理学分子生物学英文

1理学分子生物学英文第1页/共91页2MolecularBiologyoftheGene,5/E

---Watsonetal.(2004)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenome

PartIII:ExpressionoftheGenomePartIV:RegulationPartV:Methods2005-5-10第2页/共91页3Chapter16RegulationprinciplesandHowgenesareregulatedinbacteriaChapter17BasicmechanismofgeneexpressionineukaryotesChapter18ThemechanismofRNAiandtheroleofmiRNAindevelopmentandcancer第3页/共91页4Chapter18:RNAiandmiRNAregulationMolecularBiologyCourse第4页/共91页5OutlinesThebackgroundanddiscoveryofmiRNAsRNAidiscoveryandmechanismmiRNAbiogenesisandregulationmiRNArolesindevelopment,celldifferentiationandvirusmiRNAincancersiRNAapplication第5页/共91页6Topic1:ThebackgroundanddiscoveryofmiRNAsCHAPTER18RNAiandmiRNAregulation一、miRNA发现的背景和miRNA发现第6页/共91页7后基因组时代的分子生物学MaintenanceoftheGenome

(基因组保持)ExpressionoftheGenome (基因组表达)Regulation(调控)第7页/共91页8DNADoubleHelix(1953)FrancisH.CrickJamesD.Watson生命科学20世纪的里程碑第8页/共91页中心法则－TheCentralDogma(1953-1956)是分子生物学的基本框架DNARNAProteinReplication复制Transcription转录Translation翻译第9页/共91页10后基因组时代的中心法则RNAprocessingGeneregulationDNArepairandrecombination基因组的保持基因组的表达第10页/共91页11后基因组时代的基因调控：RNA调控MostoftheRNAtranscribedfromyourgenomedoesn’tmakeprotein.CarinaDennistalkstotherevolutionarieswhobelievethatitfunctionsingene-regulatorynetworksthatunderliethecomplexityofhigherorganisms.第11页/共91页12含有30亿对碱基的人类基因组仅含有2－3万个蛋白质基因，是果蝇的两倍，啤酒酵母的4倍。显而易见，生物的复杂性不由编码蛋白质的数目决定。人类基因组的蛋白质编码区的总和占总基因组长度为1－2％，那么其他98％的基因组有什么功能呢？当然，在这98％的非蛋白质编码基因组序列里，约24％为插入编码序列的内含子序列；人类基因平均每个基因有7个内含子。但这么冗长的内含子序列有什么生物学功能呢？人类基因组草图带给科学家们的困惑第12页/共91页13人类基因组绝大部分都被转录成RNA，细胞内非编码RNA的数量是编码RNA的上百倍。这促使许多科学家认为生物体复杂性被隐藏在它们所输出的非编码RNA内，而非编码序列内。

第13页/共91页14ThediscoveryofmiRNAs

miRNAwasfirstdiscoveredin1993byVictorAmbrosatHarvard(lin-4)ThesecondmiRNALet-7wasdiscoveredin2000byFrankSlackasapostdocatHarvard

