10.-基因转录后水平调控

基因转录后水平调控Post-transcriptionalProcessingofRNANucleusDNAPrimaryRNAtranscriptMaturemRNAPolIItranscriptionMaturemRNAProteinNuclearprocessingCappingSplicingPolyadenylationExportTranslationDegradationGeneExpression:Multiple,SpatiallyandTemporallyDistinctStepsCarriedoutbyDistinctCellularMachineryCytoplasm-RegulationCanBeatSeveralDifferentLevels-DynamicProteinAssociationGeneExpression:ComplexNetworkofCoupledInteractionsManiatis,T.,andReed,R.(2002).Nature416,499-506.mRNAtransportAlleukaryoticmRNAsareprocessedSpecificprocessforPolIItranscripts1.5’加上m7GpppN5（capping戴“帽”）•ProtectionofmRNAfromdegradation•EnhancementofmRNA’stranslatability•TransportofmRNAoutofnucleus•Propersplicingofpre-mRNA2.3’加“尾”(tailing)①Facilitatetotransportintocytosol②MaintainmRNAstability3.拼接(splicing)IncreasemRNAdiversitySplicingismediatedbysnRNPsExonsaresimilarinsizeIntronsarehighlyvariableinsizeGT-AGrule•GT-AGruledescribesthepresenceoftheseconstantdinucleotidesatthefirsttwoandlasttwopositionsofintronsofnucleargenes.•Splicesitesarethesequencesimmediatelysurroundingtheexon-intronboundaries•SplicingjunctionsarerecognizedonlyinthecorrectpairwisecombinationsSplicingofpre-mRNAoccursina“spliceosome”anRNA-proteincomplexpre-mRNAsplicedmRNAspliceosome(~100proteins+5smallRNAs)Thespliceosomeisalargeprotein-RNAcomplexinwhichsplicingofpre-mRNAsoccurs.染色质的空间变化转录RNA的加工成熟蛋白质的降解多肽链的加工修饰翻译mRNA的降解•转录水平•转录后水平•翻译水平调控•蛋白质加工水平基因转录后水平调控主要方式第一部分mRNAstructure/folding第三部分mRNAstability/degradation/decay第二部分mRNAtransport第四部分mRNAediting第五部分mRNAmethylation第六部分mRNAselectivedegradation第七部分miRNA/ncRNA(epigeneticsection)第一部分RNAstructure/foldingNobelprize1989Nobelprize2009RibozymeRibosomeThefunctionoftheRNAmoleculedependsonitsfoldedstructureRNAfunctionsStructural•e.g.,rRNA,whichisamajorstructuralcomponentofribosomesBUT-itsroleisnotjuststructural,also:CatalyticRNAintheribosomehaspeptidyltransferaseactivity•Enzymaticactivityresponsibleforpeptidebondformationbetweenaminoacidsingrowingpeptidechain•Also,manysmallRNAsareenzymesribozymes”RegulatoryRecentlydiscoveredimportantnewrolesforRNAsInnormalcells:•indefense-esp.inplants•innormaldevelopmente.g.,siRNAs,miRNA•RNAi•RNAaptamersAstools:•forgenetherapyortomodifygeneexpressionHierarchicalorganizationofRNAmoleculesPrimarystructureSecondaryStructure5’to3’listofcovalentlylinkednucleotides,namedbytheattachedbaseCommonlyrepresentedbyastringSoverthealphabetΣ={A,C,G,U}GGAUUGCCGGAUAAUGCAGCUUINTERNALLOOPHAIRPINLOOPBULGESTEMDANGLINGENDS5’3’RNA“tertiaryinteractions”PseudoknotKissinghairpinsHairpin-bulgeInadditiontosecondarystructuralinteractionsinRNA,therearealsotertiaryinteractions.HairpinloopJunction(Multiloop)BulgeLoopSingle-StrandedInteriorLoopStemPseudoknotRNAstructurepredictionTwoprimarymethodsforRNAsecondarystructureprediction:-Co-variationanalysis(comparativesequenceanalysis)Takesintoaccountconservedpatternsofbasepairsduringevolution(morethan2sequences)-Minimumfree-energymethodDeterminestructureofcomplementaryregionsthatareenergeticallystableFreeEnergy(E)=E(CG)+E(CG)+…..ComputingRNAsecondarystructure:Minimumfree-energymethod•Workinghypothesis:ThenativesecondarystructureofaRNAmoleculeistheonewiththeminimumfreeenergy•Restrictions:–Noknots–Noclosebasepairs–Basepairs:A-U,C-GandG-UProteinstructuresRNAstructuresTotal72000Total2000DuetothelimitedamountofdataPredictingRNAtertiarystructureisalmostimpossiblePARS:ParallelanalysisofRNAstructureDMS:dimethylsulphate第二部分mRNAtransportCytoplasmicfilamentCytoplasmNucleusCytoplasmicringInnerringBasketDistalringTheNuclearPoreComplexNPC:alltrafficbetweennucleus&cytoplasminhighereukaryotesoccursviaNPCMoreonthenuclearporecomplexNPCsize:diameter=120nm,8-foldrotationalsymmetry,MW=125milliondaltons,composedof50-100differentproteinscallednucleoporins(Nups)SmallmoleculescandiffusefreelythroughthenuclearporeHowever,largemolecularrequiresactivetransport!mRNAexport-formationofanexportcompetentmRNP•Whyexportasaprotein/DNAcomplex?RNAsaretoobigandlackthesignalstointeractw/nuclearexportreceptors•Specific“adaptor”proteinsmustfirstbindtotheRNAandchaperonethismoleculetotheexportreceptor,which,inturn,guidestheRNAacrosstheNPCNuclearlocalizationsequenceisrequiredforproteinnuclearimportFunctionalNLSisrequiredfornuclearlocalization(mutationinNLSresultsincytosoliclocalization)NLS:consistsofeitheroneortwostretchesofbasicaminoacidsPathoftransportingmRNAtothenuclearporecomplexSomeEukaryoticmRNAsAreLocalizedtoSpecificRegionsofaCellLocalizationofAsh1mRNA.QuestionsremainaboutmRNAtransport-HowaremRNAsubstratesreleasedinthecytoplasm?-AretheremultiplemRNAexportpathways?-Howisprocessingandtransportmechanistically“coupled”?-IsmRNAexportregulated?etc.*mRNAsareunstablemolecules*ImportantcontributortomRNAabundance*Initiatedbydeadenylationcatalyzedbypoly(A)nucleases.*Followedeitherbydecappingand5′to3′exonucleasedigestion,orby3′to5′exonucleasedigestion.Exonucleasesareunidirectional.第三部分mRNAstability/degradation/decay/turnover5’cap5’-UTR3’-UTRAAAAAAAorfstemloopAREexon-exonjunctioncodingregiondeterminantElementsofanmRNAthataffectitsstabilityAUGUAGprema