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Transcriptomics轉錄組學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、轉錄組學研究全部RNA的表達及功能轉錄組(transcriptome)指特定狀態(tài)下一種細胞或組織所能轉錄出來的所有RNA的總和?!ň幋aRNA,即mRNA和非編碼RNA(non-codingRNA,ncRNA)轉錄組學(transcriptomics):是在整體水平上研究細胞基因轉錄情況及轉錄調控規(guī)律的科學。RNA組學(RNomics):是分析、鑒定非信使小RNA(smallnon-messengerRNA,snmRNA)在特定狀態(tài)下表達情況、功能及其與蛋白質的相互作用。轉錄組的特點:受到內外多種因素的調節(jié),因而是動態(tài)可變的。能夠揭示不同物種、不同個體、不同細胞、不同發(fā)育階段及不同生理病理狀態(tài)下的基因差異表達信息。ObservingthetranscriptomeFocussedExperimentalApproaches:NorthernBlottingAnalysisRT-PCR(quantitativeorsemi-quantitative)HighthroughputApproaches:ClosedSystemProfiling: MicroarrayexpressionprofilingOpenSystemProfiling: Serialanalysisofgeneexpression(SAGE) MassivelyParallelSignatureSequencing(MPSS)微陣列(microarray)SAGEMPSS研究技術(一)微陣列是大規(guī)?;蚪M表達譜研究的主要技術大規(guī)模表達譜或全景式表達譜(globalexpressionprofile):是生物體(組織、細胞)在某一狀態(tài)下基因表達的整體狀況。微陣列或基因芯片(DNAchip):利用光導化學合成、照相平板印刷以及固相表面化學合成等技術,在固相表面合成成千上萬個寡核苷酸探針,并與放射性同位素或熒光物標記的來自不同細胞、組織或整個器官的DNA或mRNA反轉錄生成的第一鏈cDNA進行雜交,然后用特殊的檢測系統對每個雜交點進行定量分析。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在轉錄物水平研究細胞或組織基因表達模式SAGE的基本原理:利用錨定酶(anchoringenzyme,AE)和位標酶(taggingenzyme,TE)切割DNA分子的特定位置(一般近3’端),分離SAGE標簽(長約14bp,可藉此鑒定基因組中的所有基因),并將這些標簽串聯起來,然后對其進行測序特點:可全面提供生物體基因表達譜信息可用來定量比較不同狀態(tài)下組織或細胞的所有差異表達基因AnchoringEnzymeNlaIII,recognitionsite:The3’terminusofadaptorAandB

arebothTCCRACTAG,wherearecognitionsiteofTaggingEnzymeMmeIflankedwith

NlaIIIHuM,PolyakK.NatureProtocols2006TaggingEnzymeMmeIrecognitionsite:HuM,PolyakK.NatureProtocols2006SAGEAdvantages:Potential‘open’systemmethod–newtranscriptscanbeidentifiedAccuracyofunambiguoustranscriptobservationDigitaloutputofdataQuantitativeandqualitativeinformationDisadvantages:CharacterisingnoveltranscriptsisoftencomputationallydifficultfromshorttagsequencesTagspecificity(recentlyincreasedlengthto21bp)Lengthoftagscanvary(TEenzymeactivityvariablewithtemperature)AsubsetoftranscriptsdonotcontainenzymerecognitionsequenceSensitivetoasubsetofalternativesplicevariants(三)MPSS是以基因測序為基礎的基因表達譜分析新技術MPSS的原理:一個含有能夠特異識別轉錄子的信息標簽序列(10~20bp)與長的連續(xù)分子連接在一起,測出mRNA的一端包含一個10至20個堿基的標簽序列。每一標簽序列在樣品中的頻率(拷貝數)代表了與該標簽序列相應的基因表達水平?;虮磉_水平是以計算mRNA拷貝數為基礎,是一個數字表達系統。只要將病理和對照樣品分別進行測定,即可進行嚴格的統計檢驗,能測定表達水平較低、差異較小的基因,而且不必預先知道基因的序列。四、RNA組學研究全部snmRNA人類基因組序列特點:2萬2.5萬個基因,與蛋白質合成有關的序列占整個基因組的2%左右,其余98%的基因組序列沒有得到注釋。RNA組學研究范疇: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簡介★

(1)長度為21nt左右核苷酸的內源性單鏈小分子RNA;(2)存在65nt左右的發(fā)夾結構前體;(3)基因座位于蛋白質基因間隔區(qū);(4)其DNA序列在近源物種間高度保守?!飉iRNA具有十分重要的調控功能,它們主要參與基因轉錄后水平的調控。能夠通過與靶mRNA特異性的堿基配對引起靶mRNA的降解(植物中較為常見)或者抑制其翻譯(動物中較為常見),從而影響了靶mRNA的表達?!锬壳鞍l(fā)現miRNA是一個龐大的小分子調控RNA家族,廣泛存在于各種動植物中,參與細胞增殖和分化、細胞凋亡、胚胎發(fā)育、形態(tài)建成以及疾病發(fā)生等一系列重要的生命過程。

最近發(fā)現一系列與腫瘤發(fā)生相關的和人類病毒編碼的miRNA,揭示miRNA在哺乳動物基因表達調控中具有重要作用。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

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