(Ruvkunlab)VictorAmbrosGaryRuvkun第14页/共91页15ThefirstdiscoeredmiRNA:lin-4RuvkunG,WightmanB,HaI.The20yearsittooktorecognizetheimportanceoftinyRNAs.Cell.2004Jan23;116(2Suppl):S93-6.LeeR,FeinbaumR,AmbrosV.AshorthistoryofashortRNA.Cell.2004Jan23;116(2Suppl):S89-92Thoughttobeanodditynotageneralphenomenon第15页/共91页16BreakthroughwithBlastNofthesecondmiRNA(stRNA)let-7PasquinelliAE,ReinhartBJ,SlackF,MartindaleMQ,KurodaMI,MallerB,HaywardDC,BallEE,DegnanB,MullerP,SpringJ,SrinivasanA,FishmanM,FinnertyJ,CorboJ,LevineM,LeahyP,DavidsonE,RuvkunG.Conservationofthesequenceandtemporalexpressionoflet-7heterochronicregulatoryRNA.Nature.2000Nov2;408(6808):86-9.第16页/共91页17microRNAshadbeenneglectedforsomanyyearsbecauseoftheirsmallsize.OOPs!第17页/共91页18第18页/共91页19ThenumberoftheidentifiedmiRNAsisgrowingrapidlyinrecentyears.Over5000miRNAshavebeenfounduntiltheAugustofthisyear(ThemiRBaseSequenceDatabase).ThesemiRNAsarefromprimates,rodents,birds,fish,worms,flies,plantsandviruses.Thedataarefreelyavailabletoallthroughthewebinterfaceathttp://microrna.sanger.ac.uk/sequences/andinflatfileformfromftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.第19页/共91页20第20页/共91页21Topic2:RNAinterferenceanditsmechanism二、RNA干扰及其机制CHAPTER18RNAiandmiRNAregulation第21页/共91页221Double-strandedRNAinhibitsexpressionofgeneshomologoustothatRNA.[phenomena-现象]双链RNA抑制含其同源序列基因的表达第22页/共91页232006年的诺贝尔生理学奖获得者：AndrewZ.FireCraigC.Mello第23页/共91页24Fig2.AnalysisofRNA-interferenceeffectsinindividualcells.FluorescencemicrographsshowprogenyofinjectedanimalsfromGFP-reporterstrainPD4251(aC.elegansstrainexpressingGFPfluorescenceprotein)(使用外源导入的报告基因).Younglarva(幼虫)Adult(成虫)adultbodywallathighmagnification(高放大倍数的成虫体壁)ControldsRNAds-gfpRNA第24页/共91页25Fig3.Effectsofmex-3RNAinterferenceonlevelsoftheendogenousmRNA(insituhybridizationinembryos)(胚胎的原位杂交).adultbodywallathighmagnification(高放大倍数的成虫体壁)Nohybridizationandstaining+hybridization(endogenousmex-3RNA)+antisense+hybridization+dsmex-3RNA+hybridization第25页/共91页26ThediscoveryofRNAiexplainsthevirus-inducedgenesilencinginplants(植物病毒引起的基因沉默).

Mostplantviruseshavesingle-strandedRNAgenomes,whicharereleasedfromtheproteincoatoftheirvirusparticlesastheyenteracell.TheirgenomicRNAisthenreplicatedbythevirusencodedRNA-dependentRNApolymerasetoproducesenseandantisenseRNA,whichcanhybridizetoformdsRNAandtriggeranRNAiresponseagainsttheirownsequences.第26页/共91页272.ShortinterferingRNA(siRNAs)areproducedfromdsRNAanddirectmachinerythatswitchoffgenesinvariousway.[Mechanism-机制]从双链RNA产生的小干扰RNA可以指导用不同机制关闭基因的细胞机器第27页/共91页28Thequestiontobeaddressedis“WhyexogenousdsRNAcaninhibitexpressionofgeneshomologoustothatRNA?”第28页/共91页29Figure17-30RNAisilencingExogenousdsRNA外源双链RNA第29页/共91页30ThetargetsoftheRNAi-directedgenesilencingDegradationofthetargetmRNA(引起靶标mRNA的降解),InhibitionoftranslationofthetargetmRNA(抑制靶标mRNA的翻译),Silencingthegenetranscriptionfromthetargetpromoter(引起靶标启动子的转录沉默).第30页/共91页31TheheartoftheRNAimechanismDicer:anRNaseIII-likemultidomainribonucleasethatfirstprocessesinputdsRNAintosmallfragmentscalledshortinterferingRNAs(siRNAs)ormicroRNAs(miRNA).DicerthenhelpsloaditssmallRNAproductsintoRISC.RISC

(RNAinducedsilencingcomplexes)(RNA诱导的沉默复合体):alargemultiproteincomplexthatdirecttheboundsiRNAormiRNAtoitstargetandinhibitthetargetgeneexpression.第31页/共91页32Dicer:Structuralorganization:---APAZdomain,bindstheendofthedsRNA---TwoRNaseIIIdomains---Othernon-conserveddomains.贾第鞭毛虫第32页/共91页33ThecrystalstructureoftheGiardiaintactDicerenzymeshowsthatthePAZdomain,amodulethatbindstheendofdsRNA,isseparatedfromthetwocatalyticRNaseIIIdomainsbyaflat,positivelychargedsurface. The65angstromdistancebetweenthePAZandRNaseIIIdomainsmatchesthelengthspannedby25basepairsofRNA.Thus,DiceritselfisamolecularrulerthatrecognizesdsRNAandcleavesaspecifieddistancefromthehelicalend.第33页/共91页34RISC:thekeycomponentisArgonaute(AGO)Argonaute(AGO):AlargeproteinfamilythatconstituteskeycomponentsofRISCs.---AGOproteinsarecharacterizedbytwouniquedomains,PAZandPIWI,whosefunctionsarenotfullyunderstood.CurrentevidencesuggeststhatthePAZdomainbindsthe3’-endtwo-nucleotideoverhangsofthesiRNAduplex,whereasthePIWIdomainofsomeAGOproteinsconferssliceractivity.PAZandPIWIdomainsarebothessentialtoguidetheinteractionbetweenthesiRNAandthetargetmRNAforcleavageortranslationalrepression.---DistinctAGOmembershavedistinctfunctions.Forexample,humanAGO2programsRISCstocleavethemRNAtarget,whereasAGO1andAGO3donot.第34页/共91页35第35页/共91页36AmodelforsiRNA-guided

