RNA剪切.ppt

WelcomeEachofYoutoMyMolecularBiologyClass MolecularBiologyoftheGene 5 E Watsonetal 2004 PartI ChemistryandGeneticsPartII MaintenanceoftheGenomePartIII ExpressionoftheGenomePartIV RegulationPartV Methods 4 3 05 Ch12 MechanismsofTranscriptionCh13 RNASplicingCh14 TranslationCh15 TheGeneticcode 4 3 05 CHAPTER13RNASplicing MolecularBiologyCourse Figure13 1 Primarytranscript Mostoftheeukaryoticgenesaremosaic 嵌合体 consistingofinterveningsequencesseparatingthecodingsequenceExons 外显子 thecodingsequencesIntrons 内含子 theinterveningsequencesRNAsplicing theprocessbywhichintronsareremovedfromthepre mRNA Alternativesplicing 可变剪接 somepre mRNAscanbesplicedinmorethanoneway generatingalternativemRNAs 60 ofthehumangenesaresplicedinthismanner Topic1 THECHEMISTRYOFRNASPLICING CHAPTER13RNASplicing SequenceswithintheRNADetermineWhereSplicingOccurs ThechemistryofRNAsplicing Thebordersbetweenintronsandexonsaremarkedbyspecificnucleotidesequenceswithinthepre mRNAs Figure13 2 Theconsensussequencesforhuman 5 splicesite 5 剪接位点 theexon intronboundaryatthe5 endoftheintron3 splicesite 3 剪接位点 theexon intronboundaryatthe3 endoftheintronBranchpointsite 分枝位点 anAclosetothe3 endoftheintron whichisfollowedbyapolypyrimidinetract Pytract TheintronisremovedinaFormCalledaLariat 套马索 astheFlankingExonsarejoined Twosuccessivetransesterification Step1 TheOHoftheconservedAatthebranchsiteattacksthephosphorylgroupoftheconservedGinthe5 splicesite Asaresult the5 exonisreleasedandthe5 endoftheintronformsathree wayjunctionstructure ThechemistryofRNAsplicing Figure13 3 Three wayjunction Thestructureofthree wayjunction Figure13 4 Thisfigurehasanerror Intron 5 end Step2 TheOHofthe5 exonattacksthephosphorylgroupatthe3 splicesite Asaconsequence the5 and3 exonsarejoinedandtheintronisliberatedintheshapeofalariat Figure13 3 ExonsfromdifferentRNAmoleculescanbefusedbyTrans splicing Trans splicing theprocessinwhichtwoexonscarriedondifferentRNAmoleculescanbesplicedtogether ThechemistryofRNAsplicing Trans splicing Figure13 5 Notalariat Topic2THESPLICESOMEMACHINERY CHAPTER13RNASplicing RNAsplicingiscarriedoutbyalargecomplexcalledspliceosome Theabovedescribedsplicingofintronsfrompre mRNAaremediatedbythespliceosome Thespliceosomecomprisesabout150proteinsand5snRNAs ManyfunctionsofthespliceosomearecarriedoutbyitsRNAcomponents Thespliceosomemachinery ThefiveRNAs U1 U2 U4 U5 andU6 100 300nt arecalledsmallnuclearRNAs snRNAs ThecomplexesofsnRNAandproteinsarecalledsmallnuclearribonuclearproteins snRNP pronounces snurps ThespliceosomeisthelargestsnRNP andtheexactmakeupdiffersatdifferentstagesofthesplicingreaction ThreerolesofsnRNPsinsplicing1 Recognizingthe5 splicesiteandthebranchsite 2 Bringingthosesitestogether 3 Catalyzing orhelpingtocatalyze theRNAcleavage RNA RNA RNA proteinandprotein proteininteractionsareallimportantduringsplicing Figure13 6 RNA RNAinteractionsbetweendifferentsnRNPs andbetweensnRNPsandpre mRNA Topic3SPLICINGPATHWAYS CHAPTER13RNASplicing Assembly rearrangement andcatalysiswithinthespliceosome thesplicingpathway Fig 13 8 Assemblystep11 U1recognize5 splicesite 2 OnesubunitofU2AFbindstoPytractandtheothertothe3 splicesite TheformersubunitsinteractswithBBPandhelpsitbindtothebranchpoint 3 Early E complexisformed Splicingpathways Assemblystep21 U2bindstothebranchsite andthenAcomplexisformed 2 Thebase pairingbetweentheU2andthebranchsiteissuchthatthebranchsiteAisextruded Figure13 6 ThisAresidueisavailabletoreactwiththe5 splicesite Figure13 8 Ecomplex Acomplex Figure13 6b Assemblystep31 U4 U5andU6formthetri snRNPParticle 2 Withtheentryofthetri snRNP theAcomplexisconvertedintotheBcomplex Figure13 8 Acomplex Bcomplex Assemblystep4U1leavesthecomplex andU6replacesitatthe5 splicesite