转录组学transcriptomicswxj课件

Transcriptomics转录组学Transcriptome:Anevolvingdefinition(Thepopulationof)mRNAsexpressedbyagenomeatanygiventime(Abbott,1999)Thecompletecollectionoftranscribedelementsofthegenome.(Affymetrix,2004)TranscribedelementsmRNAs:35,913transcripts(includingalternativesplicedvariants)

Non-codingRNAstRNAs(497genes)rRNAs(243genes)snmRNAs(smallnon-messengerRNAs)microRNAsandsiRNAs(smallinterferringRNAs)snoRNAs(smallnucleolarRNAs)snRNAs(smallnuclearRNAs)Pseudogenes(~2,000)Transcriptomics

DefinitionThestudyofcharacteristicsandregulationofthefunctionalRNAtranscriptpopulationofacell/sororganismataspecifictime.ScopethepopulationoffunctionalRNA

transcripts.themechanismsthatregulatetheproductionofRNAtranscriptsdynamicsofthetrancriptome(time,celltype,genotype,externalstimuli)一、转录组学研究全部RNA的表达及功能转录组（transcriptome）指特定状态下一种细胞或组织所能转录出来的所有RNA的总和。——包括编码RNA，即mRNA和非编码RNA(non-codingRNA,ncRNA)转录组学（transcriptomics）：是在整体水平上研究细胞基因转录情况及转录调控规律的科学。RNA组学（RNomics）：是分析、鉴定非信使小RNA（smallnon-messengerRNA,snmRNA）在特定状态下表达情况、功能及其与蛋白质的相互作用。转录组的特点：受到内外多种因素的调节，因而是动态可变的。能够揭示不同物种、不同个体、不同细胞、不同发育阶段及不同生理病理状态下的基因差异表达信息。ObservingthetranscriptomeFocussedExperimentalApproaches:NorthernBlottingAnalysisRT-PCR(quantitativeorsemi-quantitative)HighthroughputApproaches:ClosedSystemProfiling: MicroarrayexpressionprofilingOpenSystemProfiling: Serialanalysisofgeneexpression(SAGE) MassivelyParallelSignatureSequencing(MPSS)微阵列（microarray）SAGEMPSS研究技术（一）微阵列是大规模基因组表达谱研究的主要技术大规模表达谱或全景式表达谱（globalexpressionprofile）：是生物体（组织、细胞）在某一状态下基因表达的整体状况。微阵列或基因芯片（DNAchip）:利用光导化学合成、照相平板印刷以及固相表面化学合成等技术，在固相表面合成成千上万个寡核苷酸探针，并与放射性同位素或荧光物标记的来自不同细胞、组织或整个器官的DNA或mRNA反转录生成的第一链cDNA进行杂交，然后用特殊的检测系统对每个杂交点进行定量分析。Experimentaloverview:HybridizationWashingScancy5channelScancy3channel“Overlayimages”Quantifypixelintensities.CellpopulationACellpopulationBRNAextractionAABBReversetranscriptionAABBKlenowlabelincorporationSampleBlabelledwithcy3dyeSampleAlabelledwithcy5dyeLimitofDetection:1in30,000transcripts

~20transcripts/cellRed–increaseofCy5sampletranscriptsGreen–increaseofCy3sampletranscriptsYellow–equalabundanceAffymetrixGeneChip®Limits:1:100,000transcripts~5transcripts/cellAffymetrix:GeneExpressionArrays Transcripts/GenesArabidopsisGenome 24,000C.elegansGenome 22,500DrosophilaGenome 18,500E.coliGenome 20,366HumanGenomeU133Plus 47,000