mRNAcleavagebyArgonaute第36页/共91页37ThemultiplefunctionsofRNAi第37页/共91页38Topic3:miRNAbiogenesisandregulation三、miRNA生成和调控CHAPTER18RNAiandmiRNAregulation第38页/共91页391.MicroRNA(miRNA)&itsprocessing微小RNA及其加工第39页/共91页40MicroRNA(miRNA):

Atypeofnon-codingsmallRNA(~21–23nucleotides)producedbyDicerfromastem-loopstructuredRNAprecursor(~70-90ntsong)(结构和来源).miRNAsarewidelyexpressedinanimalandplantcellsandfunctionsintheformofRNA–proteincomplexes,termedmiRISCs.miRNAshavebeenimplicatedinthecontrolofdevelopmentbecausetheyleadtothedestructionortranslationalsuppressionoftargetmRNAswithhomologytothemiRNA(生物学功能和机制).第40页/共91页41ThemiRNAgenesandStructureofpri-miRNAsPri-miRNAsbearthe5’capand3’poly(A)tails第41页/共91页42miRNAprocessingPri-miRNA(miRNA初级转录产物)Drosha(1)pre-miRNA(miRNA前体)Dicer(2)miRNAExportin5(Exp5)transportspre-miRNAtothecytoplasm第42页/共91页43Atypicalmetazoanpri-miRNAconsistsofastemofapproximately33bp,withaterminalloopandflankingsegments.Theterminalloopisunessential,whereastheflankingssRNAsegmentsarecriticalforprocessing.Thecleavagesiteisdeterminedmainlybythedistance(approximately11bp)fromthestem-ssRNAjunction.第43页/共91页44Hanetal.,Cell125,887–901,June2,2006第44页/共91页45HumanDroshaandDicersharethesameRNaseIIIdomainsanddsRNAbindingdomain.第45页/共91页462.MicroRNA(miRNA)targetsandregulation.第46页/共91页47AcomparisonbetweenmiRNAandsiRNA第47页/共91页48RNAsilencingindifferentorganisms第48页/共91页49ThreestrategiesofmiRNAandtargetrecognition(targetsarelocatingin3’UTRs).第49页/共91页50Topic4:miRNArolesindevelopment,celldifferentiationandvirusCHAPTER18RNAiandmiRNAregulation四、MicroRNA在发育中的调控作用，及其他作用第50页/共91页51VictorR.Ambros秀丽线虫C.elegans1.miRNAinC.elegansdevelopment第51页/共91页52lin-4andlet-7miRNAscontrolthedevelopmentaltimeofC.elegans.第52页/共91页53Expressionoflin-4allowsC.eleganstoproceedtothelatedevelopmentalstage第53页/共91页54lin-4

bindsitstargetmRNAsbyimperfectbasepairing.第54页/共91页552.miRNAsinvertebratedevelopment:

TherearealotunknownbecausethethelackofefficientmethodstouncoverthetargetsofmiRNAs.第55页/共91页56Figure2.ExpressionofmiR-124aandmiR-1inZebrafish,Medaka,Mouse,andFly.

miR-124aisrestrictedlyexpressedinthebrainandthespinalcordinfishandmouseortotheventralnervecordinthefly.TheexpressionofmiR-1isrestrictedtothemusclesandtheheartinthemouse.青鳉斑马鱼小鼠果蝇LearningthemiRNAfunctionfromitsexpressionpattern第56页/共91页573.miRNAcontrolsplantphenotypes(控制植物表型特征)Jaw-miRNA控制拟南芥叶形变化(Nature,2003)第57页/共91页58(Science2004)3种miRNA控制造血干细胞向淋巴细胞的分化过程4.miRNAcontrolsthedifferentiationofthehematopoieticstemcell(调控造血干细胞的分化)第58页/共91页595.SomevirusesencodemiRNAs(有些病毒编码miRNAs)第59页/共91页60第60页/共91页61Topic5:miRNAincancer五、微小RNA在癌症发生中的作用CHAPTER18RNAiandmiRNAregulation第61页/共91页62miRNAsinhuman:

Thereareabout500miRNAsfromhumanhavebeenfoundandannotated.Theyarenamedashas-miRx.第62页/共91页63miRNAexpressionpatternchangesduringoncogenesis,andisuniqueforeachcancer.微小RNA在癌症发生中表达谱的变化第63页/共91页64第64页/共91页65Figure3,ComparisonbetweennormalandtumorsamplesrevealsglobalchangesinmiRNAexpression.第65页/共91页66OnemechanismofmiRNAcontrollingoncogeneexpression

微小RNA调控癌基因表达的一种机制。第66页/共91页67c-Mycisahelix–loop–helixleucinezippertranscriptionfactorthatregulatesanestimated10–15%ofgenesinthehumanandDrosophilagenomes.c-MycactivatesexpressionofaclusterofsixmiRNAsonhumanchromosome13.(Figure1)E2F1isthetranscriptionfactor,whichisatargetofc-Mycthatpromotescellcycleprogression.ExpressionofE2F1isnegativelyregulatedbytwomiRNAsinthiscluster,miR-17-5pandmiR-20a.(Figure1)第67页/共91页68第68页/共91页69Used2’-O-methylAntisenseoligonucleotidestodownregulatethelevelofmiR-17-5pandmiR-20a,andthenanalyzedtheprotein(B-Western)andmRNAlevels(C-Northen)ofE2F1.第69页/共91页70SomemicroRNAsarepotentialoncogenes

有些微小RNA可能是致癌基因。第70页/共91页71B-细胞淋巴瘤第71页/共91页72Figure1.Themir-17–92clustershowsincreasedexpressioninB-celllymphomasamplesandcelllines.

Thelevelofmir-17–92pri-miRNAwasdeterminedbyreal-timequantitativeRT-PCRin46lymphomasand47colorectalcarcinomas,andcomparedtolevelsfoundincorrespondingnormaltissuesfromfiveindividuals.第72页/共91页73Figure2.Overexpressionofthemir-17–19bclusteracceleratesc-myc-inducedlymphomagenesisinmice.第73页/共91页74Topic6:siRNAapplicationCHAPTER18RNAiandmiRNAregulation六、siRNA的应用第74页/共91页75siRNAapplicationinmammalianTransfectexogenoussiRNAintocells(transientexpression)Chemicalsynthesis:expensiveInvitrotranscriptionofpre-miRNAwithT7promoter.InvitrotranscriptionoflongdsRNAbythatarethencleavedbyE.coliRNaseIIIorRNaseIII-likeDICER.ExpressionofsiRNAinculturedcellsorinanimalmodelssiRNAproducedwithpolIIIpromoterfromthetransfectedDNAplasmids.第75页/共91页76TranscriptionfromRNAPIIIpromotersofU6andH1arewellcharacterized.RNAPIIItranscriptionusesawell-definedterminationsignal(TTTTT)andtheproductshavenoextrasequence.2.Transcriptionfromthesepromotersisveryefficientinvarioustissues.ExpressionofhairpinRNA(shRNA)usingPolIIIpromoters第76页/共91页77第77页/共91页78AmammalianexpressionvectordesignedtodirecttheintracellularsynthesisofsiRNAs.第78页/共91页79CreateinducedphenotypesthatcanbeobservedoverlongtimespansCreateastablyengineeredcellscanbeassayedeitherinvitroorinvivo,perhapstestingtheangiogenic(血管生成)

ormetastatic(转移)

potentialsoftumorcellsinxenograftmodels(异种移植模型)。Createhypomorphicalleles(亚等位基因)rapidlyintransgenicmice.第79页/共91页804.CombineshRNAswithexistinghigh-efficiencygenedeliveryvehiclestocreatebonafideRNAi-basedtherapeutics.Forexample,ultimately,tosilenceadisease-causingmutantallelespecifically.第80页/共91页81ResearchApplicationsofRNAi:Anewstrategyofreversegenetics&anovelwayofgeneknock-outItcanbeusedinreversegenetics(反向遗传学)toidentifythecellularorbiologicalfunctionofagene.Itcanbecombined