WelcomeEachofYoutoMyMolecularBiologyClass1MolecularBiologyoftheGene,5/E

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

PartIII:ExpressionoftheGenomePartIV:RegulationPartV:Methods2005-5-102Chapter16RegulationprinciplesandHowgenesareregulatedinbacteriaChapter17BasicmechanismofgeneexpressionineukaryotesChapter18ThemechanismofRNAiandtheroleofmiRNAindevelopmentandcancer3Chapter8:RNAinregulationThemechanismofRNAiandtheroleofmiRNAindevelopmentandcancerMolecularBiologyCourse4Topic1:RNAinterferenceanditsmechanismCHAPTER18RNAiandmiRNAindevelopmentandcancergenesis一、RNA干擾及其機(jī)制51Double-strandedRNAinhibitsexpressionofgeneshomologoustothatRNA.[phenomena-現(xiàn)象]雙鏈RNA抑制含其同源序列基因的表達(dá)62006年的諾貝爾生理學(xué)獎獲得者：AndrewZ.FireCraigC.Mello7Fig2.AnalysisofRNA-interferenceeffectsinindividualcells.FluorescencemicrographsshowprogenyofinjectedanimalsfromGFP-reporterstrainPD4251(aC.elegansstrainexpressingGFPfluorescenceprotein)(使用外源導(dǎo)入的報告基因).Younglarva(幼蟲)Adult(成蟲)adultbodywallathighmagnification(高放大倍數(shù)的成蟲體壁)ControldsRNAds-gfpRNA8Fig3.Effectsofmex-3RNAinterferenceonlevelsoftheendogenousmRNA(insituhybridizationinembryos)(胚胎的原位雜交).adultbodywallathighmagnification(高放大倍數(shù)的成蟲體壁)Nohybridizationandstaining+hybridization(endogenousmex-3RNA)+antisense+hybridization+dsmex-3RNA+hybridization9Anplantimmunesystem:Virus-inducedgenesilencing(植物病毒引起的基因沉默).

Mostplantviruseshavesingle-strandedRNAgenomes,whicharereleasedfromtheproteincoatoftheirvirusparticlesastheyenteracell.TheirgenomicRNAisthenreplicatedbythevirusencodedRNA-dependentRNApolymerasetoproducesenseandantisenseRNA,whichcanhybridizetoformdsRNAandtriggeranRNAiresponseagainsttheirownsequences.102.ShortinterferingRNA(siRNAs)areproducedfromdsRNAanddirectmachinerythatswitchoffgenesinvariousway.[Mechanism-機(jī)制]從雙鏈RNA產(chǎn)生的小干擾RNA可以指導(dǎo)用不同機(jī)制關(guān)閉基因的細(xì)胞機(jī)器11Thequestiontobeaddressedis“WhyexogenousdsRNAcaninhibitexpressionofgeneshomologoustothatRNA?”12Figure17-30RNAisilencingExogenousdsRNA外源雙鏈RNA13ThetargetsoftheRNAi-directedgenesilencingDegradationofthetargetmRNA(引起靶標(biāo)mRNA的降解),InhibitionoftranslationofthetargetmRNA(抑制靶標(biāo)mRNA的翻譯),Silencingthegenetranscriptionfromthetargetpromoter(引起靶標(biāo)啟動子的轉(zhuǎn)錄沉默).14TheheartoftheRNAimechanismDicer:anRNaseIII-likemultidomainribonucleasethatfirstprocessesinputdsRNAintosmallfragmentscalledshortinterferingRNAs(siRNAs)ormicroRNAs(miRNA).DicerthenhelpsloaditssmallRNAproductsintoRISC.RISC

