基础分子生物第八章

David

BaulcombeForthediscoveryofRNAinterference–genesilencingbydsRNAandsmallRNARNAinterference(RNAi)RISC:RNA-inducedsilencingcomplexDicer-like(DCL)inplantsCarthewandSontheimer,Cell(2009)•SomedsRNAshaveviralorigin•GenomicrepetitivesequencesalsoaresourceofsiRNA•Someevenregulateothergenes(ta-siRNAfortrans-acting)•Somearenaturalsense-antisenseduplex•TransgeneBiogenesis---Source(longstem)PP19-nt双链2-nt3’末端突出5’-磷酸基3’-OH:在动物中3’OH被阻碍后便失去活性植物siRNAs3’甲基化，可能保护或稳定了siRNA分为引导链与乘客链（Guidevspassengerstrands）具有双链的不对称性TuschlT.GeneDev15:188(2001)小干扰RNA(ShortinterferingRNA,siRNA)是引发RNAi的充分必要条件，并且为Dicer的产物OHOHsiRNAs是Dicer作用的产物

DicersbelongtoClassIIIofRNaseIII,afamilyofendoribonucleasesthatshowspecificityfordouble-strandedRNA(dsRNA)----PAZdomainsplustwoRNaseIIIdomains.dsRBD:dsRNAbindingdomainThePAZdomain110aadomainfoundinPiwi,Ago,Zwille&DicerproteinsAbindingpocketinPAZaccommodatesthe2ntoverhangNointeractionsfoundbetweenthe2ntswiththepocket,suggestingthatthepocketaccommodatesallnucleotidecombinationsMaetal.Nature429,318-322(2004)ModelforDicercatalysisThePAZdomainbindsthe2

nt3′overhangofadsRNAterminus.TheRNaseIIIdomainsformapseudo-dimer.Eachdomainhydrolyzesonestrandofthesubstrate.ThebindingsiteofthedsRBDisnotspecified.DicerasamolecularrulertoprocessdsRNAinto~25ntsiRNAThePAZdomainofDicer,amodulethatbindstheendofdsRNA,isseparatedfromthetwocatalyticribonucleaseIII(RNaseIII)domainsbyaflat,positivelychargedsurface.The65angstromdistancebetweenthePAZandRNaseIIIdomainsmatchesthelengthspannedby25basepairsofRNA.Thus,DiceritselfisamolecularrulerthatrecognizesdsRNAandcleavesaspecifieddistancefromthehelicalend.Dicerisamolecularrulerthatmea-suresandcleaves~25nucleotidesfromtheendofadsRNA.ThelengthofthesmallRNAsproducedbyDicerissetbythedistancebetweenthePAZandRNaseIIIdomains,whichislargelyafunctionofthelengthoftheconnectorhelixPrincipleof“molecularruler”RNaseIIIProteins:Dicer-1,Dicer-2,DroshadsRNA-BindingDomainProteins:R2D2,Pasha,LoquaciousArgonauteProteins:Argonaute-1,Argonaute-2,Argonaute-3,Piwi,AubergineTheKeyPlayersinRNASilencingArgonaute(Ago):CentralComponentoftheRNA-InducedSilencingComplex(RISC)CarthewandSontheimer,Cell(2009)136,642-655.MIDdomain:thestructuralbasisfor5’end-specificrecognitionofguideRNAPAZdomainfor3’endbindingofguideRNAPIWIdomainadoptsRNaseHfold,andcancleavethe‘passengerstrand’andmRNAtargetCarthewandSontheimer,Cell(2009)136,642-655.AnactivatedRISCcomplexcontainsArgonauteinassociationwiththeguidestrandofthesiRNAproducedbyDicerAgoproteinscontainPAZdomainandPIWIandMiddomainsLoading&activationofsiRNAsiRNA的装载需要一个双链RNA结合蛋白R2D2。R2D2包含两个一前一后的双链RNA结合结构域Dicer-2与R2D2形成了一个异源二聚体R2D2将与siRNA热稳定性更高的一端结合（3’端）siRNA的不对称性（siRNAasymmetry）

