and

/ / /

Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 and after4hofnocodazoleexposure. (E)RepresentativeFACS both Rae1 Michel etal.,2001).At24 h afterreleaseinnocodazole, at varioustimepoints(Wassmann andBenezra,1998; Figure 3. and Rae1 kinase activityofsynchronizedMEF cellsatindicatedtimepointsafterreleaseintonocodazole. Cdc2 kinaseactivity (indicatingthatthesynchronized cul- Mad2 proteinlevelsbyWesternblotting(4–20%polyacrylamidegel). 100 Rae1 are meansandstandarddeviationsderivedfromthreeRae1 monoclonal antibodyagainsthumanMad2thatrecognizesmouse Mad2(BDBiosciences).(B)GrowthcurvesofprimaryMEFs.Datashown analysis. Forprobes,weusedarabbitantibodyagainstmouseRae1(188–368), arabbitantibodyagainstmouseBub3(145–276),and amouse / MEFs(panels4–6).Durationsof nocodazole treatment(80ng/ml)andrecoveryintervalsareindicated. (F)CyclinB–associatedCdc2 / The mitoticcheckpointrequiresafullcomplementofRae1. MEFcelllines( / andRae1 n /

culturesshowed ahighlevelof 3foreachgenotype).(D)Representativephasecontrastimagesof Rae1 / andthreeRae1 ® profilesofpropidium-stainedunsynchronized Rae1 (A)AnalysisofRae1 tures hadreachedMphase;Fig. 3F).WhereasRae1 decline inkinase activityafter30h,confirming thatRae1 least 42hafter release,Rae1 tures maintainedahighlevel of Cdc2kinaseactivityuntilat g oftotalproteinextractfromeachMEFcelllinewasusedinthe / MEFlines.(C)Mitoticindexofnocodazole-treatedRae1 Novel roleforRae1inthemitoticcheckpoint| / andRae1 / / andRae1 / MEFlinesforRae1,Bub3,and culturesshowed amarked / / (panels1–3)and

MEFculturesbefore

Babuetal.

/ cul- / 345

Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 346 spectrum ofabnormalchromosomenumbersthanRae1 crosses, Bub3 Rae1 in Bub3 vidual embryos.Bub3protein levelswereconsistentlylower and generatedBub3 three independentRae1 blastocysts (Fig.1D)andBub3 cysts (unpublisheddata)weresimilartothoseofRae1 The growthabnormalitiesoftheseBub3-deficientblasto- blastocysts ataMendelianfrequency(unpublisheddata). and B).Bub3 amino acid191viahomologousrecombination(Fig.5, A the mouseBub3genebyinterruptingitscodingregion at cus wouldalsocauseamitoticphenotype.Weinactivated We thenaskedwhetherahaplo-insufficiencyattheBub3lo- failure andchromosomemissegregation Bub3 haplo-insufficiencycausesmitoticcheckpoint icantly increasedrateofchromosomemissegregation. findings showthatlossofasingleRae1alleleleadstosignif- Together,these Fig. 8,BandD). spreads (Fig.4B;seealso metaphases wassignificantlyhigherinRae1 MEFs atpassage5.Theaveragepercentageofaneuploid some countsonmetaphasespreadsfromRae1 on chromosomenumberstabilitybyperformingchromo- Next, wedeterminedtheeffectofRae1haplo-insufficiency chromosome missegregation Rae1 haplo-insufficiencypromotes haplo-insufficient cells. suggest thatthemitoticcheckpointisdefectiveinRae1 spindle damage.Takentogether,theaboveanalysesstrongly haplo-insufficient cellsfailtoarrestinmitosisresponse are fromthreeindependentRae1 cells inMEFculturesatpassage5.Values (A) Analysisofthepercentageaneuploid exhibit increasedchromosomalinstability. Figure 4. birth andatday8.5ofembryogenesis,butproducedBub3 this reduction did notaffectcellgrowth(unpublished data). type littermates(unpublisheddata).LikeRae1 per genotype. values fromthreeindependentMEFlines MEF lines.Datashownarecombined of wild-typeandRae1haplo-insufficient line. (B)Distributionofchromosomenumbers 50 metaphaseswerecountedforeachMEF poly(A) Bub3 hadnoimpacton the localizationandlevelof ments onE8.5outgrowthsconfirmedthatdisruptionof lier study(Kalitsisetal.,2000).Insituhybridizationexperi- 8, BandD).Inaddition,Rae1 We collectedE13.5embryos fromBub3 The JournalofCellBiology / MEFs(20 RNAinthecell(unpublished data). / Rae1 haplo-insufficientcells MEFsthanin Bub3 / / intercrossesyieldednoBub3 micewereindistinguishablefromwild- / / 2%vs.9 andBub3 MEFcultures.