U4isreleasedfromthecomplex allowingU6tointeractwithU2 Figure13 6c ThisarrangementcalledtheCcomplex Figure13 8 Figure13 6c Bcomplex Ccomplexinwhichthecatalysishasnotoccurredyet CatalysisStep1 FormationoftheCcomplexproducestheactivesite withU2andU6RNAsbeingbroughttogetherFormationoftheactivesitejuxtaposes 并置 the5 splicesiteofthepre mRNAandthebranchsite allowingthebranchedAresiduetoattackthe5 splicesitetoaccomplishthefirsttransesterfication 转酯 reaction CatalysisStep2 U5snRNPhelpstobringthetwoexonstogether andaidsthesecondtransesterificationreaction inwhichthe3 OHofthe5 exonattacksthe3 splicesite FinalStep ReleaseofthemRNAproductandthesnRNPs Figure13 8 Ccomplex 1streaction 2ndreaction Ecomplex Acomplex Bcomplex Ccomplex 没有该complex的图 splicesome mediatedsplicingreactions Figure13 8 Howdoesspliceosomefindthesplicesitesreliably Splicingpathways Twokindsofsplice siterecognitionerrorsSplicesitescanbeskipped Pseudo splicesitescouldbemistakenlyrecognized particularlythe3 splicesite Figure13 12 Reasonsfortherecognitionerrors 1 Theaverageexonis150nt andtheaverageintronisabout3 000ntlong someintronsarenear800 000nt Itisquitechallengingforthespliceosometoidentifytheexonswithinavastoceanoftheintronicsequences 2 Thesplicesiteconsensussequenceareratherloose Forexample onlyAG Gtri nucleotidesisrequiredforthe3 splicesite andthisconsensussequenceoccursevery64nttheoretically 1 BecausetheC terminaltailoftheRNApolymeraseIIcarriesvarioussplicingproteins co transcriptionalloadingoftheseproteinstothenewlysynthesizedRNAensuresallthesplicesitesemergingfromRNAPIIarereadilyrecognized thuspreventingexonskipping Twowaystoenhancetheaccuracyofthesplice siteselection 2 Thereisamechanismtoensurethatthesplicesitesclosetoexonsarerecognizedpreferentially SRproteinsbindtotheESEs exonicsplicingenhancers presentintheexonsandpromotetheuseofthenearbysplicesitesbyrecruitingthesplicingmachinerytothosesites SRproteins boundtoexonicsplicingenhancers ESEs interactwithcomponentsofsplicingmachinery recruitingthemtothenearbysplicesites Figure13 13 Ensuretheaccuracyandefficacyofconstitutivesplicing 组成性剪接 RegulatealternativesplicingTherearemanyvarietiesofSRproteins Someareexpressedpreferentiallyincertaincelltypesandcontrolsplicingincell typespecificpatterns SRproteinsareessentialforsplicing Topic4ALTERNATIVESPLICING CHAPTER13RNASplicing ManygenesinhighereukaryotesencodeRNAsthatcanbesplicedinalternativewaystogeneratetwoormoredifferentmRNAsand thus differentproteinproducts Singlegenescanproducemultipleproductsbyalternativesplicing Alternativesplicing DrosophilaDSCAMgenecanbesplicedin38 000alternativeways Figure13 13 Figure13 15 Differentwaysofalternativesplicing Alternativesplicingcanbeeitherconstitutiveorregulated Constitutivealternativesplicing morethanoneproductisalwaysmadefromapre mRNARegulativealternativesplicing differentformsofmRNAareproducedatdifferenttime underdifferentconditions orindifferentcellortissuetypes Anexampleofconstitutivealternativesplicing SplicingoftheSV40TantigenRNA Figure13 16 Alternativesplicingisregulatedbyactivatorsandrepressors Alternativesplicing Theregulatingsequences exonic orintronic splicingenhancers ESEorISE orsilencers ESSandISS Activatorsareproteinsbindtoenhancerstoenhancesplicing Repressorsareproteinsbindtosilencerstorepresssplicing SRproteinsaresplicingactivatorsandcontaintwodomains OnedomainistheRNA recognitionmotif RRM whichisresponsibleforRNAbinding TheotherdomainistheRSdomain richinarginineandserine whichmediatesinteractionsbetweentheSRproteinsandproteinswithinthesplicingmachinerytopromotesplicingatthenearbysplicesites hnRNPsaresplicingrepressorsMostsilencersarerecognizedbyhnRNP heterogeneousnuclearribonucleoprotein family TheseproteinsbindRNA