WelcomeEachofYoutoMyMolecularBiologyClass2MolecularBiologyoftheGene,5/E

---Watsonetal.(2004)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenome

PartIII:ExpressionoftheGenomePartIV:RegulationPartV:Methods2005-5-103PartIVRegulationCh16:TranscriptionalregulationinprokaryotesCh17:TranscriptionalregulationineukaryotesCh18:RegulatoryRNAsCh19:GeneregulationindevelopmentandevolutionCh20:GenomeAnalysisandSystemsBiology4Chapter18RegulatoryRNAsMolecularBiologyCourse5TOPIC1RegulationbyRNAsinBacteria.TOPIC2RNAInterferenceIsaMajorRegulatoryMechanisminEukaryotes.TOPIC3SynthesisandfunctionofmiRNAmolecules.TOPIC4TheEvolutionandExploitationofRNAi.TOPIC5RegulatoryRNAsandX-inactivation.

6PART1RegulationbyRNAsinBacteria.PART2RNAInterferenceandmiRNARegulationinEukaryotes

Chapter18RegulatoryRNAs7PART1RegulationbyRNAsinBacteriaChapter18RegulatoryRNAs8SmallRNAs(sRNA):regulationbybasepairing.Riboswitches:regulationbymetabolite-mediatedstructurechangesAttenuation:Regulationbyribosomestop-mediatedformationofterminators9SmallRNAs(sRNA)

---RegulationoftranslationinitiationandtranscriptionterminationbyalteringtheaccessibilityofRBSandtheformationofterminator,respectively.[Targets]---RegulationbybasepairingwiththetargetedsequencesonmRNAs.[Mechanism]---ActsintranssimilartomiRNA,butdoesnotrequirespecificmachineryforaction.[Mechanism]Figure

18-1102.RiboswitchesareregulatoryRNAelementsthatactasdirectsensorsofsmallmoleculemetabolitestocontrolgenetranscriptionortranslation.---RegulatetranslationinitiationandtranscriptionterminationbyalteringtheaccessibilityofRBSandtheformationofterminator,respectively.[Targets]---ResideupstreamofthetargetedmRNA,andformspecificstructuretobinditssmallmoleculeligand.[Mechanism]---Actincisbyalterationofitsownstructureuponthebindingofthesmallmetabolites.[Mechanism]11Figure

18-2.ThestructureofariboswitchinitsregulatedmRNA12代謝物代謝物Figure

18-3a.ControloftranscriptionterminationbyariboswitchFigure

18-3a.Controloftranslationinitiationbyariboswitch.13Figure

18-4.AlterationofofthestructureoftheSAMriboswitchuponthebindingofSAM(S-adenosylmethionine)14The2ndstructuresof7riboswitchesandmetabolitesthattheysense15RNARegulationinBacteria3.Attenuation(衰減作用)---Aprematuretranscriptionterminationthatswitchesoffgeneexpressionfromaminoacidbiosyntheticoperonsafterthecorrespondingaminoacidissynthesizedatanadequatelevel.[target]---Requirestheparticipationofribosomesthattranslatealeaderpeptide.Theprematuretranscriptionterminationistriggeredbyformationofanintrinsicterminatorwhenribosomereadthroughcodonsoftheaminoacidthattheoperonsynthesizes.[mechanism].16Thetrpoperonencodesfivestructuralgenesrequiredfortryptophan(色胺酸)synthesis.Thesegenesareregulatedtoefficientlyexpressonlywhentryptophanislimiting.Twolayersofregulationareinvolved:(1)transcriptionrepressionbytheTrprepressor(initiation);(2)attenuationTheTRPoperon17Fig16-19Transcriptionofthetrpoperonisprematurallystoppedifthetryptophanlevelisnotlowenough,whichresultsintheproductionofaleaderRNAof161nt.(WHY?)18Transcriptionandtranslationinbacteriaarecoupled(細菌體內的轉錄和翻譯是偶聯的).

