RNA的剪接

RNASplicingRNA的剪接Figure13-1Primarytranscript多数真核基因都是断裂的，其编码序列由非编码序列隔开Exons(外显子):thecodingsequencesIntrons(内含子):theinterveningsequencesRNAsplicing:theprocessbywhichintronsareremovedfromthepre-mRNA.Alternativesplicing(可变剪接):somepre-mRNAscanbesplicedinmorethanoneway,generatingalternativemRNAs.60%ofthehumangenesaresplicedinthismanner.Topic1:RNA剪接的化学反应RNA的序列决定剪接位点Thebordersbetweenintronsandexonsaremarkedbyspecificnucleotidesequenceswithinthepre-mRNAs.剪接应该在那里发生？Figure13-2Theconsensussequencesforhuman5’splicesite(5’剪接位点):theexon-intronboundaryatthe5’endoftheintron3’splicesite(3’剪接位点):theexon-intronboundaryatthe3’endoftheintronBranchpointsite(分枝位点):anAclosetothe3’endoftheintron,whichisfollowedbyapolypyrimidinetract(Pytract).两相邻的外显子连接起来，内含子以套马索(Lariat)的结构被除去两次转酯化反应(transesterification):Step1:TheOHoftheconservedAatthebranchsiteattacksthephosphorylgroupoftheconservedGinthe5’splicesite.Asaresult,the5’exonisreleasedandthe5’-endoftheintronformsathree-wayjunctionstructure.ThechemistryofRNAsplicingFigure13-3Three-wayjunction丁字路口结构Three-wayjunctionFigure13-4Step2:TheOHofthe5’exonattacksthephosphorylgroupatthe3’splicesite.Asaconsequence,the5’and3’exonsarejoinedandtheintronisliberatedintheshapeofalariat.Figure13-3来至不同RNA分子的外显子可以通过反式剪接(Trans-splicing)连接起来Trans-splicing:theprocessinwhichtwoexonscarriedondifferentRNAmoleculescanbesplicedtogether.Trans-splicingFigure13-5NotalariatTopic2：剪接机器THESPLICESOMEMACHINERYRNA剪接由一个大的叫剪接体(spliceosome)的复合体执行剪接体由大约150个蛋白和5snRNAs组成剪接体的许多功能都是由其RNA组分执行的5RNAs(U1,U2,U4,U5,andU6,100-300nt)叫核内小RNA(smallnuclearRNAs,snRNAs).snRNA和蛋白的复合体叫核内小分子核糖核蛋白smallnuclearribonucleoproteins(snRNP,发音“snurps”).剪接体是最大的snRNP，在剪接的不同阶段它的组成会有变化1.识别the5’splicesiteandthebranchsite.2.将这两个位点带到一起.3.催化(or帮助催化)RNA断裂.RNA-RNA,RNA-proteinandprotein-proteininteractionsareallimportantduringsplicing.snRNPs在剪接中的角色Figure13-6RNA-RNAinteractionsbetweendifferentsnRNPs,andbetweensnRNPsandpre-mRNATopic3：剪接途径SPLICINGPATHWAYS剪接体的装配,重排,和催化:thesplicingpathway(Fig.13-8)装配step11.U1recognize5’splicesite.2.OnesubunitofU2AFbindstoPytractandtheothertothe3’splicesite.TheformersubunitsinteractswithBBPandhelpsitbindtothebranchpoint.3.Early(E)complexisformed装配step21.U2bindstothebranchsite,andthenAcomplexisformed.2.Thebase-pairingbetweentheU2andthebranchsiteissuchthatthebranchsiteAisextruded(Figure13-6).ThisAresidueisavailabletoreactwiththe5’splicesite.Figure13-8EcomplexAcomplexFigure13-6b装配step31.U4,U5andU6formthetri-snRNPParticle.2.Withtheentryofthetri-snRNP,theAcomplexisconvertedintotheBcomplex.Figure13-8AcomplexBcomplex装配step4U1leavesthecomplex,andU6replacesitatthe5’splicesite.U4isreleasedfromthecomplex,allowingU6tointeractwithU2(Figure13-6c).ThisarrangementcalledtheCcomplex.Figure13-8Figure13-6cBcomplexCcomplexinwhichthecatalysishasnotoccurredyet催化Step1:FormationoftheCcomplexproducestheactivesite,withU2andU6RNAsbeingbroughttogetherFormationoftheactivesitejuxtaposesthe5’splicesiteofthepre-mRNAandthebranchsite,allowingthebranchedAresiduetoattackthe5’splicesitetoaccomplishthefirsttransesterficationreaction.催化Step2:U5snRNPhelpstobringthetwoexonstogether,andaidsthesecondtransesterificationreaction,inwhichthe3’-OHofthe5’exonattacksthe3’splicesite.最后Step:ReleaseofthemRNAproductandthesnRNPsFigure13-8CcomplexEcomplexAcomplexBcomplexsplicesome-mediatedsplicingreactionsFigure13-8自我剪接内含子(Self-splicingintrons)揭示RNA可以催化RNA剪接Self-splicingintrons:theintronitselffoldsintoaspecificconformationwithintheprecursorRNAandcatalyzesthechemistryofitsownreleaseandtheexonligationAdamsetal.,Nature2004,Crystalstructureofaself-splicinggroupIintronwithbothexonsPracticaldefinitionforself-splicingintrons:theintronsthatcanremovethemselvesfrompre-RNAsinthetesttubeintheabsenceofanyproteinsorotherRNAs.有两类型的自我剪接内含子：groupIandgroupIIself-splicingintrons.TABLE13-1RNA剪接的三种类型ClassAbundanceMechanismCatalyticMachineryNuclearpre-mRNA很普遍;被很多真核基因使用两次转酯反应;分支位点A大剪接体GroupIIintrons很少;一些真核细胞器基因和原核基因和pre-mRNA一样内含子编码的RNA酶(核酶)GroupIintrons很少;一些真核细胞核rRNA,细胞器基因,以及很少的原核基因两次转酯反应;需要外源的G参与和groupIIintrons一样II类内含子(groupIIintron)剪接的化学反应及中间产物和核前体RNA(nuclearpre-mRNA)一样.Figure13-9I类内含子(GroupIintrons)以线性形式释放而非套马索形式InsteadofusingabranchpointA,groupIintronsuseafreeGtoattackthe5’splicesite.ThisGisattachedtothe5’endoftheintron.The3’-OHgroupofthe5’exonattacksthe5’splicesite.Thetwo-steptransesterificationreactionsarethesameasthatofsplicingofthegroupIIintronandpre-mRNAintrons.GinsteadofAalinearintronaLariatintronFigure13-91.比groupIIintrons小2.有一个保守的二级结构，包含内在前导序列“internalguidesequence”，该序列可以和上游外显子的5’剪接位点碱基配对.3.三级结构有一个可以结合鸟苷酸或鸟甙的结合袋GroupIintronsII类内含子和U2-U6snRNA的结构相似性(在第一次转酯反应中)Figure13-10剪接体怎样找到正确的碱基位点？两类碱基位点识别错误Splicesitescanbeskipped.“Pseudo”splicesitescouldbemistakenlyrecognized,particularlythe3’splicesite.Figure13-12识别错误产生的原因(1)Theaverageexonis150nt,andtheaverageintronisabout3,000ntlong(someintronsarenear800,000nt)Itisquitechallengingforthespliceosometoidentifytheexonswithinavastoceanoftheintronicsequences.(2)Thesplicesiteconsensussequenceareratherloose.Forexample,onlyAGGtri-nucleotidesisrequiredfor