butlacktheRSdomains Therefore 1 Theycannotrecruitthesplicingmachinery 2 theyblocktheuseofthespecificsplicesitesthattheybind Regulatedalternativesplicing Figure13 17 Bindsateachendoftheexonandconceals 隐藏 it CoatstheRNAandmakestheexonsinvisibletothesplicingmachinery TwomodelsfortheactionofarepressorhnRNPI PTBininhibitingsplicing Figure13 18 Theoutcomeofalternativesplicing 可变剪接的结果 生物学功能 Producingmultipleproteinproducts calledisoforms Theycanhavesimilar distinctorantagonisticfunctions Onegeneencodesmultiplefunctions 2 Switchingonandofftheexpressionofagivengenethatencodesonlyonefunction Whentheexoncontainingastopisincludedtoproducenonfunctionalprotein ortheintronisincludedtopreventmRNAtransport Asmallgroupofintronaresplicedbyminorspliceosome Itsplicesintronsharboringdeterminantsequencesdistinctfromthoserecognizedbythemajorspliceosome ItisknownAT ACspliceosome TheterminioftheoriginallyidentifiedintronsthatissplicecontainAUat5 ss andACatthe3 ss Thechemicalpathwayisthesameasthemajorspliceosome butU11andU12areusedinplacesofU1andU2 respectively Alternativesplicing Figure13 19TheAT ACspliceosome Figure13 20Sequencesconservedindifferentkindsofintrons Trans splicing Topic5 Self splicingintrons自剪接内含子 CHAPTER13RNASplicing Self splicingintronsrevealthatRNAcancatalyzesplicing Self splicingintrons IntronsthatcanfoldintoaspecificconformationwithintheprecursorRNA andcatalyzethechemistryoftheirownreleaseandtheexonligation Theycanremovethemselvesfrompre RNAsintheabsenceofanyproteinsorotherRNAsinvitro Splicingpathways foldintoaspecificconformation catalyzethechemicalreactionusingmetalionsascofactors Therearetwoclassesofself splicingintrons groupIself splicingintronsgroupIIself splicingintrons Figure13 9 ThechemistryofgroupIIintronsplicingandRNAintermediatesproducedarethesameasthatofthenuclearpre mRNA ThesimilarityofthestructuresofgroupIIintronsandU2 U6snRNAcomplexformedtoprocessfirsttransesterification Figure13 10 GroupIintronsreleasealinearintronratherthanalariat Duringthe1sttransesterificationreaction groupIintronsuseafreeG insteadofusingabranchpointA toattackthe5 splicesite Asaresult thisGisattachedtothe5 endoftheintron A3 OHgroupisresultedatthe5 exon whichthenattacksthe5 splicesiteforthe2ndtransesterificationreaction ThisisthesameasthatofsplicingofthegroupIIandpre mRNAintrons Splicingpathways GinsteadofA alinearintron aLariatintron Figure13 9 SmallerthangroupIIintronsShareaconservedsecondarystructure whichincludesan internalguidesequence base pairingwiththe5 splicesitesequenceintheupstreamexon Theirtertiarystructurecontainsabindingpocketthatwillaccommodatetheguaninenucleotideornucleosidecofactor GroupIintrons Adamsetal Nature2004 Crystalstructureofaself splicinggroupIintronwithbothexons GroupIintronwebsite 2Dstructure 3Dstructure Topic6RNAEDITING CHAPTER13RNASplicing RNAeditingisanotherwayofchangingthesequenceofanmRNAattheRNAlevel I Sitespecificdeamination 位点特异性去氨反应 1 AspecificallytargetedCresiduewithinmRNAisconvertedintoUbythedeaminase 脱氨酶 Theprocessoccursonlyincertaintissuesorcelltypesandinaregulatedmanner RNAediting Figure13 25 Stopcode Inliver Inintestines Figure13 25RNAeditingbydeamination Thehumanapolipoproteingene 2 Adenosinedeaminationalsooccursincells TheenzymeADAR adenosinedeaminaseactingonRNA convertAintoInosine Insonecanbase pairwithC andthischangecanalterthesequenceoftheprotein IIGuideRNA directeduridineinsertionordeletion 1 ThisformofRNAeditingisfoundinthemitochondriaoftrypanosomes 2 MultipleUsareinsertedintospecificregionofmRNAsaftertranscription orUSmaybedeleted 3 TheadditionofUstomRNAchangescodonsandreadingframes completelyalteringthe meaning ofthemessage 4 UsareinsertedintothemessagebyguideRNAs gRNAs Havingthreeregions a