(RNAinducedsilencingcomplexes)(RNA誘導(dǎo)的沉默復(fù)合體):alargemultiproteincomplexthatdirecttheboundsiRNAormiRNAtoitstargetandinhibitthetargetgeneexpression.15Dicer:Structuralorganization:---APAZdomain,bindstheendofthedsRNA---TwoRNaseIIIdomains---Othernon-conserveddomains.賈第鞭毛蟲16ThecrystalstructureoftheGiardiaintactDicerenzymeshowsthatthePAZdomain,amodulethatbindstheendofdsRNA,isseparatedfromthetwocatalyticRNaseIIIdomainsbyaflat,positivelychargedsurface. The65angstromdistancebetweenthePAZandRNaseIIIdomainsmatchesthelengthspannedby25basepairsofRNA.Thus,DiceritselfisamolecularrulerthatrecognizesdsRNAandcleavesaspecifieddistancefromthehelicalend.17RISC:thekeycomponentisArgonaute(AGO)AGO2DicerTRBP18Argonaute(AGO):AlargeproteinfamilythatconstituteskeycomponentsofRISCs.---AGOproteinsarecharacterizedbytwouniquedomains,PAZandPIWI,whosefunctionsarenotfullyunderstood.CurrentevidencesuggeststhatthePAZdomainbindsthe3’-endtwo-nucleotideoverhangsofthesiRNAduplex,whereasthePIWIdomainofsomeAGOproteinsconferssliceractivity.PAZandPIWIdomainsarebothessentialtoguidetheinteractionbetweenthesiRNAandthetargetmRNAforcleavageortranslationalrepression.---DistinctAGOmembershavedistinctfunctions.Forexample,humanAGO2programsRISCstocleavethemRNAtarget,whereasAGO1andAGO3donot.1920AmodelforsiRNA-guided

mRNAcleavagebyArgonaute21ThemultiplefunctionsofRNAi223.MicroRNA(miRNA)&itsprocessing微小RNA及其加工23MicroRNA(miRNA):

Atypeofnon-codingsmallRNA(~21–23nucleotides)producedbyDicerfromastem-loopstructuredRNAprecursor(~70-90nts

ong)(結(jié)構(gòu)和來源).miRNAsarewidelyexpressedinanimalandplantcellsasRNA–proteincomplexes,termedmiRISCs,andhavebeenimplicatedinthecontrolofdevelopmentbecausetheyleadtothedestructionortranslationalsuppressionoftargetmRNAswithhomologytothemiRNA(生物學(xué)功能和機(jī)制).24Structureofpri-miRNAsPri-miRNAsbearthe5’capand3’poly(A)tails25miRNAprocessingPri-miRNA(miRNA初級轉(zhuǎn)錄產(chǎn)物)Drosha(1)pre-miRNA(miRNA前體)Dicer(2)miRNAExportin5(Exp5)transportspre-miRNAtothecytoplasm26HumanDroshaandDicersharethesameRNaseIIIdomainsanddsRNAbindingdomain.27ThenumberoftheidentifiedmiRNAsisgrowingrapidlyinrecentyears.Over4000miRNAshavebeenfounduntiltheOctoberofthisyear(ThemiRBaseSequenceDatabase).Release9.0(Oct,2006)ofthedatabasecontains4361entriesrepresentinghairpinprecursormiRNAs,expressing4167maturemiRNAproducts,inprimates,rodents,birds,fish,worms,flies,plantsandviruses.Thedataarefreelyavailabletoallthroughthewebinterfaceathttp://microrna.sanger.ac.uk/sequences/andinflatfileformfromftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.28Topic2:miRNAsinanimaldevelopmentandotherfunctionsCHAPTER18RNAiandmiRNAindevelopmentandcancergenesis二、MicroRNA在發(fā)育中的調(diào)控作用，及其他作用29VictorR.Ambros秀麗線蟲C.elegans1.miRNAinC.elegansdevelopment30lin-4andlet-7miRNAscontrolthedevelopmentaltimeofC.elegans.31Expressionoflin-4allowsC.eleganstoproceedtothelatedevelopmentalstage32lin-4

bindsitstargetmRNAsbyimperfectbasepairing.332.miRNAsinvertebratedevelopment:

TherearealotunknownbecausethethelackofefficientmethodstouncoverthetargetsofmiRNAs.34Figure2.Expressionof

miR-124aand

miR-1inZebrafish,Medaka,Mouse,andFly.