Volume 160,Number3,2003 / and / / 1%; Fig. 4 A; see also Fig. 1%;Fig.4A;seealso spreadsshowedabroader / blastocystsfromanear- / MEFs(Fig.5 C),but MEFlinesfromindi-

/ / / MEFsthanin andRae1 intercrosses / / miceat inter- / / / / 9 than inBub3 totic indexofBub3 Consistent withalossofmitoticcheckpointactivity,themi- tures (Fig.7A).Asexpected, Rae1 and (b)Bub3 totic phenotypeisadirectconsequenceoftheRae1mutation) Rae1 haplo-insufficiency(whichwouldbeexpectedifthemi- ectopic expressionofRae1wouldbeabletocorrectfor(a) the pMSCV-Puroretroviralvectorandusedittotestwhether Next, weclonedHAepitope–taggedmouseRae1cDNAinto of Bub3haplo-insufficientcells Overexpression ofRae1rescuesthecheckpointdefect spreads, Bub3 number instability. checkpoint andestablishesahigherrateofchromosome veal thatthelossofasingleBub3alleleperturbsmitotic Collectively,thesestudiesre- Fig. 8,BandD). 6 B;seealso nonmodal chromosomenumbersthancontrolspreads(Fig. analysis confirmedthatnocodazole-exposedBub3 the presenceofnocodazole(Fig.5,DandE).DNAprofile Rae1 point activity,themitotic index ofHA–Rae1-expressing Consistent withareestablishment ofnormalmitoticcheck- point inresponsetonocodazole-induced spindledamage. tested thesecellsfortheirabilitytoactivatethemitoticcheck- 1999), selectedtheculturesforpuromycin-resistantcells,and leased innocodazoledeclinedmuchfasterBub3 Cdc2 kinaseactivityofsynchronizedMEFsthatwerere- (Fig. 5F).Furthermore,asexpected,thecyclinB–associated leave mitosisprematurelywithoutundergoingcytokinesis Bub3 percentage ofaneuploidcellswassignificantlyhigherin Chromosome countsonmetaphasespreadsrevealedthatthe affects thefidelityofchromosomesegregationinMphase. mitotic checkpoint activity.Strikingly,HA–Rae1 wasalsoca- empty pMSCV-Puro vectorremainedunable tosustaintheir the HA–Rae1expressionvectorintotwoRae1 that Rae1hasfunctionaloverlapwithBub3).Weintroduced Bub3 Next, wedeterminedwhetherBub3haplo-insufficiency 3%; Fig. 6 A; see also Fig. 8, B and D). LikeRae1 Fig. 8,BandD). 3%;Fig.6A;seealso / / / culturesshowedasimilarincrease asRae1 MEFlinesbyretroviralgenetransfer(Kasperetal., culturesthaninBub3 / / / cells(Fig.5G). haplo-insufficiency(whichwouldindicate spreadsdisplayedabroaderspectrumof / cultureswasnotmuchincreasedin / cultures(21 / cellstransducedwith / andtwo 2%vs. / / / cells cells cul-