Therefore,synthesisoftheleaderpeptideimmediatelyfollowsthetranscriptionofleaderRNA.Theleaderpeptide

containstwotryptophancodons.Ifthetryptophanlevelisverylow,theribosomewillpauseatthesesites.RibosomepauseatthesesitesalterthesecondarystructureoftheleaderRNA,whicheliminatestheintrinsicterminatorstructureandallowthesuccessfultranscriptionofthetrpoperon.19TheleaderRNAandleaderpeptideofthetrpoperon20LowTrpHighTrpTranscriptionoftheleaderRNA.TranscriptionofthetrpoperonmRNA.RNAPol21Importanceof

attenuationAtypicalnegativefeed-backregulationUseofbothrepressionandattenuationallowsafinetuningoftheleveloftheintracellulartryptophan.Attenuationalonecanproviderobustregulation:otheraminoacidsoperonslikehisandleuhavenorepressorsandrelyentirelyonattenuationfortheirregulation.Providesanexampleofregulationwithouttheuseofaregulatoryprotein,butusingRNAstructureinstead.22PART2RNAiandmiRNAregulationChapter18RegulatoryRNAs23OutlinesThebackgroundanddiscoveryofmiRNAsRNAidiscoveryandmechanismmiRNAbiogenesisandregulationmiRNArolesindevelopment,celldifferentiationandvirusmiRNAincancersiRNAapplication24Topic1:ThebackgroundanddiscoveryofmiRNAsCHAPTER18RNAiandmiRNAregulation一、miRNA發現的背景和miRNA發現25后基因組時代的中心法則RNAprocessingGeneregulationDNArepairandrecombination基因組的保持基因組的表達26后基因組時代的基因調控：RNA調控MostoftheRNAtranscribedfromyourgenomedoesn’tmakeprotein.CarinaDennistalkstotherevolutionarieswhobelievethatitfunctionsingene-regulatorynetworksthatunderliethecomplexityofhigherorganisms.27含有30億對堿基的人類基因組僅含有2－3萬個蛋白質基因，是果蠅的兩倍，啤酒酵母的4倍。顯而易見，生物的復雜性不由編碼蛋白質的數目決定。人類基因組的蛋白質編碼區的總和占總基因組長度為1－2％，那么其他98％的基因組有什么功能呢？(1)24％的基因組是插入編碼序列的內含子序列；人類基因平均每個基因有7個內含子。但這么冗長的內含子序列有什么生物學功能呢？(2)其他74%的基因組的功能是什么？【注：90%以上的基因組都是轉錄的！】人類基因組草圖帶給科學家們的困惑28人類基因組絕大部分都被轉錄成RNA，細胞內非編碼RNA的數量是編碼RNA的上百倍。這促使許多科學家認為生物體復雜性被隱藏在它們所輸出的非編碼RNA內，而非編碼序列內。

29ThediscoveryofmiRNAs

miRNAwasfirstdiscoveredin1993byVictorAmbrosatHarvard(lin-4)ThesecondmiRNALet-7wasdiscoveredin2000byFrankSlackasapostdocatHarvard

