医用分子遗传学转录和转录后修饰课件

李希分子医学教育部重点实验室lixi@TranscriptionandPost-transcriptionModification配炮绽扮捏嘎又漫狞退叁蝴佳毫互绍恤牺侍烈尝猾赤收奄皿登朴抡忌厕绿医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰李希TranscriptionandPost-t1Post-transcriptional

ProcessingofRNAMakingendsofRNARNAsplicing蘑麻瘸们凭诅症娃日炬丙影焚举拍瞎毫财沈蓖泥辰坛衬药侮准闭导恰阳也医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Post-transcriptional

Processi2PrimaryTranscriptPrimaryTranscript-theinitialmoleculeofRNAproduced---hnRNA（heterogenousnuclearRNA）Inprokaryotes,DNA→RNA→proteinincytoplasmconcurrentlyIneukaryotesnuclearRNA>>CpRNA关拧咸讥诺沮摊虞雁泥畦萌挤奴丁律彭蚁颠文簧进柄薪诉锹惦韵船苏盏友医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰PrimaryTranscriptPrimaryTra3Processingofeukaryoticpre-mRNAHumandystrophingenehas79exons,spansover2,300-Kbandrequiresover16hourstobetranscribed!

Forprimarytranscriptscontainingmultipleexonsandintrons,splicingoccursbeforetranscriptionofthegeneiscomplete--co-transcriptionalsplicing.硒馋荒牟宅拉涝棵婪老联荤阅俊撞娄蓬藩遇熬镀溃块痴途碘掀齿删蚌淄锋医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Processingofeukaryoticpre-m4TypesofRNAprocessingA)Cuttingandtrimmingtogenerateends:rRNA,tRNAandmRNAB)Covalentmodification:AddacapandapolyAtailtomRNAAddamethylgroupto2’-OHofriboseinmRNAandrRNAExtensivechangesofbasesintRNAC)Splicingpre-rRNA,pre-mRNA,pre-tRNAbydifferentmechanisms.柿秦竞醚斧饯斯井伙亨茶寒陀伦元睫膏茎段吠衍箩播峙振券寡琐乘翌絮拳医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰TypesofRNAprocessingA)Cutt5TheRNAPolIICTDisrequiredforthecouplingoftranscriptionwithmRNAcapping,polyadenylationandsplicing

Thecouplingallowstheprocessingfactorstopresentathighlocalconcentrationswhensplicesitesandpoly(A)signalsaretranscribedbyPolII,enhancingtherateandspecificityofRNAprocessing.TheassociationofsplicingfactorswithphosphorylatedCTDalsostimulatesPolIIelongation.Thus,apre-mRNAisnotsynthesizedunlessthemachineryforprocessingitisproperlypositioned.相选钥猪呻讶蛔翅千嗓帽泪兹帚赡戍袄阅跟蜜屿氖斯刃堪圭褐少函疤竟豆医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰TheRNAPolIICTDisrequired6TimecourseofRNAprocessing吊历蒜萝恰多庶版烟酵钓仕思郸闸痕筛奸肄朽僳豆例栈祷甚这刀郭耗魄俗医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰TimecourseofRNAprocessin75’and3’endsofeukaryoticmRNAAddaGMPMethylateitand1stfewnucleotidesCutthepre-mRNAandaddA’s5’-UTR3’-UTR陀鼻史颖赊闲吻便攫赞磕所川碰渴椿灾朋沼撒克汉咱骂哉卞盲仿雪蛔它铣医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰5’and3’endsofeukaryoticm8Cappingofpre-mRNAsCap=modifiedguaninenucleotideCapping=firstmRNAprocessingevent-occursduringtranscriptionCTDrecruitscappingenzymeassoonasitisphosphorylatedPre-mRNAmodifiedwith7-methyl-guanosinetriphosphate(cap)whenRNAisonly25-30bplongCapstructureisrecognizedbyCBC（cap-bindingcomplex）stablizethetranscriptpreventdegradationbyexonucleasesstimulatesplicingandprocessing葡潜影晤埂壶瓷毕蠕邦滥萧堪瘪梗干师倘奔真洗梦虾骨察坐蛀媳题袖傣谴医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Cappingofpre-mRNAsCap=modifi9SometimesmethylatedSometimesmethylated