MouseGenome 39,000

YeastGenome 5,841(S.cerevisiae)&5,031(S.pombe)RatGenome 30,000

Zebrafish 14,900Plasmodium/Anopheles 4,300(P.falciparum)&14,900(A.gambiae)Barley(25,500),Soybean(37,500+23,300pathogen),Grape(15,700)Canine(21,700),Bovine(23,000)B.subtilis(5,000),S.aureus(3,300ORFS),Xenopus(14,400)MicroarrayandGeneChipApproachesAdvantages:RapidMethodanddataanalysiswelldescribedandsupportedRobustConvenientfordirectedandfocussedstudiesDisadvantages:ClosedsystemapproachDifficulttocorrelatewithabsolutetranscriptnumberSensitivetoalternativesplicingambiguities（二）SAGE在转录物水平研究细胞或组织基因表达模式SAGE的基本原理：利用锚定酶（anchoringenzyme，AE）和位标酶（taggingenzyme，TE）切割DNA分子的特定位置（一般近3’端），分离SAGE标签（长约14bp，可藉此鉴定基因组中的所有基因），并将这些标签串联起来，然后对其进行测序特点：可全面提供生物体基因表达谱信息可用来定量比较不同状态下组织或细胞的所有差异表达基因AnchoringEnzymeNlaIII,recognitionsite:The3’terminusofadaptorAandB

arebothTCCRACTAG,wherearecognitionsiteofTaggingEnzymeMmeIflankedwith

NlaIIIHuM,PolyakK.NatureProtocols2006TaggingEnzymeMmeIrecognitionsite:HuM,PolyakK.NatureProtocols2006SAGEAdvantages:Potential‘open’systemmethod–newtranscriptscanbeidentifiedAccuracyofunambiguoustranscriptobservationDigitaloutputofdataQuantitativeandqualitativeinformationDisadvantages:CharacterisingnoveltranscriptsisoftencomputationallydifficultfromshorttagsequencesTagspecificity(recentlyincreasedlengthto21bp)Lengthoftagscanvary(TEenzymeactivityvariablewithtemperature)AsubsetoftranscriptsdonotcontainenzymerecognitionsequenceSensitivetoasubsetofalternativesplicevariants（三）MPSS是以基因测序为基础的基因表达谱分析新技术MPSS的原理：一个含有能够特异识别转录子的信息标签序列（10～20bp）与长的连续分子连接在一起，测出mRNA的一端包含一个10至20个碱基的标签序列。每一标签序列在样品中的频率（拷贝数）代表了与该标签序列相应的基因表达水平。基因表达水平是以计算mRNA拷贝数为基础，是一个数字表达系统。只要将病理和对照样品分别进行测定，即可进行严格的统计检验，能测定表达水平较低、差异较小的基因，而且不必预先知道基因的序列。四、RNA组学研究全部snmRNA人类基因组序列特点：2万2.5万个基因，与蛋白质合成有关的序列占整个基因组的2%左右，其余98%的基因组序列没有得到注释。RNA组学研究范畴：小分子RNA，包括snRNA、snoRNA、scRNA、siRNA、miRNASmallRNACatalogues

Naqvi(2009)IntJBiolSciWithincellsthereareavarietyofdiscoveredsmallRNAsinlengthin19-30nt,recentlygoverningdiversecellularprocessessuchasdevelopment,differentiationacrosstheeukaryotickingdom.siRNA:shortinterferingRNAasdefensivemechanismtoprotecthostgenomeintegrityfromintrusionofforeignnucleicacids.miRNA(microRNA):21-30ntinlengthtranscribedfromhostgenomeloci,involvedinwidecellularprocesses,specifictodevelopmentanddifferentiation.tasiRNA(trans-actingshortinterferingRNA):21ntlengthtakenendogenoustranscriptastemplate,underRdRPactivity,followedbyDicertoproducetasiRNA.InhumanandflywithouttasiRNAitisduetoabsenttoRdRP.27SmallRNAsCataloguesrasiRNA(repeat-associatedRNA):24-26ntlongproductsofDCL3(Dicerlikeprotein,inplant)onlongdsRNAformed,usualinretro-transposonlociwithmethylation,toplayroleingametogenesisviarecruitingchromatinremodelingproteinstomodulatechromatinstatus.scnRNA(smallscanningRNA):29ntlongscnRNA,reportedfromprotozoan,derivedfrommicro-nuclei,eliminatedoriginallociofgenome,givenbirthtomacro-nuclei.lsiRNA(longsiRNA):30-40ntinlengthinducedinresponsetobacterialinfectionorgrowthcondition.piRNA(piwi-actingRNA),21-URNA28