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The Journal of Cell Biology Published January27,2003 7-kb wild-typeand5.5-kbmutant bands.(C)WesternblotanalysisofBub3proteinlevelsin Bub3 indicated. (B)Southernblotofgenomic mousetailDNA,digestedwithBamH1andhybridizedtoa 5 restriction sitesandthe5 Shown ispartoftheendogenousmouse(m)Bub3gene(top),targeting vector(middle),andthedisruptedmBub3allele(bottom). BamHI Figure 5. to mouseBub3(145–276)(Wang etal.,2001).(D)Mitoticindexofnocodazole-treatedBub3 Bub3 indicated timepointsafterrelease intonocodazole. / / cells(panels4–6)aftertheindicated treatments.(G)CyclinB–associatedCdc2kinaseactivityofsynchronized MEFculturesat andBub3 Disruption ofasingleBub3alleleleadstomitoticcheckpointdysfunction. / MEFculturesbeforeandafternocodazole treatment.(F)DNAcontentsofasynchronousBub3 DNAprobe(solidbar)usedforSouthernblotidentificationofwild-type (WT)andknockout(KO)Bub3allelesare Novel roleforRae1inthemitoticcheckpoint| (A)TargetedinactivationofthemouseBub3gene. / andBub3 / andBub3 externalprobe,revealingtheexpected / MEFcelllines.(E)Imagesof / MEFlinesusinganantibody / (panels1–3)and Babuetal.

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Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 348 Rae1 treated Bub3 index ofnocodazole-treatedRae1 activation inbothRae1andBub3 haplo-insufficientcells. Figure 7. by limitedavailabilityofBub3molecules. for Bub3-mediatedmitoticcheckpointactivitythatisaffected duced Bub3activity(Fig.7B).Thus,Rae1cancompensate pable ofrestoringthemitoticcheckpointdefectcausedbyre- Figure 6. activity isrestored. mitotic cellsover time,illustratingthattheirmitotic checkpoint cell lines.NotethatHA–Rae1-expressing culturesaccumulate three independentBub3 Values arefromthreeindependentBub3 degree ofaneuploidyinMEFculturesatpassage5. chromosome numberinstability. (combined valuesofeachgenotype). of wild-typeandBub3haplo-insufficientMEFlines culture. (B)Distributionofchromosomenumbers metaphase spreadswerecountedforeachMEF The JournalofCellBiology / /HA–Rae1 ( HA–Rae1 expressionrestoresmitotic checkpoint Bub3 / /empty vector( / n cellsdisplayincreased

2)celllines.(B)Mitoticindexofnocodazole- / MEFcultures.50 | n

Volume 160,Number3,2003

(A)Analysisofthe / 2)andBub3 /empty vector( / and / /HA–Rae1 ( n 2)and (A)Mitotic n

2) Rae1 present in13%ofmitoticfigures fromRae1 thermore, prematurelyseparated sisterchromatidswere pared fromcrossesofRae1 duction inbodymass(unpublisheddata).MEFswerepre- heterozygotes hadanormalappearanceanddisplayednore- gous mice(53among214totaloffspring).Thesecompound mice ledtonormalnumbersofRae1/Bub3doubleheterozy- WefoundthatcrossesofRae1 insufficiency. analyzed thecombinedeffectsofRae1andBub3haplo- To furtherdefinetherolesofRae1andBub3inmitosis,we accelerates geneticinstability Combined Rae1andBub3haplo-insufficiency cells andmice. and Bub3accelerates chromosomalinstability incultured defect. Therefore,combined haplo-insufficiencyofRae1 mice. Asexpected,theother genotypesdidnotshowthis higher percentagethanwild-typecells(2%)( of Rae1 was muchhigherinRae1 percentage ofanaphasefigureswithlaggingchromosomes haplo-insufficient cells(Fig.8,BandC).Inaddition,the cient cells,butessentiallynoneinmitoticfiguresfromsingle tids in and D).Weobservedprematurelyseparatedsisterchroma- turn hadabroaderspectrumthanwild-typecells(Fig.8,B heterozygous cellsthaninsinglecells,which modal chromosomenumberswasmuchlargerindouble cultures(Fig.8B).Notably,therangeofnon- insufficient measured incorrespondingRae1andBub3singlehaplo- B). Thiswassignificantlyhigherthanthe10%increases had increasedby32%relativetowild-typecultures(Fig.8 age ofaneuploidcellsindoublehaplo-insufficientcultures chromosome countsatpassage5revealedthatthepercent- type andsingleheterozygousMEFs(Fig.8A).Furthermore, haplo-insufficient MEFsgrewsignificantlyslowerthanwild- contrast, 9%ofRae1 E). Noaneuploidywasobservedinwild-typesplenocytes.In mice pergenotype)andpreparedmetaphasespreads(Fig. 8 type, Rae1 adult mice,wecollectedsplenocytesfrom5-mo-oldwild- anaphases pergenotype). To investigatethedegreeofchromosomalinstabilityin / (5%)orBub3 19% ofmitoticfiguresfromdoublehaplo-insuffi- / /Bub3 / , Bub3 / splenocytesshowedaneuploidy. Fur- / / , andRae1 andBub3 / (5%)cells,whichinturnhada / / /Bub3 andBub3 / / / /Bub3 splenocytesand37% cells(15%)thanin / / mice.Double / andBub3 mice( / n /Bub3

