分子生物学英文课件.ppt

WelcomeEachofYoutoMyMolecularBiologyClass 2 MolecularBiologyoftheGene 5 E Watsonetal 2004 PartI ChemistryandGeneticsPartII MaintenanceoftheGenomePartIII ExpressionoftheGenomePartIV RegulationPartV Methods 2005 5 10 3 Chapter16RegulationprinciplesandHowgenesareregulatedinbacteriaChapter17BasicmechanismofgeneexpressionineukaryotesChapter18ThemechanismofRNAiandtheroleofmiRNAindevelopmentandcancer 4 Chapter18 RNAiandmiRNAregulation MolecularBiologyCourse 5 OutlinesThebackgroundanddiscoveryofmiRNAsRNAidiscoveryandmechanismmiRNAbiogenesisandregulationmiRNArolesindevelopment celldifferentiationandvirusmiRNAincancersiRNAapplication 6 Topic1 ThebackgroundanddiscoveryofmiRNAs CHAPTER18RNAiandmiRNAregulation 一 miRNA发现的背景和miRNA发现 7 后基因组时代的分子生物学 MaintenanceoftheGenome 基因组保持 ExpressionoftheGenome 基因组表达 Regulation 调控 8 DNADoubleHelix 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 DNA RNA Protein Replication复制 Transcription转录 Translation翻译 10 后基因组时代的中心法则 RNAprocessing Generegulation DNArepairandrecombination 基因组的保持 基因组的表达 11 后基因组时代的基因调控 RNA调控 MostoftheRNAtranscribedfromyourgenomedoesn tmakeprotein CarinaDennistalkstotherevolutionarieswhobelievethatitfunctionsingene regulatorynetworksthatunderliethecomplexityofhigherorganisms 12 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 是果蝇的两倍 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 13 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 14 ThediscoveryofmiRNAs miRNAwasfirstdiscoveredin1993byVictorAmbrosatHarvard lin 4 ThesecondmiRNALet 7wasdiscoveredin2000byFrankSlackasapostdocatHarvard Ruvkunlab VictorAmbros GaryRuvkun 15 ThefirstdiscoeredmiRNA lin 4 RuvkunG WightmanB HaI The20yearsittooktorecognizetheimportanceoftinyRNAs Cell 2004Jan23 116 2Suppl S93 6 LeeR FeinbaumR AmbrosV AshorthistoryofashortRNA Cell 2004Jan23 116 2Suppl S89 92 Thoughttobeanodditynotageneralphenomenon 16 BreakthroughwithBlastNofthesecondmiRNA stRNA let 7 PasquinelliAE ReinhartBJ SlackF MartindaleMQ KurodaMI MallerB HaywardDC BallEE DegnanB MullerP SpringJ SrinivasanA FishmanM FinnertyJ CorboJ LevineM LeahyP DavidsonE RuvkunG Conservationofthesequenceandtemporalexpressionoflet 7heterochronicregulatoryRNA Nature 2000Nov2 408 6808 86 9 17 microRNAshadbeenneglectedforsomanyyearsbecauseoftheirsmallsize OOPs 18 19 ThenumberoftheidentifiedmiRNAsisgrowingrapidlyinrecentyears Over5000miRNAshavebeenfounduntiltheAugustofthisyear ThemiRBaseSequenceDatabase ThesemiRNAsarefromprimates rodents birds fish worms flies plantsandviruses Thedataarefreelyavailabletoallthroughthewebinterfaceathttp microrna sanger ac uk sequences andinflatfileformfromftp ftp sanger ac uk pub mirbase sequences 20 21 Topic2 RNAinterferenceanditsmechanism 二 RNA干扰及其机制 CHAPTER18RNAiandmiRNAregulation 22 1Double strandedRNAinhibitsexpressionofgeneshomologoustothatRNA phenomena 现象 双链RNA抑制含其同源序列基因的表达 23 2006年的诺贝尔生理学奖获得者 AndrewZ FireCraigC Mello 24 Fig2 AnalysisofRNA interferenceeffectsinindividualcells FluorescencemicrographsshowprogenyofinjectedanimalsfromGFP reporterstrainPD4251 aC elegansstrainexpressingGFPfluorescenceprotein 使用外源导入的报告基因 Younglarva 幼虫 Adult 成虫 adultbodywallathighmagnification 高放大倍数的成虫体壁 ControldsRNA ds gfpRNA 25 Fig3 Effectsofmex 3RNAinterferenceonlevelsoftheendogenousmRNA insituhybridizationinembryos 胚胎的原位杂交 adultbodywallathighmagnification 高放大倍数的成虫体壁 Nohybridizationandstaining hybridization endogenousmex 3RNA antisense hybridization dsmex 3RNA hybridization 26 ThediscoveryofRNAiexplainsthevirus inducedgenesilencinginplants 植物病毒引起的基因沉默 Mostplantviruseshavesingle strandedRNAgenomes whicharereleasedfromtheproteincoatoftheirvirusparticlesastheyenteracell TheirgenomicRNAisthenreplicatedbythevirusencodedRNA dependentRNApolymerasetoproducesenseandantisenseRNA whichcanhybridizetoformdsRNAandtriggeranRNAiresponseagainsttheirownsequences 27 2 ShortinterferingRNA siRNAs areproducedfromdsRNAanddirectmachinerythatswitchoffgenesinvariousway Mechanism 机制 从双链RNA产生的小干扰RNA可以指导用不同机制关闭基因的细胞机器 28 Thequestiontobeaddressedis WhyexogenousdsRNAcaninhibitexpressionofgeneshomologoustothatRNA 29 Figure17 30RNAisilencing ExogenousdsRNA外源双链RNA 30 ThetargetsoftheRNAi directedgenesilencingDegradationofthetargetmRNA 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 31 TheheartoftheRNAimechanismDicer anRNaseIII likemultidomainribonucleasethatfirstprocessesinputdsRNAintosmallfragmentscalledshortinterferingRNAs siRNAs ormicroRNAs miRNA DicerthenhelpsloaditssmallRNAproductsintoRISC RISC RNAinducedsilencingcomplexes RNA诱导的沉默复合体 alargemultiproteincomplexthatdirecttheboundsiRNAormiRNAtoitstargetandinhibitthetargetgeneexpression 32 Dicer Structuralorganization APAZdomain bindstheendofthedsRNA TwoRNaseIIIdomains Othernon conserveddomains 贾第鞭毛虫 33 ThecrystalstructureoftheGiardiaintactDicerenzymeshowsthatthePAZdomain amodulethatbindstheendofdsRNA isseparatedfromthetwocatalyticRNaseIIIdomainsbyaflat positivelychargedsurface The65angstromdistancebetweenthePAZandRNaseIIIdomainsmatchesthelengthspannedby25basepairsofRNA Thus DiceritselfisamolecularrulerthatrecognizesdsRNAandcleavesaspecifieddistancefromthehelicalend 34 RISC thekeycomponentisArgonaute AGO Argonaute AGO AlargeproteinfamilythatconstituteskeycomponentsofRISCs AGOproteinsarecharacterizedbytwouniquedomains PAZandPIWI whosefunctionsarenotfullyunderstood CurrentevidencesuggeststhatthePAZdomainbindsthe3 endtwo nucleotideoverhangsofthesiRNAduplex whereasthePIWIdomainofsomeAGOproteinsconferssliceractivity PAZandPIWIdomainsarebothessentialtoguidetheinteractionbetweenthesiRNAandthetargetmRNAforcleavageortranslationalrepression DistinctAGOmembershavedistinctfunctions Forexample humanAGO2programsRISCstocleavethemRNAtarget whereasAGO1andAGO3donot 35 36 AmodelforsiRNA guidedmRNAcleavagebyArgonaute 37 ThemultiplefunctionsofRNAi 38 Topic3 miRNAbiogenesisandregulation 三 miRNA生成和调控 CHAPTER18RNAiandmiRNAregulation 39 1 MicroRNA miRNA itsprocessing 微小RNA及其加工 40 MicroRNA miRNA Atypeofnon codingsmallRNA 21 23nucleotides producedbyDicerfromastem loopstructuredRNAprecursor 70 90ntsong 结构和来源 miRNAsarewidelyexpressedinanimalandplantcellsandfunctionsintheformofRNA proteincomplexes termedmiRISCs miRNAshavebeenimplicatedinthecontrolofdevelopmentbecausetheyleadtothedestructionortranslationalsuppressionoftargetmRNAswithhomologytothemiRNA 生物学功能和机制 41 ThemiRNAgenesandStructureofpri miRNAs Pri miRNAsbearthe5 capand3 poly A tails 42 miRNAprocessing Pri miRNA miRNA初级转录产物 Drosha 1 pre miRNA miRNA前体 Dicer 2 miRNA Exportin5 Exp5 transportspre miRNAtothecytoplasm 43 Atypicalmetazoanpri miRNAconsistsofastemofapproximately33bp withaterminalloopandflankingsegments Theterminalloopisunessential whereastheflankingssRNAsegmentsarecriticalforprocessing Thecleavagesiteisdeterminedmainlybythedistance approximately11bp fromthestem ssRNAjunction 44 Hanetal Cell125 887 901 June2 2006 45 HumanDroshaandDicersharethesameRNaseIIIdomainsanddsRNAbindingdomain 46 2 MicroRNA miRNA targetsandregulation 47 AcomparisonbetweenmiRNAandsiRNA 48 RNAsilencingindifferentorganisms 49 ThreestrategiesofmiRNAandtargetrecognition targetsarelocatingin3 UTRs 50 Topic4 miRNArolesindevelopment celldifferentiationandvirus CHAPTER18RNAiandmiRNAregulation 四 MicroRNA在发育中的调控作用 及其他作用 51 VictorR Ambros 秀丽线虫C elegans 1 miRNAinC elegansdevelopment 52 lin 4andlet 7miRNAscontrolthedevelopmentaltimeofC elegans 53 Expressionoflin 4allowsC eleganstoproceedtothelatedevelopmentalstage 54 lin 4bindsitstargetmRNAsbyimperfectbasepairing 55 2 miRNAsinvertebratedevelopment TherearealotunknownbecausethethelackofefficientmethodstouncoverthetargetsofmiRNAs 56 Figure2 ExpressionofmiR 124aandmiR 1inZebrafish Medaka Mouse andFly miR 124aisrestrictedlyexpressedinthebrainandthespinalcordinfishandmouseortotheventralnervecordinthefly TheexpressionofmiR 1isrestrictedtothemusclesandtheheartinthemouse 青鳉 斑马鱼 小鼠 果蝇 LearningthemiRNAfunctionfromitsexpressionpattern 57 3 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 59 5 SomevirusesencodemiRNAs 有些病毒编码miRNAs 60 61 Topic5 miRNAincancer 五 微小RNA在癌症发生中的作用 CHAPTER18RNAiandmiRNAregulation 62 miRNAsinhuman Thereareabout500miRNAsfromhumanhavebeenfoundandannotated Theyarenamedashas miRx 63 miRNAexpressionpatternchangesduringoncogenesis andisuniqueforeachcancer 微小RNA在癌症发生中表达谱的变化 64 65 Figure3 ComparisonbetweennormalandtumorsamplesrevealsglobalchangesinmiRNAexpression 66 OnemechanismofmiRNAcontrollingoncogeneexpression微小RNA调控癌基因表达的一种机制 67 c Mycisahelix loop helixleucinezippertranscriptionfactorthatregulatesanestimated10 15 ofgenesinthehumanandDrosophilagenomes c MycactivatesexpressionofaclusterofsixmiRNAsonhumanchromosome13 Figure1 E2F1isthetranscriptionfactor whichisatargetofc Mycthatpromotescellcycleprogression ExpressionofE2F1isnegativelyregulatedbytwomiRNAsinthiscluster miR 17 5pandmiR 20a Figure1 68 69 Used2 O methylAntisenseoligonucleotidestodownregulatethelevelofmiR 17 5pandmiR 20a andthenanalyzedtheprotein B Western andmRNAlevels C Northen ofE2F1 70 SomemicroRNAsarepotentialoncogenes有些微小RNA可能是致癌基因 71 B 细胞淋巴瘤 72 Figure1 Themir 17 92clustershowsincreasedexpressioninB celllymphomasamplesandcelllines Thelevelofmir 17 92pri miRNAwasdeterminedbyreal timequantitativeRT PCRin46lymphomasand47colorectalcarcinomas andcomparedtolevelsfoundincorrespondingnormaltissuesfromfiveindividuals 73 Figure2 Overexpressionofthemir 17 19bclusteracceleratesc myc inducedlymphomagenesisinmice 74 Topic6 siRNAapplication CHAPTER18RNAiandmiRNAregulation 六 siRNA的应用 75 siRNAapplicationinmammalian TransfectexogenoussiRNAintocells transientexpression Chemicalsynthesis expensiveInvitrotranscriptionofpre miRNAwithT7promoter InvitrotranscriptionoflongdsRNAbythatarethencleavedbyE coliRNaseIIIorRNaseIII likeDICER ExpressionofsiRNAinculturedcellsorinanimalmodelssiRNAproducedwithpolIIIpromoterfromthetransfectedDNAplasmids 76 TranscriptionfromRNAPIIIpromotersofU6andH1arewellcharacterized RNAPIIItranscriptionusesawell definedterminationsignal TTTTT andtheproductshavenoextrasequence 2 Transcriptionfromthesepromotersisveryefficientinvarioustissues ExpressionofhairpinRNA shRNA usingPolIIIpromoters 77 78 AmammalianexpressionvectordesignedtodirecttheintracellularsynthesisofsiRNAs 79 CreateinducedphenotypesthatcanbeobservedoverlongtimespansCreateastablyengineeredcellscanbeassayedeitherinvitroorinvivo perhapstestingtheangiogenic 血管生成 ormetastatic 转移 potentialsoftumorcellsinxenograftmodels 异种移植模型 Createhypomorphicalleles 亚等位基因 rapidlyintransgenicmice 80 4 CombineshRNAswithexistinghigh efficiencygenedeliveryvehiclestocreatebonafideRNAi basedtherapeutics Forexample ultimately tosilenceadisease causingmutantallelespecifically 81 ResearchApplicationsofRNAi Anewstrategyofreversegenetics anovelwayofgeneknock out Itcanbeusedinreversegenetics 反向遗传学 toidentifythecellularorbiologicalfunctionofagene