(Ruvkunlab)VictorAmbrosGaryRuvkun30ThefirstdiscoveredmiRNAlin-4in1993RuvkunG,WightmanB,HaI.The20yearsittooktorecognizetheimportanceoftinyRNAs.Cell.2004Jan23;116(2Suppl):S93-6.LeeR,FeinbaumR,AmbrosV.AshorthistoryofashortRNA.Cell.2004Jan23;116(2Suppl):S89-92Thoughttobeanodditynotageneralphenomenon31BreakthroughwithBlastNofthesecondmiRNA(stRNA)let-7PasquinelliAE,ReinhartBJ,SlackF,MartindaleMQ,KurodaMI,MallerB,HaywardDC,BallEE,DegnanB,MullerP,SpringJ,SrinivasanA,FishmanM,FinnertyJ,CorboJ,LevineM,LeahyP,DavidsonE,RuvkunG.Conservationofthesequenceandtemporalexpressionoflet-7heterochronicregulatoryRNA.Nature.2000Nov2;408(6808):86-9.3233ThenumberoftheidentifiedmiRNAsisgrowingrapidlyinrecentyears.Over5000miRNAshavebeenfounduntiltheAugustof2007(ThemiRBaseSequenceDatabase).ThesemiRNAsarefromprimates,rodents,birds,fish,worms,flies,plantsandviruses.Thedataarefreelyavailabletoallthroughthewebinterfaceathttp://microrna.sanger.ac.uk/sequences/andinflatfileformfromftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.3435microRNAshadbeenneglectedforsomanyyearsbecauseoftheirsmallsize.Theunderlyingreasonis:peopleneverdreamthatsmallRNAswillhaveimportantbiologicalroles.36Topic2:RNAinterferenceanditsmechanism二、RNA干擾及其機制CHAPTER18RNAiandmiRNAregulation371Double-strandedRNAinhibitsexpressionofgeneshomologoustothatRNA.[phenomena-現象]雙鏈RNA抑制含其同源序列基因的表達382006年的諾貝爾生理學獎獲得者：AndrewZ.FireCraigC.Mello39Fig2.AnalysisofRNA-interferenceeffectsinindividualcells.FluorescencemicrographsshowprogenyofinjectedanimalsfromGFP-reporterstrainPD4251(aC.elegansstrainexpressingGFPfluorescenceprotein)(使用外源導入的報告基因).Younglarva(幼蟲)Adult(成蟲)adultbodywallathighmagnification(高放大倍數的成蟲體壁)ControldsRNAds-gfpRNA40Fig3.Effectsofmex-3RNAinterferenceonlevelsoftheendogenousmRNA(insituhybridizationinembryos)(胚胎的原位雜交).adultbodywallathighmagnification(高放大倍數的成蟲體壁)Nohybridizationandstaining+hybridization(endogenousmex-3RNA)+antisense+hybridization+dsmex-3RNA+hybridization41ThediscoveryofRNAiexplainsthevirus-inducedgenesilencinginplants(植物病毒引起的基因沉默).

Mostplantviruseshavesingle-strandedRNAgenomes,whicharereleasedfromtheproteincoatoftheirvirusparticlesastheyenteracell.TheirgenomicRNAisthenreplicatedbythevirusencodedRNA-dependentRNApolymerasetoproducesenseandantisenseRNA,whichcanhybridizetoformdsRNAandtriggeranRNAiresponseagainsttheirownsequences.422.ShortinterferingRNA(siRNAs)areproducedfromdsRNAanddirectmachinerythatswitchoffgenesinvariousway.[Mechanism-機制]從雙鏈RNA產生的小干擾RNA可以指導用不同機制關閉基因的細胞機器43Thequestiontobeaddressedis“WhyexogenousdsRNAcaninhibitexpressionofgeneshomologoustothatRNA?”44Figure17-30RNAisilencingExogenousdsRNA外源雙鏈RNA45ThreewaysoftheRNAi-directedgenesilencingTriggerdestructionofthetargetmRNA(引起靶標mRNA的降解),InhibittranslationofthetargetmRNA(抑制靶標mRNA的翻譯),Inducechromatinmodification(引起靶標啟動子的轉錄沉默).46TheheartoftheRNAimechanismDicer:anRNaseIII-likemultidomainribonucleasethatfirstprocessesinputdsRNAintosmallfragmentscalledshortinterferingRNAs(siRNAs)ormicroRNAs(miRNA).DicerthenhelpsloaditssmallRNAproductsintoRISC.RISC

