分子细胞生物学进展：Noncoding RNA regulation

1NoncodingRNAregulation(siRNA,miRNAandlncRNA)

2DNADoubleHelix(1953)FrancisH.CrickJamesD.Watson生命科学20世纪的里程碑中心法则－TheCentralDogma(1953-1956)是分子生物学的基本框架DNARNAProteinReplicationTranscriptionTranslation34后基因组时代的分子生物学MaintenanceoftheGenome

ExpressionoftheGenome

RegulationoftheGenome5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2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6RNAdiscriminationInthecell,genomicDNAistranscribedintovarioustypesofRNA.ButnotallRNAsaretranslatedintoproteins.Doesthisgiveprotein-codingRNAsgreatercredibilityintermsoffunction?Viewsdiffer.

310|NATURE|VOL482|16FEBRUARY20127后基因组时代的基因调控：RNA调控CarinaDennis(Editorof50YearsOfDNA)

8MostoftheRNAtranscribedfromyourgenomedoesn’tmakeprotein.RNAfunctionsingene-regulatorynetworksthatunderliethecomplexityofhigherorganisms.CarinaDennis(Editorof50YearsOfDNA)

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。9

MONIKAS.KOWALCZYK&DOUGLASR.HIGGS,2012,Nature482:310-31110OutlinesThebackgroundanddiscoveryofmiRNAsRNAidiscoveryandmechanismmiRNAbiogenesisandregulationmiRNArolesindevelopment,celldifferentiationandvirusmiRNAincancerandinflammationsiRNAapplicationlncRNARNAMethylation11Topic1:ThebackgroundanddiscoveryofmiRNAs一、miRNA发现与背景ThediscoveryofmiRNAs12miRNAwasfirstdiscoveredin1993byVictorAmbrosatHarvard(lin-4)VictorAmbrosThesecondmiRNALet-7

wasdiscoveredin2000byFrankSlackasapostdocatHarvard

(Ruvkunlab)13GaryRuvkunThefirstdiscoveredmiRNA:lin-414RuvkunG,WightmanB,HaI.The20yearsittooktorecognizetheimportanceoftinyRNAs.Cell.2004Jan23;116(2Suppl):S93-6.LeeR,FeinbaumR,AmbrosV.AshorthistoryofashortRNA.Cell.2004Jan23;116(2Suppl):S89-92ThoughttobeanodditynotageneralphenomenonBreakthroughwiththesecondmiRNAlet-715Conservationofthesequenceandtemporalexpressionoflet-7heterochronicregulatoryRNA.

Nature.2000Nov2;408(6808):86-9.16microRNAshadbeenneglectedforsomanyyearsbecauseoftheirsmallsize.OOPs!Member,WhiteheadInstitute

ProfessorofBiology,MIT

InvestigatoroftheHowardHughesMedicalInstitute1718miRBase21released(June,2014)/Thedatabasecontains28645entriesrepresentinghairpinprecursormiRNAs,expressing35828maturemiRNAproducts,in233species(primates,rodents,birds,fish,worms,flies,plantsandviruses).**Homosapiens(1881precursors,2588mature)[GRCh38]19Topic2:RNAinterferenceanditsmechanism二、RNA干扰与机制201.Double-strandedRNAinhibitsexpressionofgeneshomologoustothatRNA.双链RNA抑制含其同源序列基因的表达212006年的诺贝尔生理学奖获得者：AndrewZ.FireCraigC.Mello22Fig3.Effectsofmex-3RNAinterferenceonlevelsoftheendogenousmRNA

(insituhybridizationinembryos)Nohybridizationandstaining+hybridization(endogenousmex-3RNA)+antisense+hybridization+dsmex-3RNA+hybridization23ThediscoveryofRNAiexplainsthevirus-inducedgenesilencinginplants(植物病毒引起的基因沉默)

Mostplantviruseshavesingle-strandedRNAgenomes,whicharereleasedfromtheproteincoatoftheirvirusparticlesastheyenteracell.TheirgenomicRNAisthenreplicatedbythevirusencodedRNA-dependentRNApolymerasetoproducesenseandantisenseRNA,whichcanhybridizetoformdsRNA

andtriggeranRNAiresponseagainsttheirownsequences.242.ShortinterferingRNA(siRNAs)areproducedfromdsRNAanddirectmachinerythatswitchoffgenesinvariousway.从双链RNA产生的小干扰RNA可以指导细胞机器以不同机制来关闭基因25DegradationofthetargetmRNA(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).ThetargetsoftheRNAi-directedgenesilencing26Thequestiontobeaddressedis:“WhyexogenousdsRNAcaninhibitexpressionofgeneshomologoustothatRNA?”27ExogenousdsRNA28TheheartoftheRNAimechanismDicer:

ARNaseIII-likemultidomainribonucleasethatfirstprocessesinputdsRNAintosmallfragmentscalledshortinterferingRNAs(siRNAs)ormicroRNAs(miRNA).DicerthenhelpsloaditssmallRNAproductsintoRISC.2.RISC(RNAinducedsilencingcomplexes,RNA诱导的沉默复合体):AlargemultiproteincomplexthatdirecttheboundsiRNAormiRNAtoitstargetandinhibitthetargetgeneexpression.29DicerStructuralorganization:---APAZdomain,bindstheendofthedsRNA---TwoRNaseIIIdomains---Othernon-conserveddomains.贾第鞭毛虫30ThecrystalstructureoftheGiardiaintactDicerenzymeshowsthatthePAZdomain,amodulethatbindstheendofdsRNA,isseparatedfromthetwocatalyticRNaseIIIdomains

byaflat,positivelychargedsurface.Dicer31The65angstromdistancebetweenthePAZandRNaseIIIdomainsmatchesthelengthspannedby25basepairsofRNA.Thus,DiceritselfisamolecularrulerthatrecognizesdsRNAandcleavesaspecifieddistancefromthehelicalend.Dicer32RISC:thekeycomponentisArgonaute(AGO)AGO233AmodelforsiRNA-guidedmRNAcleavagebyArgonauteAGO234TheStructureofHumanArgonaute-2inComplexwithmiR-20aAGO235RISC:thekeycomponentisArgonaute(AGO)Argonaute(AGO)AlargeproteinfamilythatconstituteskeycomponentsofRISCs,AGO1,2,3,4.---DistinctAGOmembershavedistinctfunctions.Forexample,humanAGO2programsRISCstocleavethemRNAtarget,whereasAGO1andAGO3donot.36RISC:thekeycomponentisArgonaute(AGO)Argonaute(AGO)----AGOproteinsarecharacterizedbytwouniquedomains,PAZandPIWI,whosefunctionsarenotfullyunderstood.CurrentevidencesuggeststhatthePAZdomainbindsthe3’-endtwo-nucleotideoverhangsofthesiRNAduplex,whereasthePIWI

domainofsomeAGOproteinsconferssliceractivity.PAZandPIWIdomainsarebothessentialtoguidetheinteractionbetweenthesiRNAandthetargetmRNAforcleavageortranslationalrepression.37ThemultiplefunctionsofRNAi38Topic3:miRNAbiogenesisandregulation三、miRNA生成和调控391.MicroRNA(miRNA)&itsprocessing40MicroRNA(miRNA)

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

(~70-90nts)(结构和来源).MiRNAsarewidelyexpressedinanimalandplantcellsandfunctionsintheformofRNA–proteincomplexes,termedmiRISCs.miRNAshavebeenimplicatedinthecontrolofdevelopmentbecausetheyleadtothedestructionortranslationalsuppressionoftargetmRNAswithhomologytothemiRNA(生物学功能和机制).41ThemiRNAgenesandStructureofpri-miRNAspri-miRNAsbearthe5’capand3’poly(A)tails42miRNAprocessingpri-miRNA(初级转录产物)Drosha(1)pre-miRNA(miRNA前体)Dicer(2)

miRNAExportin5(Exp5)transportspre-miRNAtothecytoplasm43Hanetal.,2006,Cell125,887–901,44Atypicalpri-miRNAconsistsofastemofapproximately33bp,withaterminalloopandflankingsegments.Theterminalloopisunessential,whereastheflankingssRNAsegmentsarecriticalforprocessing.Thecleavagesiteisdeterminedmainlybythedistance(approximately11bp)fromthestem-ssRNAjunction.pri-miRNApre-miRNA45HumanDroshaandDicersharethesameRNaseIIIdomainsanddsRNAbindingdomain.46miRNAbiogenesisandmechanismsofaction(RueggerS,2012)472.MicroRNAtargetsandregulation48ComparisonbetweenmiRNAandsiRNA49siRNA/miRNAsilencingindifferentorganisms果蝇拟南芥酵母线虫rasiRNA=RepeatassociatedsmallinterferingRNA(rasiRNA),piRNAs50Topic4:miRNArolesindevelopment,celldifferentiationandvirus四、MicroRNA调控作用51VictorR.Ambros秀丽线虫C.elegans1.miRNAinC.elegansdevelopment52lin-4andlet-7miRNAs

controlthedevelopmentaltimeofC.elegans.53Expressionoflin-4

allowsC.eleganstoproceedtothelatedevelopmentalstage54lin-4

bindsitstargetmRNAs

byimperfectbasepairing.552.miRNAsinvertebratedevelopment:

TherearealotunknownbecausethelackofefficientmethodstouncoverthetargetsofmiRNAs.56Figure2.ExpressionofmiR-124aandmiR-1inZebrafish,Medaka,Mouse,andFly.

miR-124aisrestrictedlyexpressedinthebrainandthespinalcordinfishandmouseortotheventralnervecordinthefly.TheexpressionofmiR-1isrestrictedtothemusclesandtheheartinthemouse.青鳉斑马鱼小鼠果蝇LearningthemiRNAfunctionfromitsexpressionpattern573.miRNAcontrolsplantphenotypes(控制植物表型特征)Jaw-miRNA控制拟南芥叶形变化(Nature,2003)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)595.SomevirusesencodemiRNAs(有些病毒编码miRNAs)6061Topic5:miRNAincancer五、miRNA在癌症发生中的作用62miRNAsinhuman:

Thereareabout2588maturemiRNAsfromhumanhavebeenfoundandannotated.Theyarenamedashsa-miRxx.63miRNAexpressionpatternchangesduringoncogenesis,andisuniqueforeachcancer.微小RNA在癌症发生中表达谱的变化6465Figure3,ComparisonbetweennormalandtumorsamplesrevealsglobalchangesinmiRNAexpression.66OnemechanismofmiRNAcontrollingoncogeneexpression

微小RNA调控癌基因表达的一种机制。67c-Mycisahelix–loop–helixleucinezippertranscriptionfactorthatregulatesanestimated10–15%ofgenesinthehumanandDrosophilagenomes.c-MycactivatesexpressionofaclusterofsixmiRNAsonhumanchromosome13.(Figure1)E2F1isthetranscriptionfactor,whichisatargetofc-Mycthatpromotescellcycleprogression.ExpressionofE2F1isnegativelyregulatedbytwomiRNAsinthiscluster,miR-17-5pandmiR-20a.(Figure1)6869SomemicroRNAsarepotentialoncogenes

有些微小RNA可能是致癌基因70B-细胞淋巴瘤71Figure1.Themir-17–92clustershowsincreasedexpressioninB-celllymphomasamplesandcelllines.

The

levelofmir-17–92pri-miRNAwasdeterminedbyreal-timequantitativeRT-PCRin46lymphomasand47colorectalcarcinomas,andcomparedtolevelsfoundincorrespondingnormaltissuesfromfiveindividuals.72Figure2.Overexpressionofthemir-17–19bclusteracceleratesc-myc-inducedlymphomagenesisinmice.73ProposedschemeforthetreatmentoflivercancerwithcombinedchemotherapyandmiRNA-basedtherapy74Topic6:siRNAapplication六、siRNA的应用75siRNAapplicationinmammalianTransfectexogenoussiRNAintocells

(transientexpression)Chemicalsynthesis:expensiveInvitrotranscriptionofpre-miRNAwithT7promoter.InvitrotranscriptionoflongdsRNAbythatarethencleavedbyE.coliRNaseIIIorRNaseIII-likeDICER.ExpressionofsiRNAinculturedcellsorinanimalmodelssiRNAproducedwithpolIIIpromoterfromthetransfectedDNAplasmids.76TranscriptionfromRNAPIIIpromotersofU6andH1arewellcharacterized.RNAPIIItranscriptionusesawell-definedterminationsignal(TTTTT)andtheproductshavenoextrasequence.2.Transcriptionfromthesepromotersisveryefficientinvarioustissues.ExpressionofhairpinRNA(shRNA)usingPolIIIpromoters7778AmammalianexpressionvectordesignedtodirecttheintracellularsynthesisofsiRNAsCreateinducedphenotypesthatcanbeobservedoverlongtimespansCreateastablyengineeredcellscanbeassayedeitherinvitroorinvivo,perhapstestingtheangiogenic(血管生成)ormetastatic(转移)potentialsoftumorcellsinxenograftmodels(异种移植模型)。Createhypomorphicalleles(亚等位基因)rapidlyintransgenicmice.CombineshRNAswithexistinghigh-efficiencygenedeliveryvehiclestocreatebonafideRNAi-basedtherapeutics.Forexample,ultimately,tosilenceadisease-causingmutantallelespecifically.79ResearchApplicationsofRNAi:

Anewstrategyofreversegenetics&anovelwayofgeneknock-outItcanbeusedinreversegenetics(反向遗传学)toidentifythecellularorbiologicalfunctionofagene.Itcanbecombinedwithgenomicstoperformlarge-scalegeneticscreensaimedatgenediscovery.80TherapeuticApplicationsofRNAi:

AnewstrategytoinventionofnewdrugsandgenetherapysiRNAscanbeusedtocounterviralinfectionbyspecificallydestroyingthemRNAsofthepathogenicviruses,suchasHIVandHBV.siRNAscanbeappliedtocountercancersbyspecificallydown-regulatetheexpressionofgenesrelatedtooncogenesis.8182Topic7:Longnon-codingRNAfunctionsandregulations七、lncRNA功能和调控83RNAinunexpectedplaces:Longnon-codingRNAfunctionsindiversecellularcontextsSarahGeislerandJeffCollerLongnoncodingRNAs(lncRNA)Longerthan200nucleotides.UnlikesmallncRNAs,lncRNAscanfoldintocomplexsecondaryandhigherorderstructures.Asimpotantregulatorsindiversebilogicalprocess84LncRNAsinvolvedincancer85ClassificationofLongnoncodingRNAs(lncRNA)86BasicmodulesoflncRNAactionRNA-ProteinRNA-DNARNA-RNA