Crossregulation and functional redundancy between the splicing regulator PTB and its paralogs nPTB a.docx
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Crossregulation and functional redundancy between the splicing regulator PTB and its paralogs nPTB a.docx
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CrossregulationandfunctionalredundancybetweenthesplicingregulatorPTBanditsparalogsnPTBa
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PubMedarticlesby:
Spellman,R.
Llorian,M.
Smith,C.
MolCell.2007August3;27(3):
420–434.
doi:
10.1016/j.molcel.2007.06.016.
Copyright©2007ElsevierInc.
CrossregulationandFunctionalRedundancybetweentheSplicingRegulatorPTBandItsParalogsnPTBandROD1
RachelSpellman,1MiriamLlorian,1andChristopherW.J.Smith1
1DepartmentofBiochemistry,UniversityofCambridge,80TennisCourtRoad,CambridgeCB21GA,UK
CorrespondingauthorEmail:
cwjs1@cam.ac.uk
ReceivedDecember19,2006;RevisedMay11,2007;AcceptedJune12,2007.
Thisdocumentwaspostedherebypermissionofthepublisher.Atthetimeofthedeposit,itincludedallchangesmadeduringpeerreview,copyediting,andpublishing.TheU.S.NationalLibraryofMedicineisresponsibleforalllinkswithinthedocumentandforincorporatinganypublisher-suppliedamendmentsorretractionsissuedsubsequently.Thepublishedjournalarticle,guaranteedtobesuchbyElsevier,isavailableforfree,onScienceDirect,at:
http:
//dx.doi.org/10.1016/j.molcel.2007.06.016
ThisarticlehasbeencitedbyotherarticlesinPMC.
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Summary
Introduction
Results
Discussion
ExperimentalProcedures
SupplementalData
NoteAddedinProof
References
Summary
Amongthetargetsoftherepressivesplicingregulator,polypyrimidinetractbindingprotein(PTB)isitsownpre-mRNA,wherePTB-inducedexon11skippingproducesanRNAsubstratefornonsense-mediateddecay(NMD).ToidentifyadditionalPTB-regulatedalternativesplicingevents,weusedquantitativeproteomicanalysisofHeLacellsafterknockdownofPTB.ApartfromlossofPTB,theonlychangewasupregulationoftheneuronallyrestrictednPTB,resultingfromdecreasedskippingofnPTBexon10,asplicingeventthatleadstoNMDofnPTBmRNA.ComparedwithknockdownofPTBalone,simultaneousknockdownofPTBandnPTBledtolargerchangesinalternativesplicingofknownandnewlyidentifiedPTB-regulatedsplicingevents.Strikingly,thehematopoieticPTBparalogROD1alsoswitchedfromanonproductivesplicingpathwayuponPTB/nPTBknockdown.OurdataindicatecrossregulationbetweenPTBanditsparalogsvianonproductivealternativesplicingandalargedegreeoffunctionaloverlapbetweenPTBandnPTB.
Keywords:
RNA
Top
Summary
Introduction
Results
Discussion
ExperimentalProcedures
SupplementalData
NoteAddedinProof
References
Introduction
Alternativesplicing(AS)allowsmultiplemRNAisoformstobeproducedbyindividualgenes.Thesecommonlyencodedistinctproteinisoforms,therebyenrichingtherepertoireofproteinsavailabletoorganisms(Black,2000;Lareauet al.,2004;ManiatisandTasic,2002;Matlinet al.,2005).AScanalsoquantitativelycontrolgeneexpressionbyproducingmRNAsthataredegradedbynonsense-mediateddecay(NMD)(Greenet al.,2003;Hillmanet al.,2004),aphenomenonthatappearstobewidespreadamongsplicingregulatorsthemselves(Lareauet al.,2007;Niet al.,2007)andwhichwerefertohereasAS-NMD.
Alternativesplicingdecisionsareinfluencedbyenhancersandsilencercis-actingelementsthatmediatetheiractivitybybindingactivatorandrepressorproteins.ActivatorsarecommonlymembersoftheSRfamilyofproteins(Graveley,2000),whereashnRNPproteinsoftenactasrepressors.Oneofthebetter-investigatedmammalianrepressorproteinsispolypyrimidinetractbindingprotein(PTB,alsoknownasPTBP1andhnRNP-I,reviewedinWagnerandGarcia-Blanco,2001).PTBrepressesmanyexons,includinganumberofmuscleandneuron-specificexons(e.g.,AshiyaandGrabowski,1997;ChanandBlack,1997;Perezet al.,1997;Wollertonet al.,2001).PTBbindstosplicingsilencerswithcoresequencesconsistingofUCUU,CUCUCU,orrelatedmotifs(ChanandBlack,1997;Perezet al.,1997).Insomecases,asinglesilencerissufficienttomediatePTBrepression(Izquierdoet al.,2005;Shenet al.,2004),butmostPTB-regulatedexonshavemultiplePTBbindingsites.MostmammalscontaintwoPTBparalogsthatareexpressedinatissue-restrictedmanner.nPTB/brPTB/PTBP2isexpressedmainlyinneurons(Kikuchiet al.,2000;Markovtsovet al.,2000;Polydorideset al.,2000),whereasROD1isexpressedinhematopoieticcells(Yamamotoet al.,1999).Rodentscontainanadditionalparalog,smPTB(Goodinget al.,2003).PTBanditsparalogsshare>70%aminoacidsequenceidentityandacommonarrangementoffourRRM-typedomains.TheactivityofnPTBhasbeenmostextensivelystudiedinthecontextoftheneuron-specificsplicingoftheN1exonofSRCwhereitislessrepressivethanPTB(Markovtsovet al.,2000).Nonetheless,itislikelythatnPTBandtheotherparalogsactasrepressorsofatleastsomeexons(Polydorideset al.,2000).