miR-124aisrestrictedlyexpressedinthebrainandthespinalcordinfishandmouseortotheventralnervecordinthefly.Theexpressionof

miR-1isrestrictedtothemusclesandtheheartinthemouse.青鳉斑馬魚小鼠果蠅LearningthemiRNAfunctionfromitsexpressionpattern35miRNAcontrolssomeplantphenotype(控制植物表型特征)Jaw-miRNA控制擬南芥葉形變化(Nature,2003)36(Science2004)3種miRNA控制造血干細(xì)胞向淋巴細(xì)胞的分化過程miRNAcontrolsthedifferentiationofthehematopoieticstemcell(調(diào)控造血干細(xì)胞的分化)37SomevirusesencodemiRNAs(有些病毒編碼miRNAs)38Topic3:miRNAincancerCHAPTER18RNAiandmiRNAindevelopmentandcancergenesis三、微小RNA在癌癥發(fā)生中的作用39miRNAsinhuman:

Thereareabout500miRNAsfromhumanhavebeenfoundandannotated.Theyarenamedashas-miRx.40miRNAexpressionpatternchangesduringoncogenesis,andisuniqueforeachcancer.微小RNA在癌癥發(fā)生中表達(dá)譜的變化4142Figure3,ComparisonbetweennormalandtumorsamplesrevealsglobalchangesinmiRNAexpression.43OnemechanismofmiRNAcontrollingoncogeneexpression

微小RNA調(diào)控癌基因表達(dá)的一種機(jī)制。44c-Mycisahelix–loop–helixleucinezippertranscriptionfactorthatregulatesanestimated10–15%ofgenesinthehumanandDrosophilagenomes.c-MycactivatesexpressionofaclusterofsixmiRNAsonhumanchromosome13.(Figure1)E2F1isthetranscriptionfactor,whichisatargetofc-Mycthatpromotescellcycleprogression.ExpressionofE2F1isnegativelyregulatedbytwomiRNAsinthiscluster,miR-17-5pandmiR-20a.(Figure1)4546Used2’-O-methylAntisenseoligonucleotidestodownregulatethelevelofmiR-17-5pandmiR-20a,andthenanalyzedtheprotein(B-Western)andmRNAlevels(C-Northen)ofE2F1.47SomemicroRNAsarepotentialoncogenes

有些微小RNA可能是致癌基因。48B-細(xì)胞淋巴瘤49Figure1.Themir-17–92clustershowsincreasedexpressioninB-celllymphomasamplesandcelllines.

Thelevelofmir-17–92pri-miRNAwasdeterminedbyreal-timequantitativeRT-PCRin46lymphomasand47colorectalcarcinomas,andcomparedtolevelsfoundincorrespondingnormaltissuesfromfiveindividuals.50Figure2.Overexpressionofthemir-17–19bclusteracceleratesc-myc-inducedlymphomagenesisinmice.51Topic4:siRNAapplicationCHAPTER18RNAiandmiRNAindevelopmentandcancergenesis四、siRNA的應(yīng)用52siRNAapplicationinmammalianTransfectexogenoussiRNAintocells(transientexpression)Chemicalsynthesis:expensiveInvitrotranscriptionofpre-miRNAwithT7promoter.InvitrotranscriptionoflongdsRNAbythatarethencleavedbyE.coliRNaseIIIorRNaseIII-likeDICER.ExpressionofsiRNAinculturedcellsorinanimalmodelssiRNAproducedwithpolIIIpromoterfromthetransfectedDNAplasmids.53TranscriptionfromRNAPIIIpromotersofU6andH1arewellcharacterized.RNAPIIItranscriptionusesawell-definedterminationsignal(TTTTT)andtheproductshavenoextrasequence.2.Transcriptionfromthesepromotersisveryefficientinvarioustissues.ExpressionofhairpinRNA(shRNA)usingPolIIIpromoters5455AmammalianexpressionvectordesignedtodirecttheintracellularsynthesisofsiRNAs.56CreateinducedphenotypesthatcanbeobservedoverlongtimespansCreateastablyengineeredcellscanbeassayedeitherinvitroorinvivo,perhapstestingtheangiogenic(血管生成)

ormetastatic

(轉(zhuǎn)移)

potentialsoftumorcellsinxenograftmodels(異種移植模型)。Createhypomorphicalleles(亞等位基因)rapidlyintransgenicmice.574.CombineshRNAswithexistinghigh-efficiencygenedeliveryvehiclestocreatebonafideRNAi-basedtherapeutics.Forexample,ultimately,tosilenceadisease-causingmutantallelespecifically.58ResearchApplicationsofRNAi: