关于真核生物降解及代谢过程的计算机模拟.ppt

关于真核生物mRNA降解及代谢过程的计算机模拟,曹丹亚力桑那大学分子细胞生物学系,DanCaoandRoyParkerHowardHughesMedicalInstitute&DepartmentofMolecularCellularBiologyUniversityofArizona,ComputationalmodelingofmRNAturnover,TheCentralDogma,Whywanttomodelthisprocess?,1.Integration,Whywanttomodelthisprocess(Contd)?,Anexplanatory/descriptivetool1)correctionofmisconceptions2)helpinterpretresultcorrectly3)advicesuitableexperimentPredictionfromdiscrepanciesbetweensimulationresultandexperimentalresultAnidealsystemtostartknowledge-drivensimulationPlentyofdataavailabletoestimatetheratesfordistinctsteps,includingsteadystatedistribution,half-lives,effectsofvariousmutants,Majorassumptions,Transcriptionisazerothorderprocess,allotherstepsarefirstorderprocessesAllstepsareirreversibleNofeedbackloops,ratesforallsteps,Steadystatelevelofeachintermediate,kineticsovercertainexperimentaltimecourse,Virtualnortherngelfordirectcomparison,Transcriptionalshut-offexp.(decayfromsteadystate),Transcriptionalpulsechaseexp.,AtsteadystateInhibittranscription,Attheendofashort“pulse”Inhibittranscription,Asamplescreenshot,Determinethefitnessofthemodelwiththeinvivodecaynetwork:MFA2pG&PGK1pG,Theyrepresentaunstable(MFA2)andstable(PGK1)mRNAinyeastTheirdegradationhasbeenextensivelycharacterized,withlotsofdataavailabletoestimatetherates.E.g:transcriptionalpulsechasegelcangiveinformationfortheratesofdeadenylationanddecapping.Strongpoly(G)structureatthe3UTRcantrapthedecayintermediates,giveadditionalinformationaboutinvivoprocess.E.g:ratesofterminaldeadenylationand3to5decay,Comparisonofmodelingresultswithexperimentalobservations,Thesimulatedsteadystatepoly(A)distribution,pulsechasegelpattern,previouslycharacterizedmutants(transport,decapping,3to5decay)arealsoconsistentwithexp.observations.,ModelingofMFA2pGintranscriptionalpulsechase,Observed,Computed,ModelingofPGK1pGintranscriptionalpulsechase,Observed,Computed,Thefactthatwecanreproducetheexperimentalresultsbymodelingsuggeststhatourmodelisquiteaccurate,andwehavearelativelyrobustunderstandingoftheinvivoprocess.TheobtainingofagoodmodelforbothMFA2pGandPGK1pGallowsustofurtheranalyzethewholenetwork.Wehaveusedourmodeltoexaminehowtranscriptsrespondtoavarietyofperturbationsbyperformingaseriesofinsilicoexperiments.E.g.:increaseordecreasetherateoftransport,deadenylation,decapping,5to3exonucleolyticdecay,3to5exonucleolyticdecay,seehowthetranscriptlevel,half-life,steadystatedistributionareaffected.,Whathavewelearned?,Comparisonofinsilicomutants,DeadenylationisakeystepincontrollingmRNAturnover.Provideexplanationforwhymanydecayelementsidentifiedaffectingdeadenylation.,3to5decayrateforfulllength.Obtainedbymatchingthecalculatedtwiththeobservedtindcp1mutant.,Thecalculated3to5decayrates,Implication:3to5decaybyexosomeshowsmRNAspecificdegradationratesthataredependentonthe5structureofthemRNA,3to5decayrateforfragment.Obtainedbymatchingthecalculatedtoffragmentwiththeobservedtwhendecappingisblocked.,Half-life.Measuredbytranscriptionalshut-offexperiment.Atthepointwheretranscriptreacheshalfofitsinitiallevel.tisthoughttorepresenthowlongthemRNAispersistinthecell.Averagelifespan.Calculatedfromthesimulationfortranscriptionalpulsechaseexperiment.MoreaccuraterepresentationoftheaveragetimethemRNAispresentinthecell.,Viewonhalf-life,Half-lifeAveragelifespan.Thetraditionalwayofmeasuringt1/2mayunderestimatethelifespanofanmRNA.,ThedifferenceisduetothedistributionofmRNAatsteadystate.Certain%ofmRNAshasalreadypassedseveraldecaysteps.,WhyAveragelifespanhalf-life?,Themeasurementofmeasureahalf-lifecanpredominantlydifferentstepsinthedecaynetwork,DifferentmRNAswillbeaffecteddifferentiallybycertainchangeonspecificstep,NeedtobeverycautiouswheninterpretingmRNAspecificeffects.,Short-livedmRNAs(MFA2)aremoreresponsivetochangesontransportratethanlong-livedmRNAs(PGK1).,PossiblefactorsthatdisruptthecorrelationbetweenmRNAandproteinlevel,Theincreaseoftranscriptlevelinthetransportmutantsolelycomesfromtheincreaseinthenuclearpool.,Theincreaseoftranscriptlevelinthe5to3exomutantssolelycomesfromtheincreaseinthecap-species,WeareabletosimulateMFA2andPGK1,whichsuggeststhatwehavearelativelyrobustunderstandingaboutyeastmRNAturnover.ThisprogramcanbeadaptabletoothereukaryoticmRNAsthatfollowthesamedegradationscheme.Thisisauseful,explanatorytoolforquantitativeanalysisoftheprocessandregulationofmRNAturnoverineukaryoticcells.SomeInsilicoexperimentsperformedmightbeabletosuggestthebestexp.foraparticularpurpose.E.g:decappingmutants.Discrepanciesbetweeninsilicoresultsandrealresultsmightleadtonewinsightsfortheinvivonetwork.,Computation,Experimentation,NonsenseMediatedmRNADecay(NMD),AAAAAAA70,DNA,transcription,AUGUAAUAA,m7Gppp,Normaldecay30,NonsenseDecay3,NormalDecayandNMD,Modeling,Prediction,Exp.testing,Advantage:increasetherateofhypothesisformingandtesting,Model1.1,Model1.2,Multiplemodelsordifferentcombinationsofrateconstantswithinthesamemodelcanbeobtainedtofitwithcurrentobservations.,Model1,Model1.1andModel1.2,Model1.1TheentryintoPASisinhibitedbymorethan100fold,therateofDIDcontributestothet1/2ofA70,Model1.2TheentryintoPASisnotinhibited,therateoftransport(ormaybeotherunknownstepsupstreamofthebifurcation)issloweddown,whichisusuallyveryfastfornormaltranscript.,IfentryintoPASisnotinhibited,willseesamedecreaseonsteadystatelevel,butthehalf-lifedoesnotchangemuch.,DistinguishModel1.1andModel1.2,RelativelevelofnucleartranscriptModel1.1predicts2%nuclearModel1.2predicts90%nuclearAssumption:therate-limitingstepabovethebifurcationisindeedtransportDecappingmutantsdcp1,dcp2,Currentdataisconsistentwithmodel1.2,inwhichtheentryintoPASisnotinhibited,therateofDIDismuchhigherthanPAS,andtheremightbearatelimitingstepupstreamofthebifurcation,insilicobiology,Computationalmodelingofcellu