:引导链的5’端稳定性较差Activation---RISCcomplexassemblyAsymmetricalloading&activationofsiRNAsRLCformationInitiationofpassengerstrandcleavageand/orunwindingRISCactivation1.InRLC,Dcr-2andR2D2

(DadR),anRNAbindingproteinwithtandemdsRNAbindingmotifs,formaheterodimerandbindtodouble-strandedsiRNAs2.Dcr-2-R2D2complexsensesthefreeenergydifferencesbetweenthetwo5′endsandbindstothethermo-stableendofsiRNA3.Thedouble-strandsiRNAistransferredtoArgonauteprotein4.Cleavage-competentAGOproteinscleavethepassengerstrand,resultingintheactivationofsiRNAThesiRNAguidestrandisboundatthe5′endbytheMID/PIWIdomainsandatthe3′endbythePAZdomain.mRNAtargetsareinitiallyboundbytheseedregion(near5′end)ofthesiRNAandpairingisextendedtothe3′end.Slicercleavageismeasuredfromthe5′endofthesiRNA.ModelforSlicercatalysisMIDMIDMIDSeedregionRNaseHActivity(targetmRNAcut)CleavageSiteCleavagesite5’-endanchoringpocketsiRNAmRNAInvitroDemonstrationofSlicerActivity•HumanAgo2mixedwith2siRNAsanda500-ntRNAtarget•Productsofexpectedsizewereproduced,dependentonsiRNA,targetRNA,andMg2+Functions---mRNAdegradation5’3’InSomeOrganisms,siRNASignalIsAmplifiedandSpread•NewsiRNAsappearagainstotherregionsoftargetedmRNAs•Signalcanevenspreadtoothercellsinplantsandnemotodes•Amplificationdoesnotseemtobepresentinmammalsincis:sameastriggerRNAintrans:otherthantriggerRNA(Ago)RNA依赖的RNA聚合酶（RNA-dependentRNApolymerase，RDRP)最早克隆自被类病毒侵染的番茄中(1998,PlantCell)与RNA病毒编码的RdRP基本没有同源性在果蝇和哺乳动物基因组中不存在是产生次级小干扰RNA的放大效应的主要因子a,RNAsarenormallynotsilencedbecausetheRDRproteinsdonothaveaccesstothetemplateRNAsequence.Cap-bindingprotein(CBP)andpoly-adenosine-bindingprotein(PABP)maybeinvolvedinthisrestrictionofRDRaccess.Howeverinb,theRDRproteinisallowedaccessbecausetheRNAlacksa5'capor3'poly-adenosinetail,anddsRNAisproducedwhichentersthesiRNApathway.b,TheamplificationprocesswouldresultfromtheabilityofasingleaberrantRNAtogeneratemanymoleculesofsiRNA.cshowstheoutcomeifasmallquantityofprimarysiRNAispresentfromeitheravirus,atransposonorfromacellularRNAthroughtheprocessshowninb.TheantisensestrandofthissiRNAmayannealbybasepairingtoatargetRNAandserveasaprimerfortheRDR.TheresultingdsRNAwouldthenbecleavedbyDicerand,asinb,therewouldbeamplificationbecausemanysecondarysiRNAswouldbeproducedfromeachmoleculeofprimarysiRNA.Howdoestransgene-inducedco-suppressionarise?transgeneCo-suppression(transgeneisdrivenbyviralenhancer,usuallygeneratingaberrantRNAstotriggerdsRNA)Transgene-inducedRNAicanspreadinshortandlongdistance---cell-to-cell/systemicmovementofRNAiGFP-expressingtransgenicplant(inagrobacterium,infiltratedintoaleaf)NonGFPexpression(Co-suppression)非细胞自主(non-cell

autonomous)的RNA沉默与系统性(systemic)RNA沉默RNAi作用可以向其它细胞传递，因此受到其他细胞或组织传来的小RNA的抑制成为非细胞自主的RNA沉默，可以分为两类：细胞与细胞间的传递：包括近程与远程两种系统性传递：通过传输器官（如韧皮部）在不同组织间广泛传播。ThemobilesignalislikelytobesiRNAordsRNAAnintegratedmodelforshort-rangeandlong-rangecell-to-cellmovementofRNAsilencinginplants(onlyafewcellsaway)(dozensofcellsaway)RDR6DCL4（P:胞间连丝）Short-rangecell-to-cellmovement.

Acompanioncell-specificlongdsRNAisprocessedinto21-ntand24-ntprimary

siRNAsbyDCL4andDCL3,respectively.The24-ntsiRNAisdispensablefor

movement.

whereasthe21-ntsiRNAmovestoadjacentcellsinaprocessthatrequiresthe

unidentifiedfactorsSMD1,SMD2andSMD3.Intheabsenceofamplification,theextentofmovementisproportionaltotheinitial

amountofprimarysiRNAproducedinthecompanioncells.Movementisindependent

ofthepresenceofsiRNA-homologoustranscriptsinrecipientcells.P,plasmodesmata.(b)Long-rangecell-to-cellmovement.21-ntprimarysiRNAsenterrecipientcellsproducinghomologoussingle-stranded(ss)transcriptsthatarepronetoamplificationbyRDR6.ThesiRNAsguideRDR6toproducenewdsRNAmolecules,whicharethenprocessedinto21-ntsecondarysiRNAsbyDCL4.Extensivemovementthereforeresultsfromreiteratedshort-distancesignalingeventsinvolvingrelayamplificationof21-ntsiRNAs.RNAi的生物学意义保护基因组免受外源核酸侵入维持基因组稳定参与基因表达调控HostssmallRNAsilencingpathways(RNAi)havebeenrecognizedasessentialcomponentsofplantimmunity.Onewayplantsrespondanddefendagainstvirus(manyareRNAviruses)infectionsisthroughthesmallRNAiimmunesystem.Todealwithplantdefenseresponses,pathogenshaveevolvedsophisticatedmechanismstoavoidandcounterattackthisdefensestrategy.VSR:ViralSuppressorsofRNAsilencingAlmostallplantviralgenomesencodecertainkindofVSRs保护基因组免受外源核酸侵入(transgeneorvirus)ViralRNAsAnArmsRaceBetweenVirusandHostDingandVoinnet,Cell(2007)130,413-422•ViruscouldalsouseRNAitohijackhostdefense,forinstance,byproducingsiRNAtotargetRgenesorRNAicomponenttranscripts.•ViruscanencodeVSRtosuppresshostRNAi•thereislikelyacontinualarmsracebetweenvirusandhost•thesesiRNAcanspreadandcausesubversionofplantdefensesystem.•HostcoulduseRNAitobecomeimmunetoviruses•HostcouldevolveR(Resistance)proteintotargetandrepressVSRpiRNA-mediatedRNAiRNA-directedDNAMethylation(RdDM)维持基因组稳定(Themostcriticalsteptomaintaingenomestabilityistosilencetransposonsorothermobile

DNAelements)

RdDM主要通过siRNA介导相同DNA序列发生重新甲基化(denovomethylation)而实现转录水平的基因沉默，主要发生在植物中。引起相同序列DNA甲基化的是24ntsiRNA

RdDM的生物学意义:

阻抑不必要基因（repetitive

sequences)和有害基因(尤其是transposons)的表达，对维护基因组的稳定至关重要。Functions---RNA-DirectedDNAMethylation(RdDM)tosilencetransposonsormethylaterepetitivesequencesDCL3,24ntsiRNAs,AGO4,DRM2(denovoMTase)AGO424ntrasiRNAAllstepsoccurinthenucleusPreferentiallyoccursforCNNdenovomethylationCanonicalRdDMpathwayinplants1.PolIVmaytranscribemethylatedheterochromatinattransposonsandotherrepetitiveDNAtoproducetheprecursor

transcriptsforsiRNAs.DuringdenovoDNAmethylation,aberranttranscriptsfromthelocusmayserveastemplateforPolIVamplification.2.TheseprecursortranscriptsareconvertedintodsRNAbyRDR2.DCL3cleavesthedsRNAstoproduce24-ntsiRNAs.3.ThesesiRNAsareloadedontoAGO4-containingRISC.siRNAsintheAGO4-RISCcomplexinteractwithnascentPolVtranscripts,therebyrecruitingchromatin-modifyingcomplexes,includingDRM2andhistone-modifyingenzymes,tothetargetloci.TheAGO4-RISCcomplexcontainingsiRNAsguideDRM2forcytosinemethylationoftheDNAsequencecomplementarytothesiRNAs.BiogenesisofPiwi-interactingRNA(piRNA)inDrosophilamelanogasterping-pongamplificaitonNoDNAmethylationinDrosophila!!1.Intheprimarypathway,theprecursorsarecleavedbyanendonuclease,mostlikelyZucchini(Zuc)2.Next,cleavedfragmentsareincorporatedintoPiwiorAubergine(Aub)withthehelpofShutdown(Shu)andHeatshockprotein83(Hsp83,whichistheflyhomologueofHSP90).Fragmentswitha5′Umaybeheavilyselectedforatthisstep.3.AfterloadingintothePiwiprotein,theunknownTrimmerenzymetrimsthe3′endtofitthePiwiprotein,afterwhichHen1methylatesthemature3′end.Thiscompletesprimarybiogenesis.4.Aub,butnotPiwi,canthenenterthesecondarypathway.Aubcanrecognizeacognatetranscriptandcleaveit.The3′cleavagefragmentofthetargetedRNAcanthenbetakenupbyanotherPiwiproteinnamedArgonaute3(Ago3),againwiththehelpofShu5.Furtherdownstream,stepsareprobablyidenticaltoprimarycleavagefragmentsbiogenesis.Inturn,Ago3mayassistinloadingmoreAubproteinwithsecondarypiRNAs.6.Onlyoccursingermlines,toensuregeneticallystable.7.About140piRNAclusters,eachcontainingadistinctpoolofover20transposonsMicroRNA(miRNA)DiscoveryBiogenesisBiogenesisComplexloadingselectionComparewithsiRNAFunctionsmRNAdegradationRibosomedrop-offInitiationblockTechnicalapplicationArtificialmiRNADiscovery1993年,LeeRC等在线虫(C.elegans)中意外地发现了一种定时调控胚胎后期发育的miRNA-lin4,它是一种非编码RNA,长度为22nt。2000年,miRNA-let7的发现掀起了寻找miRNA的热潮。在线虫（C.elegans）当中,通过功能缺失突变体的筛选，找到了let-7和lin-41基因，其中let-7也是长度为22nt的非编码RNA。不同物种中的let-7基因具有序列保守性,且均可与lin-41基因的3’UTR区域互补在lin-41基因的3’UTR区域发现了let-7的互补区TranscriptionofmiRNAgenesbyRNAPolIITranscribedbypolIItopri-miRNA(primaryprecursor)Pri-miRNAcapandapoly(A)tailcontainsthe7-methylguanosinePolIIisphysicallyassociatedwithmiRNAgenepromotersmiRNAgenetranscriptionissensitiveto-amanitinPolIIdependenttranscriptionenablestemporalandspatialregulationofmiRNAproduction.BiogenesisDuandZamore,Development132,4645-4652.animalsplantsBiogenesis----miRNAmicroRNA的作用机理ReductionofmRNAstability(slicing)MostplantmiRNAsguidecleavageoftargetmRNAsOnlyfewanimalmiRNAswerereportedtocleavetargetmRNAsInhibitionofmRNAtranslationMostanimalmiRNAscausereducedtargetproteinlevelswithoutcleavingmRNA(slicing)ButrecentlymanyanimalmiRNAsalsocausereducedtargetmRNAlevels(decay)withoutcleavingmRNA(slicing)AtleastthreeexamplesofplantmiRNAsaffectingtargetproteinbutnotmRNAlevelsAnimalvsPlantmiRNA-mediatedmRNAdecayCarthewandSontheimer,Cell(2009)136,642-655.mRNA成环翻译活跃mRNA开环翻译抑制miRNAEffectsAreMediatedThroughGW182ProteinTritschleretal,Nat.Rev.CellMol.Biol.(2010)11,379-384•GW182showntobindtoAgoproteins.•GW182showntobindtopolyA-bindingprotein(PABPorPABPC1here).•GW182interrupts

the

binding

of

eIF4G

and

PABP,

thus

disrupts

mRNA

circularization.

