基礎分子生物第八章

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雙鏈2-nt3’末端突出5’-磷酸基3’-OH:在動物中3’OH被阻礙后便失去活性植物siRNAs3’甲基化，可能保護或穩(wěn)定了siRNA分為引導鏈與乘客鏈（Guidevspassengerstrands）具有雙鏈的不對稱性TuschlT.GeneDev15:188(2001)小干擾RNA(ShortinterferingRNA,siRNA)是引發(fā)RNAi的充分必要條件，并且為Dicer的產物OHOHsiRNAs是Dicer作用的產物

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的裝載需要一個雙鏈RNA結合蛋白R2D2。R2D2包含兩個一前一后的雙鏈RNA結合結構域Dicer-2與R2D2形成了一個異源二聚體R2D2將與siRNA熱穩(wěn)定性更高的一端結合（3’端）siRNA的不對稱性（siRNAasymmetry）

:引導鏈的5’端穩(wěn)定性較差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依賴的RNA聚合酶（RNA-dependentRNApolymerase，RDRP)最早克隆自被類病毒侵染的番茄中(1998,PlantCell)與RNA病毒編碼的RdRP基本沒有同源性在果蠅和哺乳動物基因組中不存在是產生次級小干擾RNA的放大效應的主要因子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)非細胞自主(non-cell

autonomous)的RNA沉默與系統(tǒng)性(systemic)RNA沉默RNAi作用可以向其它細胞傳遞，因此受到其他細胞或組織傳來的小RNA的抑制成為非細胞自主的RNA沉默，可以分為兩類：細胞與細胞間的傳遞：包括近程與遠程兩種系統(tǒng)性傳遞：通過傳輸器官（如韌皮部）在不同組織間廣泛傳播。ThemobilesignalislikelytobesiRNAordsRNAAnintegratedmodelforshort-rangeandlong-rangecell-to-cellmovementofRNAsilencinginplants(onlyafewcellsaway)(dozensofcellsaway)RDR6DCL4（P:胞間連絲）Short-rangecell-to-cellmovement.

Acompanioncell-speci?clongdsRNAisprocessedinto21-ntand24-ntprimary

siRNAsbyDCL4andDCL3,respectively.The24-ntsiRNAisdispensablefor

movement.

whereasthe21-ntsiRNAmovestoadjacentcellsinaprocessthatrequiresthe

unidenti?edfactorsSMD1,SMD2andSMD3.Intheabsenceofampli?cation,theextentofmovementisproportionaltotheinitial

amountofprimarysiRNAproducedinthecompanioncells.Movementisindependent

ofthepresenceofsiRNA-homologoustranscriptsinrecipientcells.P,plasmodesmata.(b)Long-rangecell-to-cellmovement.21-ntprimarysiRNAsenterrecipientcellsproducinghomologoussingle-stranded(ss)transcriptsthatarepronetoampli?cationbyRDR6.ThesiRNAsguideRDR6toproducenewdsRNAmolecules,whicharethenprocessedinto21-ntsecondarysiRNAsbyDCL4.Extensivemovementthereforeresultsfromreiteratedshort-distancesignalingeventsinvolvingrelayampli?cationof21-ntsiRNAs.RNAi的生物學意義保護基因組免受外源核酸侵入維持基因組穩(wěn)定參與基因表達調控HostssmallRNAsilencingpathways(RNAi)havebeenrecognizedasessentialcomponentsofplantimmunity.Onewayplantsrespondanddefendagainstvirus(manyareRNAviruses)infectionsisthroughthesmallRNAiimmunesystem.Todealwithplantdefenseresponses,pathogenshaveevolvedsophisticatedmechanismstoavoidandcounterattackthisdefensestrategy.VSR:ViralSuppressorsofRNAsilencingAlmostallplantviralgenomesencodecertainkindofVSRs保護基因組免受外源核酸侵入(transgeneorvirus)ViralRNAsAnArmsRaceBetweenVirusandHostDingandVoinnet,Cell(2007)130,413-422?ViruscouldalsouseRNAitohijackhostdefense,forinstance,byproducingsiRNAtotargetRgenesorRNAicomponenttranscripts.?ViruscanencodeVSRtosuppresshostRNAi?thereislikelyacontinualarmsracebetweenvirusandhost?thesesiRNAcanspreadandcausesubversionofplantdefensesystem.?HostcoulduseRNAitobecomeimmunetoviruses?HostcouldevolveR(Resistance)proteintotargetandrepressVSRpiRNA-mediatedRNAiRNA-directedDNAMethylation(RdDM)維持基因組穩(wěn)定(Themostcriticalsteptomaintaingenomestabilityistosilencetransposonsorothermobile