n 200

3 / /

Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 were foundinthewild-type, Rae1 old micepergenotypeforspontaneous tumors.Notumors (Fig. 9C).Ascontrols,weexamined10nontreated5-mo- B), aswastheaveragenumberoftumorspertreatedanimal increased comparedwiththeirwild-typecounterparts(Fig. 9 for possibledevelopment ofspontaneoustumors astheyage. mice pergenotype (currentlyranginginage from 2to7mo) lungtumorigenesis.Wearemonitoringover50 induced checkpoint–defective mice are predisposedtochemical- mor. Takentogether,theabove dataindicatethatmitotic morigenesis, wegavepupsfromRae1 mice haveincreasedsusceptibilitytocarcinogen-inducedtu- To determinewhethersingleanddoublehaplo-insufficient with chromosomalinstability Increased tumorformationinmice 50 metaphasespreadspermouse.100mousewerescreenedforPMSCS. various genotypes.Datashownaremeansandstandarddeviationsderivedfromthreemicepergenotype.Wecountedthechromosomes of heterozygous MEFlines(combinedvaluesofeachgenotype).(E)AneuploidyandPMSCSinprimarysplenocytesfrom5-mo-oldmice of double heterozygoteMEF(right).(D)Comparisonofchromosomenumberdistributionsbetweenwild-type,singleheterozygous,and double sister chromatidseparation(PMSCS).(C)Metaphasespreadsshowingcohesioninawild-typeMEF(left)andPMSCS ina prematureWe countedthechromosomesof50metaphasespreadsperindependentMEFline.200mitoticfigureslinewerescreenedfor able inthelungs(Fig.9A).InRae1 tive ofthemousegenotype,tumorswereexclusivelydetect- mice werekilledandscreenedfortumorformation.Irrespec- (Serrano etal.,1996).5moafterDMBAtreatment,the DMBA inacetonetothedorsalsurfaceonpost-natalday 5 crosses asingleapplicationof50 whereas 1ofthe10Rae1 Rae1 / /Bub3 / mice,theincidenceoflungtumorswas / /Bub3 l ofasolution0.5% / / micehadalungtu- , orBub3 / xBub3 / , Bub3 / / / mice, inter- , and from 13.5-d-oldembryoscollectedRae1 lines pergenotype.(B)ChromosomeanalysisofMEFs(atpassage5) means andstandarddeviationsderivedfromthreeindependentMEF Rae1 aneuploidy. Figure 8. fertilized byBub3 harvested fromRae1 deviations derivedfromthreeindependentMEFlinespergenotype. accumulation ofpoly(A) nuclear accumulationofpoly(A) Nup98 (amotifthatbindsto andinhibitsRae1)resultsin observation thatoverexpressionoftheGLEBSmotif mRNA exportinhighereukaryotes.However,theprevious the combineddatawouldbethatRae1isnotimplicatedin munication). Themoststraightforwardinterpretationof pleted byRNAinterference(E.Izaurralde,personalcom- RNA occursinhumancellswhichRae1expressionisde- pombe Although inactivatingmutationsinthe Rae1 andglobalmRNAexport Discussion with thisfinding,nonuclearaccumulationofpoly(A) tial forbulkmRNAexportinmammaliancells.Consistent not inducesuchadefect,indicatingthatRae1isessen- phy etal.,1996),similargeneticexperimentsinmicedo Rae1 ispresent inthecellasaRae1–GLEBS complex. haps suchcompensatory actionmaynotbe possiblewhen sated forbyanothertransport factorwhenitisabsent.Per- that Rae1isanmRNAexport factorthatcanbecompen- lian mRNAexportpathway. Oneattractivepossibilityis chard etal.,1999)arguesfor aroleofRae1inthemamma- / , Bub3 and Novel roleforRae1inthemitoticcheckpoint| Rae1 synergizeswithBub3inthedevelopmentof (A)GrowthcurvesofMEFsderivedfromwild-type, S. cerevisiae / , andRae1 / males.Datashownaremeansandstandard / xBub3 homologuesofRae1causenuclear / RNA(Brownetal.,1995;Mur- /Bub3 / intercrosses).Datashownare / RNAinHeLacells(Prit- 13.5-d-oldembryos(all / Schizosaccharomyces femalesthatwere Babuetal.