MicroRNA简介★

(1)长度为21nt左右核苷酸的内源性单链小分子RNA；(2)存在65nt左右的发夹结构前体；(3)基因座位于蛋白质基因间隔区；(4)其DNA序列在近源物种间高度保守。★miRNA具有十分重要的调控功能，它们主要参与基因转录后水平的调控。能够通过与靶mRNA特异性的碱基配对引起靶mRNA的降解（植物中较为常见）或者抑制其翻译（动物中较为常见），从而影响了靶mRNA的表达。★目前发现miRNA是一个庞大的小分子调控RNA家族，广泛存在于各种动植物中，参与细胞增殖和分化、细胞凋亡、胚胎发育、形态建成以及疾病发生等一系列重要的生命过程。

★

最近发现一系列与肿瘤发生相关的和人类病毒编码的miRNA，揭示miRNA在哺乳动物基因表达调控中具有重要作用。30Rana(2007)JCellPhysiol*SmallRNAbiogenesis-RISCformation-RNAi&ItsMechanism-CellPhenotypy*

31SmallRNABiogenesis(RNAPolII,Drosha&Dicer)RISCFormation(Dicer,Ago,Proteins,GuideRNA)RNAInterference&Mechanism(MetabolismofGenomicDNA,RNA&Protein)CellularProcesses(CellPhenotypy&ID,Development,GeneExpression,ProteinTranslation,genomestability)OverviewRNAInterference32AModelforHumanmiRISC(againstEndogenes)Rana(2007)NatureRevMolCellBiol33StepsinHumansiRISCFunction(againstExo-nuclearacid)Rana(2007)NatureRevMolCellBiolHumanRISC:siRNA-guideStrandDicer,Argonautes,TRBP&PACTTRBP:HIV-1transactivationresponsiveelement(TAR)RNA-bindingprotein.PACT:doublestrandedRNA-bindingprotein*OverviewonRNAInterferenceGenomiclocitranscribedbyRNAPolII,IIIandIVtoformdouble-strandedRNAs(dsRNA)orviralRNAdependentRNApolymerase(RdRP)togeneratedsRNA.DsRNAtrimmedbyRNaseIII(DroshainnuclearandDicerincytoplasm)tosmallduplexRNAwith19-30bp.AsinglestrandedRNAunwoundbyArgonauteasguideRNAandloadedintoRISC(RNAinducedsilencingcomplex).ComplementarytotargetRNAbyguideRNAinRISCandtriggeredRNAinterference.Doubled-StrandedRNATranscribedfromgenomebyRNAPolII,III&IVorViralRdRPSmallDuplexRNA(19-30nt)trimmedByRNaseIII(Drosha&Dicer)Single-StrandedGuideRNALoadedtoRISCRISCRecognizingTargetRNAtoTriggerRNAiRNAInterferenceTargetRNAtobedegradation.TargetRNAtobetranslationalrepressionordestabilization.TargetRNAtobetranscriptionalrepression.ThegenomiclocusoftargetRNAtobecomeheterochromatinordegradation.34*BiogenesisofmiRNAsandsiRNAsBartel(2004)Cell36*BiogenesisofmiRNAsandsiRNAsBartel(2004)CellSiRNADuplexSiRNAGuideStrandtoloadintoRISCRISCtoRecognizeTargetRNAtotriggerRNAiDicerTrimmingAgo,HelicaseMaturingRNAinterferingAgo,P-bodyPri-miRNAPre-miRNAPre-miRNATranscribingPolII,III&IVDroshaProcessingExportin-5ExitnucleusMiRNAGenomeLociDNA:degradation&heterochromatinRNA:mRNAdegradation&transcriptionblockProtein:translationblockRNAiStructureloci:genomicsegmentmirrorrepeat.Convergenttranscription:twopromoterstolocateeachsidesofonesegment,simultaneoustranscription.Oneversionoftransposontoaligninverselytotheother.Bidirectionaltranscription:twopromoterstolocateeachsideofthegene.Trans-interaction:geneforward-transcript(sensetranscript)tointeractwithpseudogenereverse-transcript(antisensetranscript).Pseudogeneduplicationandorientationopposite.RdRPtodirectdsRNAgenerationinplants,fungiandCelegans,virus,notinhuman.37GenerationofdsRNAasPrecursorforsiRNA&miRNAZamore(2009)NatRevGenet43215638BiogenesesofsiRNA,miRNAandpiRNA,andTheirPathwaysZamore(2009)NatRevGenet