DNAelements)

RdDM主要通過siRNA介導相同DNA序列發(fā)生重新甲基化(denovomethylation)而實現轉錄水平的基因沉默，主要發(fā)生在植物中。引起相同序列DNA甲基化的是24ntsiRNA

RdDM的生物學意義:

阻抑不必要基因（repetitive

sequences)和有害基因(尤其是transposons)的表達，對維護基因組的穩(wěn)定至關重要。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等在線蟲(C.elegans)中意外地發(fā)現了一種定時調控胚胎后期發(fā)育的miRNA-lin4,它是一種非編碼RNA,長度為22nt。2000年,miRNA-let7的發(fā)現掀起了尋找miRNA的熱潮。在線蟲（C.elegans）當中,通過功能缺失突變體的篩選，找到了let-7和lin-41基因，其中l(wèi)et-7也是長度為22nt的非編碼RNA。不同物種中的let-7基因具有序列保守性,且均可與lin-41基因的3’UTR區(qū)域互補在lin-41基因的3’UTR區(qū)域發(fā)現了let-7的互補區(qū)TranscriptionofmiRNAgenesbyRNAPolIITranscribedbypolIItopri-miRNA(primaryprecursor)Pri-miRNAcapandapoly(A)tailcontainsthe7-methylguanosinePolIIisphysicallyassociatedwithmiRNAgenepromotersmiRNAgenetranscriptionissensitiveto-amanitinPolIIdependenttranscriptionenablestemporalandspatialregulationofmiRNAproduction.BiogenesisDuandZamore,Development132,4645-4652.animalsplantsBiogenesis----miRNAmicroRNA的作用機理ReductionofmRNAstability(slicing)MostplantmiRNAsguidecleavageoftargetmRNAsOnlyfewanimalmiRNAswerereportedtocleavetargetmRNAsInhibitionofmRNAtranslationMostanimalmiRNAscausereducedtargetproteinlevelswithoutcleavingmRNA(slicing)ButrecentlymanyanimalmiRNAsalsocausereducedtargetmRNAlevels(decay)withoutcleavingmRNA(slicing)AtleastthreeexamplesofplantmiRNAsaffectingtargetproteinbutnotmRNAlevelsAnimalvsPlantmiRNA-mediatedmRNAdecayCarthewandSontheimer,Cell(2009)136,642-655.mRNA成環(huán)翻譯活躍mRNA開環(huán)翻譯抑制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.

動物miRNAs只與它們的靶位點保持很低的互補性：僅為miRNAs中2-7個核苷酸(成為種子區(qū)seedsequence)，且決定了miRNA的功能。WithinmiRNAtargetsitesofinvertebratemiRNAs,residuesthatpairwithnucleotides2-7ofthemiRNAsareconservedinorthologousmRNAsofotherspecies.Nucleotide2-7ofthemiRNAarethemostconservedamonghomologousmetazoanmiRNAs.Experimentalevidencealsoindicatesthatnucleotides2-7insiRNAsaremoreimportantthanothersinguidingcleavagemicroRNA靶作用位點——在動物當中的預測和驗證PairingtotheseedisnecessaryAdditionalpairingatnt12-17enhancesmiRNAtargetingBindingsitelocationpreference：LocalAUrichregion

植物miRNAs和靶mRNA具很高序列互補性，因此可以利用類似于siRNA介導的剪切機制去剪切靶mRNAsmiRNA基因敲除突變體miRNAs基因過表達突變體在內源啟動子作用下表達miRNA-resistanttargets約半數預測的保守miRNA的靶位點都存在于轉錄因子的mRNAs中(轉錄因子只占基因的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的生物學功能PlantmiRNAsManyactincelldifferentiationanddevelopmentalpatterningbytargetingtranscriptionfactormRNAsOthermiRNAstargetnon-transcriptionfactormRNAsandmayplayaroleinphysiologicalprocessesorstressresponsesEssentialfunctionsofmiRNAsillustratedbytheembryolethalphenotypeofdcl1nullmutantsAnimalmiRNAsDevelopmentalpatterningEScellslackingDicerareviablebutcannotdifferentiateinvitroandinvivo.DicerknockoutzebrafishlackingbothmaternalandzygoticDicerhaveintactpatterninginthefirst24hbutfailtocontinuewithmorphogenesis