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Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 350 cient cellsexhibit astrikinglysimilarmitotic phenotype, segregation. In addition,weshowthatBub3 haplo-insuffi- sults inamitoticcheckpoint defectandchromosomemis- In thisstudy,weshowthathaplo-insufficiency ofRae1re- Mechanism ofcheckpointregulation byRae1 port ofonlyasmallsubset mRNAs. does notexcludethepossibilitythatRae1isessentialforex- Lastly, wewouldliketoemphasizethatourgeneticstudy Rae1 mice. Figure 9. wild-type micebyWilcoxonranksumtest. mouse ( Chi-squared test).(C)Theaveragenumberoflungadenomasper significantly increasedrelativetowild-typemice(P incidence ofthecheckpoint-defectivegroupmiceasawholeis with wild-typemicebyChi-squaredtest.Wenotethatthetumor as percentageincidence.TheasteriskindicatesP tumors. (B)Theoccurrenceoflungtumorsin5-mo-oldmiceplotted lung tumor(right;10 The JournalofCellBiology / (A)GrossphotographoflungtumorsaDMBA-treated /Bub3 SEM). TheasterisksindicateP DMBA-induced tumorformationincheckpoint-defective / mouse(left),andasectionofRae1 , hematoxylin |

Volume 160,Number3,2003 eosin). Arrowspointoutthe 0.05comparedwith 0.05compared 0.05by / /Bub3 /

the formationofcompensatoryRae1–BubR1. correct Bub3haplo-insufficientcellsisunlikelytoinvolve 2001), themechanismbywhichRae1overexpressionactsto cause Rae1doesnotinteractwithBubR1(Wangetal., (Sudakin etal.,2001;TangFang,2002).Be- sol seemtoplayacriticalroleininhibitingtheAPC(Fig.10) and Bub3–BubR1complexespositionedinthemitoticcyto- 10). Bub3alsointeractswithBubR1(Tayloretal.,1998), the functionsofmissingBub3–Bub1complexes(Fig. Rae1–Bub1 complexesthatwouldbecapableofperforming result fromtheassemblyof“unbound”Bub1moleculesinto cells byoverexpressionofHA-taggedRae1couldthensimply rection ofthemitoticcheckpointinBub3haplo-insufficient fulfill redundantfunctionsatunattachedkinetochores.Cor- cells couldbethatRae1–Bub1andBub3–Bub1complexes typic similaritybetweenRae1andBub3haplo-insufficient (Wang etal.,2001),onepossibleexplanationforthepheno- Bub1 complexescoexistinprometaphase-arrestedHeLacells (Wang etal.,2001).GiventhatbothRae1–Bub1andBub3– Bub1 servesasasharedbindingmotifforRae1andBub3 with Bub1.WehaveshownthattheGLEBSsequenceof Bub3 alsotargetstounattachedkinetochoresandinteracts naling eventsasaproteincomplex.Interestingly,likeRae1, possible thatRae1andBub1mightparticipateinsuchsig- tosis andcanbindtoRae1(Wangetal.,2001),itisfurther protein Bub1alsotargetstounattachedkinetochoresinmi- phase waitsignalsatthatposition.Giventhecheckpoint sible thatRae1mightplayaroleintheproductionofana- ochores attheonsetofmitosis(Wangetal.,2001),itispos- Because Rae1hasbeenshowntolocalizeunattachedkinet- Rae1 beabletocompensateforBub3haplo-insufficiency? lates theactivityofmitoticcheckpointandhowmay function aswell. both proteinshaveacritical,nonredundantphysiological Rae1 andBub3miceareembryonicallylethalillustratesthat erozygous knockoutmiceareviablewhereashomozygous vation mayberedundant,thefindingthatcompoundhet- the criticalfunctionsofRae1andBub3incheckpointacti- insufficiency stronglysupportsthisconclusion.Although only forRae1haplo-insufficiencybutalsoBub3haplo- The findingthatectopicexpressionofRae1cancorrectnot suggesting thatRae1mayactinananalogouswaytoBub3. that hypomorphic mutationsinthe supported bya previousstudyofBasuetal. (1999) showing idea thatthemechanismof synergy mayinvolveBub1is Bub1 complexesatunattached kinetochores(Fig.10).The 2001), suchasonethatinvolves Rae1–Bub1andBub3– a commonstepofthecheckpoint pathway(Hartmanetal., insufficiency suggeststhatRae1 andBub3mightbeactingat Bub3? ThefactthatRae1can compensateforBub3haplo- could bethemechanismofsynergybetweenRae1and cal forthepreventionofchromosomalmissegregation.What combination ofthetwomitoticcheckpointactivitiesiscriti- ing indicatesthatRae1cooperateswithBub3andthe single haplo-insufficiencyofeithergene(Fig.8).Thisfind- much moreseverechromosomalinstabilityphenotypethan Combined haplo-insufficiencyofRae1andBub3causes a Synergy betweenRae1andBub3 What thencouldbethemechanismbywhichRae1regu- Drosophila

