Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA 英文参考文献.docVIP

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Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA 英文参考文献.doc

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Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA 英文参考文献

RoleofPolypyrimidineTractBindingProteinin MediatingInternalInitiationofTranslationofInterferon RegulatoryFactor2RNA DebojyotiDhar¤a,MusturiVenkataramana¤b,AnandPonnuswamy,SaumitraDas* DepartmentofMicrobiologyandCellBiology,IndianInstituteofScience,Bangalore,Karnataka,India Abstract Background:Earlierwehavereportedtranslationalcontrolofinterferonregulatoryfactor2(IRF2)byinternalinitiation(Dhar etal,NucleicAcidsRes,2007).TheresultsimpliedpossibleroleofIRF2incontrollingtheintricatebalanceofcellulargene expressionunderstressconditionsingeneral.HerewehaveinvestigatedthesecondarystructureoftheInternalRibosome EntrySiteofIRF2RNAanddemonstratedtheroleofPTBproteininribosomeassemblytofacilitateinternalinitiation. Methodology/PrincipalFindings:WehaveprobedtheputativesecondarystructureoftheIRF259UTRRNAusingvarious enzymaticandchemical modification agentstoconstrain thesecondary structure predictedfromRNA foldingalgorithm Mfold.TheIRESactivitywasfoundtobeinfluencedbytheinteractionoftrans-actingfactor,polypyrimidinetractbinding protein(PTB).Deletionof25ntsfromthe39terminus ofthe59untranslatedregionresultedinreducedbindingwithPTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putativecontactpointsofPTBontheIRF2–59UTRusingprimerextensioninhibitionassay.MajorityofthePTBtoe-prints werefoundtoberestrictedtothe39endoftheIRES.Additionally,CircularDichroism(CD)spectraanalysissuggestedchange in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silencedforPTBgeneexpression,resultedinreducedbindingbyothertrans-actingfactors.Finally,wehavedemonstrated thatadditionofrecombinantPTBenhancesribosomeassemblyonIRF2IRESsuggestingpossibleroleofPTBinmediating internalinitiationoftranslationofIRF2RNA. Conclusion/Significance:ItappearsthatPTBbindingtomultiplesiteswithinIRF259UTRleadstoaconformationalchange intheRNAthatfacilitatebindingofothertrans-actingfactorsto