Gene-X-ppt(Gene10-基因十)--Chapter21

Chapter21RNASplicingandProcessing21.1Introduction•pre-mRNA–ThenucleartranscriptthatisprocessedbymodificationandsplicingtogiveanmRNA.•RNAsplicing–TheprocessofexcisingintronsfromRNAandconnectingtheexonsintoacontinuousmRNA.FIGURE01:EukaryoticmRNAismodified,processed,andtransported21.1Introduction•heterogeneousnuclearRNA(hnRNA)–RNAthatcomprisestranscriptsofnucleargenesmadebyRNApolymeraseII;ithasawidesizedistributionandlowstability.•hnRNP–TheribonucleoproteinformofhnRNA(heterogeneousnuclearRNA),inwhichthehnRNAiscomplexedwithproteins.–Pre-mRNAsarenotexporteduntilprocessingiscomplete;thustheyarefoundonlyinthenucleus.21.2The5′EndofEukaryoticmRNAIsCapped•A5′capisformedbyaddingaGtotheterminalbaseofthetranscriptviaa5′–5′link.•Thecappingprocesstakesplaceduringthetranscription,whichmaybeimportantfortranscriptionreinitiation.FIGURE02:EukaryoticmRNAhasamethylated5’cap21.2The5′EndofEukaryoticmRNAIsCapped•The5′capofmostmRNAismonomethylated,butsomesmallnoncodingRNAsaretrimethylated.•ThecapstructureisrecognizedbyproteinfactorstoinfluencemRNAstability,splicing,export,andtranslation.21.3NuclearSpliceJunctionsAreShortSequences•Splicesitesarethesequencesimmediatelysurroundingtheexon–intronboundaries.Theyarenamedfortheirpositionsrelativetotheintron.•The5′splicesiteatthe5′(left)endoftheintronincludestheconsensussequenceGU.•The3′splicesiteatthe3′(right)endoftheintronincludestheconsensussequenceAG.21.3NuclearSpliceJunctionsAreShortSequences•TheGU-AGrule(originallycalledtheGT-AGruleintermsofDNAsequence)describestherequirementfortheseconstantdinucleotidesatthefirsttwoandlasttwopositionsofintronsinpre-mRNAs.FIGURE03:TheendsofnuclearintronsaredefinedbytheGU-AGrule21.3NuclearSpliceJunctionsAreShortSequences•ThereexistminorintronsrelativetothemajorintronsthatfollowtheGU-AGrule.•MinorintronsfollowageneralAU-ACrulewithadifferentsetofconsensussequencesattheexon–intronboundaries.21.4SpliceJunctionsAreReadinPairs•Splicingdependsonlyonrecognitionofpairsofsplicejunctions.•All5′splicesitesarefunctionallyequivalent,andall3′splicesitesarefunctionallyequivalent.•Additionalconservedsequencesatboth5′and3′splicesitesdefinefunctionalsplicesitesamongnumerousotherpotentialsitesinthepre-mRNA.FIGURE04:Correctsplicingremovesthreeintronsbypairwiserecognitionofthejunctions21.5Pre-mRNASplicingProceedsthroughaLariat•Splicingrequiresthe5′and3′splicesitesandabranchsitejustupstreamofthe3′splicesite.•Thebranchsequenceisconservedinyeastbutlesswellconservedinmulticellulareukaryotes.•Alariatisformedwhentheintroniscleavedatthe5′splicesite,andthe5′endisjoinedtoa2′positionatanAatthebranchsiteintheintron.21.5Pre-mRNASplicingProceedsthroughaLariat•Theintronisreleasedasalariatwhenitiscleavedatthe3′splicesite,andtheleftandrightexonsarethenligatedtogether.FIGURE05:Splicingproceedsthroughalariat21.6snRNAsAreRequiredforSplicing•smallcytoplasmicRNAs(scRNA;scyrps)–RNAsthatarepresentinthecytoplasm(andsometimesarealsofoundinthenucleus).•smallnuclearRNA(snRNA;snurps)–OneofmanysmallRNAspeciesconfinedtothenucleus;severalofthemareinvolvedinsplicingorotherRNAprocessingreactions.•smallnucleolarRNA(snoRNA)–AsmallnuclearRNAthatislocalizedinthenucleolus.21.6snRNAsAreRequiredforSplicing•ThefivesnRNPsinvolvedinsplicingareU1,U2,U5,U4,andU6.•Togetherwithsomeadditionalproteins,thesnRNPsformthespliceosome.FIGURE07:Thespliceosomeisalargeparticle21.6snRNAsAreRequiredforSplicing•AllthesnRNPsexceptU6containaconservedsequencethatbindstheSmproteinsthatarerecognizedbyantibodies(anti-SM)generatedinautoimmunedisease.•splicingfactor–AproteincomponentofthespliceosomethatisnotpartofoneofthesnRNPs.•transesterification–Areactionthatbreaksandmakeschemicalbondsinacoordinatedtransfersothatnoenergyisrequired.21.7CommitmentofPre-mRNAtotheSplicingPathway•U1snRNPinitiatessplicingbybindingtothe5′splicesitebymeansofanRNA–RNApairingreaction.•Thecommitmentcomplex(orEcomplex)containsU1snRNPboundatthe5′splicesiteandtheproteinU2AFboundtoapyrimidinetractbetweenthebranchsiteandthe3′splicesite.FIGURE10:Formationofthecommitmentcomplex21.7CommitmentofPre-mRNAtotheSplicingPathway•Inmulticellulareukaryoticcells,SRproteinsplayanessentialroleininitiatingtheformationofthecommitmentcomplex.•Pairingsplicesitescanbeaccomplishedbyintrondefinitionorexondefinition.FIGURE11:Tworouteforinitialrecognitionof5’and3’splicesites21.8TheSpliceosomeAssemblyPathway•Thecommitmentcomplexprogressestopre-spliceosome(theAcomplex)inthepresenceofATP.•RecruitmentofU5andU4/U6snRNPsconvertsthepre-spliceosometothematurespliceosome(theB1complex).•TheB1complexisnextconvertedtotheB2complexinwhichU1snRNPisreleasedtoallowU6snRNAtointeractwiththe5′splicesite.21.8TheSpliceosomeAssemblyPathway•U4dissociatesfromU6snRNPtoallowU6snRNAtopairwithU2snRNAtoformthecatalyticcenterforsplicing.•Bothtransesterificationreactionstakeplaceintheactivatedspliceosome(theCcomplex).•Thesplicingreactionisreversibleatallsteps.FIGURE12:Splicingreactionproceedsthroughdiscretestages21.9AnAlternativeSpliceosomeUsesDifferentsnRNPstoProcesstheMinorClassofIntrons•Analternativesplicingpathwayusesanot