Bub1gene

Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 expression ofMad2 arepronetodevelopspontaneous lung dition, aprevious studyhasshownthatmice withreduced predisposes micetocarcinogen-induced lungtumors.Inad- study showsthatsubnormalexpression ofRae1and/orBub3 causing subnormalexpression of itscomponents.Thepresent molecular backupsystemwith exquisitesensitivitytoevents pears thatthemammalianmitotic spindlecheckpointisa chromosomal instability(Michel etal.,2001).Thus,itap- insufficiency disturbsthemitoticcheckpointandcauses Similar toRae1andBub3haplo-insufficiency,Mad2haplo- Mitotic checkpointdefectsandtumorigenesis tic action(HerschlagandJohnson,1993). Rae1 andBub3maynotbearequirementfortheirsynergis- et al.,2001)suggeststhataphysicalinteractionbetween protein complexescontainingbothRae1andBub3(Wang Rae1/Bub3 mutants.Thefactthatwewereunabletodetect cause aphenotypethathasremarkablesimilaritytoof the componentsofcheckpointisshowninmodel. when anaphasewaitsignalsarepresent.Forsimplicity,onlypartof checkpoint complexthatactstoinhibittheAPCactivatorCdc20 with BubR1andlocalizestothecytosoliccomponentofmitotic producing anaphasewaitsignals.UnlikeRae1,Bub3alsointeracts proteins acttoactivatethemitoticcheckpoint,potentiallyby together withBub1,justlikeBub3.There,Rae1–Bub1andBub3–Bub1 Rae1 targetstounattachedkinetochoresattheonsetofmitosis Figure 10. Model forRae1functioninmitosis. Weproposethat bp fortheknockoutallele. sulting PCRfragmentsmeasured516bpforthewild-typealleleand650 combination 1and3(5 3), thosespecificfortheknockoutRae1allelewereamplifiedwithprimer CGCCCTTGG-3 tinely processedforhistopathologicalconfirmation. overt tumorsusingadissectionmicroscope.Harvestedwererou- ously described(Serranoetal.,1996).Allmajororganswerescreenedfor ES cellclonesthroughstandardprocedures.DMBAtreatmentwasasprevi- genomic DNA(Fig.5,AandB).Mutantmicewerederivedfromtargeted gene activityandchromosomalinstability. sights intothecausalrelationshipbetweenmitoticcheckpoint tors suchasRae1andBub3shouldprovideimportantin- tumors fordecreasedexpressionofmitoticcheckpointregula- carcinomas (Shichirietal.,2002).Furtheranalysisofhuman ically down-regulatedinasubstantialproportionofhuman point genesBub1andBubR1showedthattheyareepigenet- Consistent withthisidea,arecentstudyofthemitoticcheck- cers withchromosomalinstabilitythanpointmutations. that theseeventsmayactuallyplayamoreactiveroleincan- checkpoint genescouldbedown-regulated,itisconceivable chromosomes areplausiblemechanismsbywhichmitotic al., 