39siRNAiPathwayNaqvi(2009)IntJBiolSci1AnnotationFig:siRNAiPathway40RNAdependentRNApolymerase(RdRP)activityonaberranttranscriptsortranscripthavingfullorpartialcomplementarity.ThesearerecognizedandprocessedbynuclearDicer(differentfromtheoneinvolvedinmicroRNApathway)andthissiRNA-Dicercomplexisthenexportedtocytoplasm.ThesiRNA-DicercomplexthenrecruitsArgonautethatunwindtheduplextoformsi-RISC/RITS.TranscriptsbearingcomplementarysequencestoguidesiRNAstrandarecleavedbyRNaseactivityofArgonaute2.Toconferimmunity,siRNAs-DicercomplexmayalsotrafficinsystemicfashionthatisachievedbySystemicRNAInterference-Defective(SID-1;inanimals)/PhloemSmallRNAbindingprotein-1(PSRP1;inplants).TheexogenoussiRNApathwayfollowsparalleltoendogenouspathway,butdiffersinthefactthatthecytoplasmicDicergeneratesthesiRNAduplexes.TheRITScomplexleadtotranscriptionalgenesilencingthatinvolvesvariousproteins.Naqvi(2009)IntJBiolSci41miRNAiPathwayNaqvi(2009)IntJBiolSciAnnotationFigmiRNAiPathway42Afterbeingtranscribed,thepri-miRNAsstem-loopstructureisacteduponbyDrosha(thatalsoconferstomiRNAstrandandtargetspecificity)andgeneratespre-miRNA.Sometimes,theseprecursorsareeditedbyAdenosineDeaminaseActingonRNA(ADARs)atspecificpositions(generally+4and+44)changingadeninetoinosine.Inplants,theDCL1generatesmiRduplexinthenucleusthatismethylatedatterminalbasesbyHEN1.ThesearethentransportedtocytoplasmwiththeassistanceofExportin-5/HASTY.Fromhere,Dicercomesintoplay(inanimals)andgeneratesmiRNAduplexesthatwillbeincorporatedintomicroRibo-Nucleo-Protein(mi-RNP)complex.AftertheremovalofpassengerstrandmaturemiRNAthenguidesthefunctionalproteincomplextothetargets.Inmammals,miRNAsbearingnuclearsignalsequencescantrafficbacktothenucleus.DependingupontheproteinsassociatedwithmiRNAleadstoeithercleavageoftargetmRNAormodulatethetranslationturnoverby(g)translationactivationorrepressionofrespectivemRNAs.TherepressedmRNAsaretransferredtostructurescalledP-bodies.43EukaryoteNaqvi(2009)IntJBiolSci44MechanismforGenomeEliminationKurth(2009)RNABiolRNAi-GenomeEliminationinTetrahymenaIES:internaleliminationsequenceAnnotationFig:siRNAmediatedgenomicDNAelimination45ProductionofdoublestrandedRNA(thinlines)bybidirectionaltranscriptionofgenomicDNA.ProductionofscnRNAsbydicer-likeproteinDcl1p(yellow);AssociationofscnRNAswithArgonauteTwi1p(green)inthecytoplasm;Scanningintheparentalmacronucleus.TheRNAhelicaseEma1p(red)isrequiredfortheassociationofTwi1pwithnon-codingRNA;HeterochromatinformationandIESeliminationinthedevelopingnewmacronucleus.(histone

