基礎(chǔ)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(dòng)物中3’OH被阻礙后便失去活性植物siRNAs3’甲基化，可能保護(hù)或穩(wěn)定了siRNA分為引導(dǎo)鏈與乘客鏈（Guidevspassengerstrands）具有雙鏈的不對(duì)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(chǎn)物OHOHsiRNAs是Dicer作用的產(chǎn)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(gè)雙鏈RNA結(jié)合蛋白R(shí)2D2。R2D2包含兩個(gè)一前一后的雙鏈RNA結(jié)合結(jié)構(gòu)域Dicer-2與R2D2形成了一個(gè)異源二聚體R2D2將與siRNA熱穩(wěn)定性更高的一端結(jié)合（3’端）siRNA的不對(duì)稱性（siRNAasymmetry）

:引導(dǎo)鏈的5’端穩(wěn)定性較差A(yù)ctivation---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(méi)有同源性在果蠅和哺乳動(dòng)物基因組中不存在是產(chǎn)生次級(jí)小干擾RNA的放大效應(yīng)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(xì)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(xì)胞傳遞，因此受到其他細(xì)胞或組織傳來(lái)的小RNA的抑制成為非細(xì)胞自主的RNA沉默，可以分為兩類：細(xì)胞與細(xì)胞間的傳遞：包括近程與遠(yuǎn)程兩種系統(tǒng)性傳遞：通過(guò)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(xué)意義保護(hù)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(dá)調(diào)控HostssmallRNAsilencingpathways(RNAi)havebeenrecognizedasessentialcomponentsofplantimmunity.Onewayplantsrespondanddefendagainstvirus(manyareRNAviruses)infectionsisthroughthesmallRNAiimmunesystem.Todealwithplantdefenseresponses,pathogenshaveevolvedsophisticatedmechanismstoavoidandcounterattackthisdefensestrategy.VSR:ViralSuppressorsofRNAsilencingAlmostallplantviralgenomesencodecertainkindofVSRs保護(hù)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(guò)siRNA介導(dǎo)相同DNA序列發(fā)生重新甲基化(denovomethylation)而實(shí)現(xiàn)轉(zhuǎn)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(xué)意義:

阻抑不必要基因（repetitive

sequences)和有害基因(尤其是transposons)的表達(dá)，對(duì)維護(hù)基因組的穩(wěn)定至關(guān)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(chóng)(C.elegans)中意外地發(fā)現(xiàn)了一種定時(shí)調(diào)控胚胎后期發(fā)育的miRNA-lin4,它是一種非編碼RNA,長(zhǎng)度為22nt。2000年,miRNA-let7的發(fā)現(xiàn)掀起了尋找miRNA的熱潮。在線蟲(chóng)（C.elegans）當(dāng)中,通過(guò)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(zhǎng)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(bǔ)在lin-41基因的3’UTR區(qū)域發(fā)現(xiàn)了let-7的互補(bǔ)區(qū)TranscriptionofmiRNAgenesbyRNAPolIITranscribedbypolIItopri-miRNA(primaryprecursor)Pri-miRNAcapandapoly(A)tailcontainsthe7-methylguanosinePolIIisphysicallyassociatedwithmiRNAgenepromotersmiRNAgenetranscriptionissensitiveto-amanitinPolIIdependenttranscriptionenablestemporalandspatialregulationofmiRNAproduction.BiogenesisDuandZamore,Development132,4645-4652.animalsplantsBiogenesis----miRNAmicroRNA的作用機(jī)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(kāi)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(dòng)物miRNAs只與它們的靶位點(diǎn)保持很低的互補(bǔ)性：僅為miRNAs中2-7個(gè)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(diǎn)——在動(dòng)物當(dāng)中的預(yù)測(cè)和驗(yàn)證PairingtotheseedisnecessaryAdditionalpairingatnt12-17enhancesmiRNAtargetingBindingsitelocationpreference：LocalAUrichregion

植物miRNAs和靶mRNA具很高序列互補(bǔ)性，因此可以利用類似于siRNA介導(dǎo)的剪切機(jī)制去剪切靶mRNAsmiRNA基因敲除突變體miRNAs基因過(guò)表達(dá)突變體在內(nèi)源啟動(dòng)子作用下表達(dá)miRNA-resistanttargets約半數(shù)預(yù)測(cè)的保守miRNA的靶位點(diǎn)都存在于轉(zhuǎn)錄因子的mRNAs中(轉(zhuǎn)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(xué)功能PlantmiRNAsManyactincelldifferentiationanddevelopmentalpatterningbytargetingtranscriptionfactormRNAsOthermiRNAstargetnon-transcriptionfactormRNAsandmayplayaroleinphysiologicalprocessesorstressresponsesEssentialfunctionsofmiRNAsillustratedbytheembryolethalphenotypeofdcl1nullmutantsAnimalmiRNAsDevelopmentalpatterningEScellslackingDicerareviablebutcannotdifferentiateinvitroandinvivo.DicerknockoutzebrafishlackingbothmaternalandzygoticDicerhaveintactpatterninginthefirst24hbutfailtocontinuewithmorphogenesis