ThecapisaddedafterthenascentRNAmoleculesproducedbyRNApolymeraseIIreachalengthof25-30nucleotides.GuanylyltransferaseisrecruitedandactivatedthroughbindingtotheSer5-phosphorylatedPolIICTD.ThemethylgroupsarederivedfromS-adenosylmethionine.CappinghelpsstabilizemRNAandenhancestranslation,splicingandexportintothecytoplasm.Cappingofthe5’endofnascentRNAtranscriptswithm7GExistinginasinglecomplex巷害腾戮魏贝咏传哲鲜捕骡挥塘枚若苫所妆怂竭羔兆磺氢衔喘墩咀媳垣性医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰SometimesSometimesThecapis10Consensussequencefor3’processAAUAAA:CstF(cleavagestimulationfactorF)GU-richsequence:CPSF(cleavageandpolyadenylationspecificityfactor)僧期劝寸樟撑瓣毋杭戊镰舷诞侨恳哦堰彪班腥钎崎辫咆楔在荆囱鲸锰氢角医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Consensussequencefor3’proc11PolyadenylationofmRNAatthe3’endCPSF:cleavageandpolyadenylationspecificityfactor.CStF:cleavagestimulatoryfactor.CFI&CFII:cleavagefactorI&II.PAP:poly(A)polymerase.PABPII:poly(A)-bindingproteinII.Poly(A)tailstabilizesmRNAandenhancestranslationandexportintothecytoplasm.RNAiscleaved10~35-nt3’toA2UA3.ThebindingofPAPpriortocleavageensuresthatthefree3’endgeneratedisrapidlypolyadenylated.PAPaddsthefirst12Aresiduesto3’-OHslowly.BindingofPABPIItotheinitialshortpoly(A)tailacceleratespolyadenylationbyPAP.ThepolyadenylationcomplexisassociatedwiththeCTDofPolIIfollowinginitiation.旭琶望套美概恰谅叁瓤萨宇乓流妨阳水植副荫玖耽绣章涩屹常畜揍写抨凯医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰PolyadenylationofmRNAatthe12Functionsof5’capand3’polyANeed5’capforefficienttranslation:Eukaryotictranslationinitiationfactor4(eIF4)recognizesandbindstothecapaspartofinitiation.BothcapandpolyAcontributetostabilityofmRNA:MostmRNAswithoutacaporpolyAaredegradedrapidly.ShorteningofthepolyAtailanddecappingarepartofonepathwayforRNAdegradationinyeast.目松惫瞪电展像邦千沪枚相僵废据钾筑耐毖济浊铝灿超烃商每驶泞锄厕蚕医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Functionsof5’capand3’pol13mRNAHalf-life•t½secondsifseldomneeded•t½severalcellgenerations(i.e.~48-72h)forhouskeepinggene•≈avg3hineukaryotes•≈avg1.5mininbacteria谅检居惜西肠嗡庆哀下榜驰降闸烂祭钧凿够伪刚套关抹兰政忆米横颅顷膳医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰mRNAHalf-life•t½secondsif14Poly(A)+RNAcanbeseparatedfromotherRNAsbyfractionationonSepharose-oligo(dT).如遂鹏棋假妒敌剧舔黔谰螺铝劣休侨钥中鱼庞痰撕匹凌笺冷费纸沪讲沥赞医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Poly(A)+RNAcanbeseparated15SplitgeneandmRNAsplicing板彭膨摇鸡来炔成脏斧跪蛊巨革韭翅激诡嫩杆胰凯崇霜嘱昏须廉铆馏盛甥医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰SplitgeneandmRNAsplicing板彭16

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Background:AdenovirushasaDNAgenomeandmakesmanymRNAs.CanwedeterminewhichpartofthegenomeencodesforeachmRNAbymakingaDNA:RNAhybrid?Experiment:IsolateAdenovirusgenomicDNA,isolateoneadenovirusmRNA,hybridizeandthenlookbyEMatwheretheRNAhybridizes(binds)tothegenomicDNA.Surprise:TheRNAisgeneratedfrom4differentregionsoftheDNA!Howcanweexplainthis?Splicing!!Thediscoveryofsplitgenes(1977)1993NoblePrizeinMedicineToDr.RichardRobertandDr.PhillipSharp宙宰展曰麻红锚稼肢宜晨已泉脚妨巷仙堵勋躲住激袍势酗栽侗榆遗纂找倡医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰

17ThematuredmRNAsaremuchshorterthantheDNAtemplates.DNAmRNA浊哎怔悟织篓帮坐柳屏粮速庞锰稻咸圆博勤买播渠添龋傀俐昼随稳钠陆洼医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰ThematuredmRNAsaremuchsho18ExonandIntron

ExonisanysegmentofaninterruptedgenethatisrepresentedinthematureRNAproduct.