2001).However,asepigeneticeventsorlossesofwhole mosomal instability(Cahilletal.,1998,1999a;Hernando totic checkpointgenesarerareinhumantumorswithchro- studies haveshownthatinactivatingpointmutationsinmi- expression andtumorprogressionarecausallyrelated.Several these datasuggestthatdecreasedmitoticcheckpointprotein tumors afterlonglatencies(Micheletal.,2001).Together, Rae1 genewereamplifiedbyusingprimer1(5 detail (vanDeursenetal.,1996).Sequencesspecificforthewild-type growths forgenotypeanalysiswasperformedaspreviouslydescribedin microscope slidesfor2–5d.Processingofembryosandembryonicout- males at3.5dpost-coitumandculturedinDMEwith15%FCSon10-well for 0,2,6,12,or 24 h.Subsequently,cellswereimmunostained withan ng/ml nocodazole (Micheletal.,2001)wasaddedto the culturemedium clones wereidentifiedbySouthernblottingusinga5 al., 1996),interruptingtheBub3geneataminoacid191.TargetedEScell 1.4-kb SacIfragmentwasreplacedbyapGKneocassette(vanDeursenet contained an The Rae1targetingvectorcontaineda Generation ofknockoutmiceandDMBAtumorigenictreatment Materials andmethods croscope slides(5,000cellsperchamber of0.4cm To determinethemitoticindex,MEF cellswereplatedinchamberedmi- Mitotic indexandDNAprofileanalysis performed atpassage4.Atday0,10 nance ofMEFsoccurredaccordingtoa3T9schedule.Growthcurveswere casses aspassage1andthefirstreplating2.Routinemainte- mijo etal.,1997).Wecountedtheseedingoftrypsinizedembryocar- MEFs wereisolatedfrom13.5-d-oldembryosaspreviouslydescribed(Ka- Generation andcultureofMEFs Blastocysts wererecoveredfromtheuterinehornsofpluggedRae1 In vitrocultureandPCRgenotypingofblastocysts reentry intothecellcycle. MEFs weretrypsinizedandreseeded inDMEwith10%FBStoallowtheir tured inDMEcontaining0.1%FBS and0.04%BSAfor18h.Quiescent brief, confluentcultureswerewashedthreetimeswithPBSandthencul- MEFs weresynchronizedaspreviouslydescribed(Chengetal.,1999). In eter dish,andduplicatecultureswerecountedat24-hintervalsthereafter. ing a3 1996). WeidentifiedtargetedEScellclonesbySouthernblotanalysisus- tagenesis ofEScellswasaspreviouslydescribed(vanDeursenetal., IRES-lacZ-neo selectioncassette(vanDeursenetal.,1996).Targetingmu- fragment inwhichauniqueEcoRVsitewasusedtoinsertpromoterless probeonBamHI-cutgenomicDNA(Fig.1B).TheBub3construct Novel roleforRae1inthemitoticcheckpoint| 8.5-kb Bub3129Sv/JgenomicDNAfragmentinwhicha ) andprimer2(5 -GGGCTGACCGCTTCCTCGTGCTTT-3 -TACATCATTCGCCCATGATCCTGC- 5 MEFswereplatedper3.5-mm-diam- 9-kb Rae1129Sv/JgenomicDNA -GCCTTGTGCAAATGC- probeonBamHI-cut 2 ). Thenextday,200 Babuetal. ). There- /