PTBitselfissubjecttoalternativesplicing.Inclusionorskippingofexon9producesthePTB1andPTB4isoforms,whichcanvaryinactivity(RobinsonandSmith,2006;Wollertonet al.,2001).Incontrast,exon11skippingproducesaframeshiftedmRNAthatisdegradedbyNMD(Wollertonet al.,2004).PTBproteinitselfpromotesPTBexon11skippinginanautoregulatoryfeedbackloop.TheequivalentnPTBexonisalsoskipped(Rahmanet al.,2002;Tateiwaet al.,2001)andhasasimilararrangementofpotentialregulatoryelements,suggestingthatitcouldalsoberegulatedbyPTBand/ornPTB(Goodinget al.,2006;Wollertonet al.,2004).IndeednPTBexon10lieswithinoneofthe genomicultraconservedelementsthathavebeenassociatedwithAS-NMDevents(Lareauet al.,2007;Niet al.,2007).
Arangeofglobalapproacheshaverecentlybeenharnessedwiththeaimofdeciphering“cellularcodes”composedofparticularcomplementsofsplicingregulatorsand“RNAcodes”composedofparticulararrangementsofregulatorysequencemodulesthattogetherdefinecell-specificprogramsofsplicing(reviewedinBlencowe,2006;Matlinet al.,2005).AtypicalapproachinvolvesidentificationofthesetofRNAsboundbyaregulatoryfactor(e.g.,Uleet al.,2003)orthesetofsplicingeventsaffectedbythefactor.ThelattercanbeidentifiedbyperturbingthecellularlevelsofthesplicingregulatorandthenanalyzingRNAwithsplice-sensitivemicroarrays(Blanchetteet al.,2005;Johnsonet al.,2003;Panet al.,2004;Relogioet al.,2005;Sugnetet al.,2006;Uleet al.,2005).SuchanalysishasbeencarriedoutforDrosophilaSRandhnRNPproteins(Blanchetteet al.,2005)andthemammalianneuron-specificNovaproteins(Uleet al.,2005).Asacomplementaryapproach,wedecidedtousequantitativegel-basedproteomicstoanalyzetheconsequencesofPTBknockdown.Ideally,alterationsinthelevelsofalternativelysplicedisoformswouldappearaspairsofreciprocallyvaryingspotson2Dgels.AlterationsinindividualspotswouldbeconsistentwithAS-NMDevents(Wollertonet al.,2004)orwithPTB'sdocumentedrolesatotherlevelsofgeneexpression,including3′endprocessing(Castelo-Brancoet al.,2004;Moreiraet al.,1998),regulationoftranslation(Mitchellet al.,2005),andRNAstability(Hamiltonet al.,2003;Pautzet al.,2006).
DespitetheweightofevidenceforthewidespreadrolesofPTB,weobservedverylittleeffectofPTBRNAiupontheHeLacellproteome.ThesingleexceptionwasupregulationoftheusuallyneuronalnPTB,whichresultedfromalargeincreaseinnPTBexon10inclusion.InmodelsystemsofPTB-regulatedsplicing,doubleknockdownofbothPTBandtheupregulatednPTBcausedgreaterchangesinASthanknockdownofPTBalone.Strikingly,wefoundthatinclusionofcassetteexon2ofROD1,whichisessentialforgenerationofanmRNAwithanopenreadingframeinitiatingatthefirstAUGcodon,wasalsoupregulatedbyknockdownofPTBandnPTBtogether.Moreover,proteomicanalysisshowednumerousalterationsinproteinexpressionuponPTB+nPTBdouble-knockdowncellsandallowedustoidentifynovelPTB/nPTB-regulatedevents.OurdataindicatethatnonproductiveASisusedinbothautoregulationandcrossregulationofPTB,nPTB,andROD1andthatnPTBisabletoreplacemanyofthefunctionsofPTBinHeLacells.
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Summary
Introduction
Results
Discussion
ExperimentalProcedures
SupplementalData
NoteAddedinProof
References
Results
RNAiagainstPTBResultsinanIncreaseinnPTBProteinLevels
OurinitialaimwastocarryoutaproteomicanalysisofHeLacellsinwhichPTBhadbeenknockeddown.RNAiwasperformedusingaPTB-specificshort-interferingRNA(siRNA)duplex,P1,andacontrolduplex,C2(Wollertonet al.,2004),resultingin~80%–90%PTBknockdown(Figure 1A,toppanel,anddatanotshown).Sampleswereanalyzedby2D-differencegelelectrophoresis(DiGE)(Tongeet al.,2001;Unluet al.,1997).ThecontrolproteinsamplewaslabeledwithCy3fluorescentdye,andthePTB-RNAisamplewithCy5.Thetwosampleswerethenpooledandseparatedona2DgeloverapIrangeof3–10(Figure 1B),andtwofluorescentimagesweretaken.Afterfalsecoloringofthemergedimages,proteinswhoselevelshavechangedbetweenthetwosamplesappearredorgreen,whereasyellowspotsindicateproteinswhoselevelshaveremainedconstant.DespiteourexpectationthatknockdownofPTBwouldcausenumerouschangesintheHeLaproteome,veryfewup-ordownregulatedproteinswereobserved.Theonlyconsistentchangesthatwereobservedwithreciprocaldyelabeling,biologicalandtechnicalrepeats,andwithasecondPTBsiRNA(P2)wereagroupofspotsinthehighpI(>9)and55
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