•open

mRNA

undergoes

deadenylation

and

then

decapping.

动物miRNAs只与它们的靶位点保持很低的互补性：仅为miRNAs中2-7个核苷酸(成为种子区seedsequence)，且决定了miRNA的功能。WithinmiRNAtargetsitesofinvertebratemiRNAs,residuesthatpairwithnucleotides2-7ofthemiRNAsareconservedinorthologousmRNAsofotherspecies.Nucleotide2-7ofthemiRNAarethemostconservedamonghomologousmetazoanmiRNAs.Experimentalevidencealsoindicatesthatnucleotides2-7insiRNAsaremoreimportantthanothersinguidingcleavagemicroRNA靶作用位点——在动物当中的预测和验证PairingtotheseedisnecessaryAdditionalpairingatnt12-17enhancesmiRNAtargetingBindingsitelocationpreference：LocalAUrichregion

植物miRNAs和靶mRNA具很高序列互补性，因此可以利用类似于siRNA介导的剪切机制去剪切靶mRNAsmiRNA基因敲除突变体miRNAs基因过表达突变体在内源启动子作用下表达miRNA-resistanttargets约半数预测的保守miRNA的靶位点都存在于转录因子的mRNAs中(转录因子只占基因的6%)如何研究miRNA的功能？NucleicAcidsResearch,2010,Vol.38,No.206883–6894DRM1/2Functions---RNA-DirectedDenovoMethylation(RdDM)Trans-actingsmallinterferingRNA(ta-siRNA)fourfamiliesofta-siRNA-generatingloci(TASgenes)in

Arabidopsis:AllTAStranscriptsarenon-coding!TAS1:TAS1a,TAS1b,TAS1cTAS2:TAS2TAS4:TAS4TAS3:TAS3a,TAS3b,TAS3cOne-hitpathway(miR173targetsTAS1/2;miR828targetsTAS4)Two-hitpathway(miR390targetsTAS4)ProductionmodelsFunctions---triggersiRNAformation(tasiRNA)SofaronlyfoundinplantsTASpathwaysinplants22ntmiRNA(1)21ntmiRNA(2)pathwayA:onemiRNAguidescleavageatthe5‘endofthemRNAtranscriptpathwayB:

twomiRNAbindingsitesactivateta-siRNAproduction.InpathwayA,aninitialmiRNAprecursorisprocessedbyDCL1andtheresultingmiRNAstrandguidescleavageofanon-protein-codingTAStranscript.InpathwayA,miR173ormiR828bindstothetranscriptandguidescleavagemediatedbyAGO1.RDR6synthesizesadouble-strandedRNAfragmentthatissubsequentlyprocessedbyDCL4intoaphased,21-ntregisterstartingatthemiRNAcleavagesite.OnestrandoftheresultingduplexthentargetsacomplementarymRNAintrans.InpathwayB,miR390bindstothetranscriptattwosites.InArabidopsis,the3’miR390siteiscleavedbyAGO7,whilethe5’miR390isrequiredbutnotcleaved.RDR6synthesizesadouble-strandedRNAfragmentthatissubsequentlyprocessedbyDCL4intoaphased,21-ntregisterfromthe3’miR390cleavagesite.miRNA的生物学功能PlantmiRNAsManyactincelldifferentiationanddevelopmentalpatterningbytargetingtranscriptionfactormRNAsOthermiRNAstargetnon-transcriptionfactormRNAsandmayplayaroleinphysiologicalprocessesorstressresponsesEssentialfunctionsofmiRNAsillustratedbytheembryolethalphenotypeofdcl1nullmutantsAnimalmiRNAsDevelopmentalpatterningEScellslackingDicerareviablebutcannotdifferentiateinvitroandinvivo.DicerknockoutzebrafishlackingbothmaternalandzygoticDicerhaveintactpatterninginthefirst24hbutfailtocontinuewithmorphogenesis