(RNAinducedsilencingcomplexes)(RNA誘導的沉默復合體):alargemultiproteincomplexthatdirecttheboundsiRNAormiRNAtoitstargetandinhibitthetargetgeneexpression.47Dicer:Structuralorganization:---APAZdomain,bindstheendofthedsRNA---TwoRNaseIIIdomains---Othernon-conserveddomains.賈第鞭毛蟲48ThecrystalstructureoftheGiardiaintactDicerenzymeshowsthatthePAZdomain,amodulethatbindstheendofdsRNA,isseparatedfromthetwocatalyticRNaseIIIdomainsbyaflat,positivelychargedsurface. The65angstromdistancebetweenthePAZandRNaseIIIdomainsmatchesthelengthspannedby25basepairsofRNA.Thus,DiceritselfisamolecularrulerthatrecognizesdsRNAandcleavesaspecifieddistancefromthehelicalend.49RISC:thekeycomponentisArgonaute(AGO)Argonaute(AGO):AlargeproteinfamilythatconstituteskeycomponentsofRISCs.---AGOproteinsarecharacterizedbytwouniquedomains,PAZandPIWI,whosefunctionsarenotfullyunderstood.CurrentevidencesuggeststhatthePAZdomainbindsthe3’-endtwo-nucleotideoverhangsofthesiRNAduplex,whereasthePIWIdomainofsomeAGOproteinsconferssliceractivity.PAZandPIWIdomainsarebothessentialtoguidetheinteractionbetweenthesiRNAandthetargetmRNAforcleavageortranslationalrepression.---DistinctAGOmembershavedistinctfunctions.Forexample,humanAGO2programsRISCstocleavethemRNAtarget,whereasAGO1andAGO3donot.5051AmodelforsiRNA-guided

mRNAcleavagebyArgonaute52Eulalioetal.Ce1l132,2008MechanismsofmiRNA-MediatedTranslationalInhibition53(A)Inhibitionoftranslationelongation:miRNAsrepresstranslationoftargetmRNAsbyblockingtranslationelongationorbypromotingprematuredissociationofribosomes(ribosomedrop-off)(B)Co-translationalproteindegradation:Theproteinisnormallytranslatedafterwhichitisimmediatelydegradedproteolytically.(C)Competitionforthecapstructure:ArgonauteproteinscompetewitheIF4Eforbindingtothecapstructure.(D)Inhibitionofribosomalsubunitjoining:ArgonauteproteinsrecruiteIF6,whichpreventsthelargeribosomalsubunitfromjoiningthesmallsubunit.54TranscriptionalGeneSilencingbyDirectingChromatinModificationFigure18-13.SilencingofthecentromereinS.pombe(裂殖酵母)55RNAsilencingindifferentorganisms56ThemultiplefunctionsofRNAi57Topic3:miRNAbiogenesisandregulation三、miRNA生成和調控CHAPTER18RNAiandmiRNAregulation581.MicroRNA(miRNA)&itsprocessing微小RNA及其加工59MicroRNA(miRNA):

Atypeofnon-codingsmallRNA(~21–23nucleotides)producedbyDicerfromastem-loopstructuredRNAprecursor(~70-90ntsong)(結構和來源).miRNAsarewidelyexpressedinanimalandplantcellsandfunctionsintheformofRNA–proteincomplexes,termedmiRISCs.miRNAshavebeenimplicatedinthecontrolofdevelopmentbecausetheyleadtothedestructionortranslationalsuppressionoftargetmRNAswithhomologytothemiRNA(生物學功能和機制).60ThemiRNAgenesandStructureofpri-miRNAsPri-miRNAsbearthe5’capand3’poly(A)tails61miRNAprocessingPri-miRNA(miRNA初級轉錄產物)Drosha(1)pre-miRNA(miRNA前體)Dicer(2)miRNAExportin5(Exp5)transportspre-miRNAtothecytoplasm62Atypicalmetazoanpri-miRNAconsistsofastemofapproximately33bp,withaterminalloopandflankingsegments.Theterminalloopisunessential,whereastheflankingssRNAsegmentsarecriticalforprocessing.Thecleavagesiteisdeterminedmainlybythedistance(approximately11bp)fromthestem-ssRNAjunction.63Hanetal.,Cell125,887–901,June2,200664HumanDroshaandDicersharethesameRNaseIIIdomainsanddsRNAbindingdomain.652.MicroRNA(miRNA)targetsandregulation.66AcomparisonbetweenmiRNAandsiRNA67ThreestrategiesofmiRNAandtargetrecognition(targetsarelocatingin3’UTRs).68Topic4:miRNArolesindevelopment,celldifferentiationandvirusCHAPTER18RNAiandmiRNAregulation四、MicroRNA在發育中的調控作用，及其他作用69VictorR.Ambros秀麗線蟲C.elegans1.miRNAinC.elegansdevelopment70lin-4andlet-7miRNAscontrolthedevelopmentaltimeofC.elegans.71Expressionoflin-4allowsC.eleganstoproceedtothelatedevelopmentalstage72lin-4

bindsitstargetmRNAsbyimperfectbasepairing.732.miRNAsinvertebratedevelopment:

TherearealotunknownbecausethethelackofefficientmethodstouncoverthespecifictargetsofmiRNAs.74Figure2.ExpressionofmiR-124aandmiR-1inZebrafish,Medaka,Mouse,andFly.

miR-124aisrestrictedlyexpressedinthebrainandthespinalcordinfishandmouseortotheventralnervecordinthefly.TheexpressionofmiR-1isrestrictedtothemusclesandtheheartinthemouse.青鳉斑馬魚小鼠果蠅LearningthemiRNAfunctionfromitsexpressionpattern753.miRNAcontrolsplantphenotypesandmore(控制植物表型特征)Jaw-miRNA控制擬南芥葉形變化(Nature,2003)76(Science2004)3種miRNA控制造血干細胞向淋巴細胞的分化過程4.miRNAcontrolsthedifferentiationofthehematopoieticstemcell(調控造血干細胞的分化)775.SomevirusesencodemiRNAs(有些病毒編碼miRNAs)78Virus-relatedmiRNAregulation79Topic5:miRNAincancer五、微小RNA在癌癥發生中的作用CHAPTER18RNAiandmiRNAregulation80miRNAsinhuman:

Thereareover600miRNAsfromhumanhavebeenfoundandannotatedsofar.Theyarenamedashas-miRx.81miRNAexpressionpatternchangesduringoncogenesis,andisuniqueforeachcancer.微小RNA在癌癥發生中表達譜的變化8283Figure3,ComparisonbetweennormalandtumorsamplesrevealsglobalchangesinmiRNAexpression.84OnemechanismofmiRNAcontrollingoncogeneexpression

微小RNA調控癌基因表達的一種機制。85c-Mycisahelix–loop–helixleucinezippertranscriptionfactorthatregulatesanestimated10–15%ofgenesinthehumanandDrosophilagenomes.c-MycactivatesexpressionofaclusterofsixmiRNAs(mir-17-92cluster)onhumanchromosome13.(Figure1)E2F1isthetranscriptionfactor,whichisatargetofc-Mycthatpromotescellcycleprogression.ExpressionofE2F1isnegativelyregulatedbytwomiRNAsinthiscluster,miR-17-5pandmiR-20a.(Figure4)8687Used2’-O-methylAntisenseoligonucleotidestodownregulatethelevelofmiR-17-5pandmiR-20a,andthenanalyzedtheprotein(B-Western)andmRNAlevels(C-Northen)ofE2F1.88SomemicroRNAsarepotentialoncogenes

有些微小RNA可能是致癌基因。89B-細胞淋巴瘤90Figure1.Themir-17–92clustershowsincreasedexpressioninB-celllymphomasamplesandcelllines.

Thelevelofmir-17–92pri-miRNAwasdeterminedbyreal-timequantitativeRT-PCRin46lymphomasand47colorectalcarcinomas,andcomparedtolevelsfoundincorrespondingnormaltissuesfromfiveindividuals.91Figure2.Overexpressionofthemir-17–19bclusteracceleratesc-myc-inducedlymphomagenesisinmice.92Topic6:siRNAapplicationCHAPTER18RNAiandmiRNAregulation六、siRNA的應用93siRNAapplicationinmammalianTransfectexogenoussiRNAintocellsChemicalsynthesis:expensiveGenerateesiRNA(enzymaticproducedsiRNA)frominvitrotranscribedlongdsRNAbyE.coliRNaseIIIorRNaseIII-likeDICER.ExpressionofsiRNAinculturedcellsorinanimalmodelssiRNAproducedwithpolIIIpromoterfromthetransfectedDNAplasmids.94TranscriptionfromRNAPIIIpromotersofU6andH1arewellcharacterized.RNAPIIItranscriptionusesawell-definedterminationsignal(TTTTT)andtheproductshavenoextra