methylation:purple;chromodomainproteins:darkgreen;hypotheticalexcisase:orange;IES:internaleliminationsequence,repeat-seqortransposon-like).46

RNAi-SilentChromatinEstablishedviaTargetingNascentSenseRNAbyanti-RNA

Morris(2010)CurrOpinMolTherRISCtoRecognizeGenomeLociviaguidanceofsiRNA

Gene-SilencingComplextoBeRecruitedChromatintobemodified(i)H3K9me3,H3K27me3(ii)DNAme(iii)RemodelersHeterochromatintoEstablish&Maintain1.Target2.Recruit3.Modify4.Heterochromatin47AModelforSmall&LongncRNAsDirectedTranscriptionRepression

Morris(2009)EpigeneticssiRISCtotargetthegenepromoterviaDNAseqorRNAseq.RecruitHDAC&DNMTtomethylateDNA&histone

Directtotranscriptionrepression.LongncRNASmallncRNAMethylationofDNA&histonesLong&SmallncRNAstoTriggerTranscriptionalRepressionviaMethylationofDNA&Histones

Morris(2009)Epigenetics48Longantisensenon-codingRNAsexpressedatbidirectionallytranscribedgenes.ThesenseandantisensencRNAstrandscouldpaireachotherforming2ndstructuredncRNAs,i.e.RNAduplex.Those2ndstructuredncRNAsinteractwithparticularsitesinthepromoterofsensestrandatthebidirectionallytranscribedgeneandalsoinfluencetherecruitmentofAgo-1,DNMT3aandHDAC-1tothistargetsite.SmallsyntheticantisensencRNAscanbedesignedtotakeadvantageoftheendogenousmechanismandalsoutilizethesamepathwaytotranscriptionallysilencegeneexpression.TheendresultofeithersmallorlongantisensencRNAtranscriptionsilencingisthetargetedepigeneticremodelingoftheparticularRNAtargetedgenomeloci.49siRNA-RISCSuppressiononProteinSynthesisCarthewRW(2009)Cell50miRNAsPatternsVsCellTypesinTissues&CancersLee(2008)RNAPearsonCoefficient(miR-1,9,137)miRNAsPattern-Specificityincancercelllines(37celllines).miRNAsPattern-Specificityinnormaltissues(22tissues).miRNAsPattern-SpecificitywithnormalliverVslivercancer.miRNAsPattern-SpecificitywithnormalpancreasVspancreascancer.miRNACancerCellLines(37)-Pattern(copy/cell)miRNATissues(22)-Pattern(copy/cell)pre-miRNAmaturemiRNA51HeterochronicphenotypesofsomeC.elegansmutants.Tworepresentativecelllineages,VandP,showthetransformationscausedbytheabsence(0)orcontinuousactivity(gf)oftwoheterochronicgenes,lin-4andlin-14.VcellsnormallydividetwiceintheL2stage(arrowhead)anddifferentiateattheendoftheL4stage(arrow),buttheseeventsoccuronestageearlyinaprecociousmutantandnotatallintheretardedmutants.PcellsshowacompletelydifferentoverallpatternfromtheVlineage,buttheirfatesarelikewisechangedintheheterochronicmutants—inthiscase,throughalterationofcell-cyclelength(graybar).miRNAVsDevelopment:Lin14,orLin4VsH