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Downloaded from from Downloaded on November 17, 2010 17, November on jcb.rupress.org The Journal of Cell Biology Published January27,2003 352 Blajeski, A.L.,V.A.Phan,T.J.Kottke,and S.H.Kaufmann.2002.G(1)andG(2) Basu, J.,H.Bousbaa,E.Logarinho,Z. Li, B.C.Williams,C.Lopes,C.E.Sunkel, Bailer, S.M.,S.Siniossoglou,A.Podtelejnikov,Hellwig,M.Mann,and E. Drug selectionwasin1.25 taining retroviruses,aspreviouslydescribedindetail(Kasperetal.,1999). then transducedthemwithpMSCV-Puro-HA–Rae1orpMSCV-Purocon- pact onthemitoticcheckpointactivityinMEFlinesused;Fig.7),and ized themwithsimianvirus40largeTantigen(thisviralproteinhasnoim- lected onewild-type,twoRae1 (Blajeski etal.,2002).ForRae1correctionexperiments,werandomlyse- pidium iodide,andsubjectedtoflowcytometryasdescribedpreviously in 95%ethanol,treatedwith1mg/mlRNaseA,stainedpro- and nonadherentMEFcellswerepooled,washedwithice-coldPBS,fixed phospho-histone H3–positivecells).ForDNAcontentanalysis,adherent cells perwelltocalculatethemitoticindex(definedaspercentageofanti– number ofHoechst-stainednucleiandtheanti-H3–positive Hoechst staining.Usingafluorescencemicroscope,wecountedboththe cells) aspreviouslydescribed(Kasperetal.,1999).DNAwasvisualizedby antibody againstphospho-histoneH3(acommonlyusedmarkerformitotic was usedin1:500dilution. scribed (vanDeursenetal.,1996).Anti-Tapantiserum(Braun1999) Bachi, A.,I.C.Braun,J.P.Rodrigues,N.Pante,K.Ribbeck,C.VonKobbe, U. References Accepted: 19December2002 Revised: 19December2002 Submitted: 13November2002 National InstitutesofHealthgrants RO1 CA77262-01 andRO1CA96985-1. and anti-Tapantibody. cussions. WethankElisaIzaurraldeforprovidingunpublishedRNAidata and FergusCouchforcriticallyreviewingourmanuscripthelpfuldis- We areverygratefultoourcolleaguesRickBram,PaulBrindle,TimYen, Immunostainings andpoly(A) Immunofluorescence andstainingforpoly(A) mann andBenezra,1998). kinase assaysonhistoneH1substratewereaspreviouslydescribed(Wass- al., 1999).ImmunoprecipitationofcyclinB–associatedCdc2andinvitro Western blotanalyseswereperformedaspreviouslydescribed(Kasperet and invitrokinaseassay Western blotanalysis,immunoprecipitation, To preparemetaphasespreadsfromMEFs,1–2 Karyotype analyses using a100 in 5%Giemsasolution,andanalyzedonanOlympusAX70microscope iquots weredroppedontoprewettedmicroscopeslides,stainedfor10min acetic acid),washed,andfinallyresuspendedin for 30min.CellswerefixedinCarnoy’ssolution(75%methanol,25% cells wereharvested,suspendedin5ml0.075MKCl,andincubatedatRT cultured for6–7hat37 U/ml), PHA(5 rpm for5min,resuspendedin4mlofRPMIcontaining10%FBS,IL-2(10 slides. Releasedcellsweresuspendedin5mlPBS,centrifugedat1,000 spleens werefreshlycollectedandmincedbetweentwomicroscope prepare metaphasespreadsfromsplenocytes(Rudolphetal.,1999), were treatedwith0.05 X. Wu,J.R.Babu,K.Jeganathan,andJ.vanDeursenweresupportedby The JournalofCellBiology cancer cells. cell-cycle arrestfollowing microtubuledepolymerizationin humanbreast Drosophila gene bub1causechromosomemissegregation andfailtoblockapoptosisin and M.L.Goldberg.1999.Mutations in theessentialspindlecheckpoint gle2p. served motifwhichconstitutesadockingsiteforthemRNAtransportfactor Hurt. 1998.Nup116pandnup100pareinterchangeablethroughacon- promotes exportofspecificCTE-bearingRNAsubstrates. The C-terminaldomainofTAPinteractswiththenuclearporecomplexand Kutay, M.Wilm,D.Gorlich,Carmo-Fonseca,andE.Izaurralde.2000. EMBO J. objective. . J.CellBiol. g/ml), conA(5 J. Clin.Invest. 17:1107–1119. g/ml colcemid(GIBCOBRL)for4–5hat37 C. Aftercolcemidtreatment,MEForsplenocyte 146:13–28. g/ml puromycin(Sigma-Aldrich)for5d. 110:91–99. | RNAdetectionwereaspreviouslyde- / g/ml), andcolcemid(0.05

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