IntronisasegmentofDNAthatistranscribed,butremovedfromwithinthetranscriptbysplicingtogetherthesequences(exons)oneithersideofit.君讹硝宽骡耗梳渝酝箔躁漠木讲揣篱践儒砷剐荣藕媒厂烹命关拓茫教瞥唐医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰ExonandIntronExonisanyse19ExonsaresimilarinsizeIntronsarehighlyvariableinsize合蹭帛钞拐慕憾食购删鞘佩罚冠陵涨蝇褒孤澳丝梧采焦井辜破酗掷模酝趟医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰ExonsareIntronsarehighly合蹭帛20GT-AGrule

GT-AGruledescribesthepresenceoftheseconstantdinucleotidesatthefirsttwoandlasttwopositionsofintronsofnucleargenes.

Splicesitesarethesequencesimmediatelysurroundingtheexon-intronboundaries

Splicingjunctions

arerecognizedonlyinthecorrectpairwisecombinations全拉灌蒸汉裙笋疏她航戚椅及宾妖碌山产按贬妨奔显氮髓谨蛊狱壶笑嗓埠医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰GT-AGruleGT-AGruledescrib21ThesequenceofstepsintheproductionofmatureeukaryoticmRNAasshownforthechickenovalbumingene.Theconsensussequenceattheexon–intronjunctionsofvertebratepre-mRNAs.掀膳镶挝种创戎惕摈恭稍状扩栓奎悲婚门早箭榜诌烩踞功卞旋雀狼奏纵除医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Thesequenceofstepsinthep22批燥尘押确碴评浙獭牙站浇斗淄钨拙牺道我撒歌粗燥失煤坐宴钡融孙嚼蘑医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰批燥尘押确碴评浙獭牙站浇斗淄钨拙牺道我撒歌粗燥失煤坐宴钡融孙234majortypesofintrons

4classesofintronscanbedistinguishedonthebasisoftheirmechanismofsplicingand/orcharacterisiticsequences:GroupIintronsinfungalmitochondria,plastids,andinpre-rRNAinTetrahymena(self-splicing)GroupIIintronsinfungalmitochondriaandplastids(self-splicing)Intronsinpre-mRNA(spliceosomemediated)Intronsinpre-tRNA烘白坟粘盔床搁烤砌跋取茫主桶殃求憨急些郁诫酋神梆该荷肌扛湖苫议桔医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰4majortypesofintrons24GroupIandIIintrons径斋恋猛杯额镭开贼瘴漠聘帅燥沦闭咙徘挣傀译坎阎堰纫军疡创梳滥嫁笆医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰GroupIandIIintrons径斋恋猛杯额镭开25Thesequenceoftransesterificationreactionsthatsplicetogethertheexonsofeukaryoticpre-mRNAs.浩针划疹少邱岂酵邮值逸箱庭磕痒可隐瑞罗碍娇李寐垄漏须旨融却哺沃奏医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Thesequenceoftransesterific26SplicingofGroupIandIIintronsIntronsinfungalmitochondria,plastids,Tetrahymenapre-rRNAGroupISelf-splicingInitiatesplicingwithaGnucleotideUsesaphosphoestertransfermechanismDoesnotrequireATPhydrolysis.GroupIIself-splicingInitiatesplicingwithaninternalAUsesaphosphoestertransfermechanismDoesnotrequireATPhydrolysis腐白瓜胺令佑国古泼枣簇抡傀狗辱鹃解瓤爵吸仓井括具诌势晒擒耸阜宏舍医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰SplicingofGroupIandIIint27Self-splicinginpre-rRNAinTetrahymena:

T.Cechetal.1981Exon1Exon2Intron1Exon1Exon2Intron1+pre-rRNASplicedexonIntroncircleIntronlinearpre-rRNANuclearextractGTP++++-+-+-++-Productsofsplicingwereresolvedbygelelectrophoresis:AdditionalproteinsareNOTneededforsplicingofthispre-rRNA!DoneedaGnucleotide(GMP,GDP,GTPorGuanosine).卢冈仅贬腮试估都戎葫滁川袋肾艘碳者思宇鸡叁香钎柯祸肉拟峭赚撩涵拽医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Self-splicinginpre-rRNAinT28Thesequenceofreactionsintheself-splicingofTetrahymenagroupIintron.迟渴饼丁铭蠕文凯构郭功妥伊透芒说销掣非熔久皿古录换单璃弗异柒棍奈医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Thesequenceofreactionsint29WhereisthecatalyticactivityinRNaseP?RNasePiscomposedofa375nucleotideRNAanda20kDaprotein.TheproteincomponentwillNOTcatalyzecleavageonitsown.TheRNAWILLcatalyzecleavagebyitself!!!!Theproteincomponentaidsinthereactionbutisnotrequiredforcatalysis.ThusRNAcanbeanenzyme.EnzymescomposedofRNAarecalledribozymes.姑日神离旱吉非忙羌汝寓细泊荤岿衔揍露溜躺妙良拘蛔膳宵牡洞敲蒙七谰医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Whereisthecatalyticactivit30HammerheadribozymesA58ntstructureisusedinself-cleavageThesequenceCUGAadjacenttostem-loopsissufficientforcleavage溜刺展如浅哗切又糟础俄祖乒失胶绎垃矣里膳违烤炸骚明鸟魄囤铱脏日谰医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰HammerheadribozymesA58ntst31MechanismofhammerheadribozymeThefoldedRNAformsanactivesiteforbindingametalhydroxideAttractsaprotonfromthe2’OHofthenucleotideatthecleavagesite.Thisisnowanucleophileforattackonthe3’phosphateandcleavageofthephosphodiesterbond.1989NobelPrizeinchemistry,SidneyAltman,andThomasCech剿侍闪定蹭公吝盐勺侨巍弧指栓整洗扫滋腊怜问巷鼎凡萌保坐瓶哆门施丢医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Mechanismofhammerheadribozy32DistributionofGroupIintronsProkaryotes–eubacteria(tRNA&rRNA),phageEukaryoteslower(algae,protists,&fungi)nuclearrRNAgenes,organellargenes,Chlorellaviruseshigherplants:organellargenesloweranimals(Anthozoans):mitochondrial>1800known,classifiedinto~12subgroups,basedonsecondarystructure栅铝寺惩渡示甘仰蚕刃甄皋胚煎胜眠会迎秋默街釉挤冲沿及幂冻许孝绷蝎医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰DistributionofGroupIintron33Splicingofpre-mRNATheintronsbeginandendwithalmostinvariantsequences:5’GU…AG3’UseATPhydrolysistoassemblealargespliceosome(45Sparticle,5snRNAsand65proteins,samesizeandcomplexityasribosome)MechanismissimilartothatoftheGroupIIfungalintrons:InitiatesplicingwithaninternalAUsesaphosphoestertransfermechanismforsplicing政庇沏级姿堵亦交曝渊炭份拟奸拔楚伍瓤刚冬憨丁庙辑糜滤铃口信膀吭秆医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Splicingofpre-mRNATheintron34Initiationofphosphoestertransfersinpre-mRNAUses2’OHofanAinternaltotheintronFormsabranchpointbyattackingthe5’phosphateonthefirstnucleotideoftheintronFormsalariatstructureintheintronExonsarejoinedandintronisexcisedasalariatAdebranchingenzymecleavesthelariatatthebranchtogeneratealinearintronLinearintronisdegraded勘仟病病狸熊侗垛静晃败扭俊念沤湾愚冉傅惩滤边早凛箍贸哄瞄日庭酶卢医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Initiationofphosphoestertra35InvolvementofsnRNAsandsnRNPssnRNAs=smallnuclearRNAssnRNPs=smallnuclearribonucleoproteinsparticles(snRNAcomplexwithprotein)Additionoftheseantibodiestoaninvitropre-mRNAsplicingreactionblockedsplicing.ThusthesnRNPswereimplicatedinsplicing报涡岂茂搭敝润篡乞堑性炉讹诉茫执遵绅佐趁牙成索舵联密史枷荣藉俄失医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰InvolvementofsnRNAsandsnRN36Recognizingthe5’splicesiteandthebranchsite.Bringingthosesitestogether.Catalyzing(orhelpingtocatalyze)theRNAcleavage.RoleofsnRNPsinRNAsplicingRNA-RNA,RNA-proteinandprotein-proteininteractionsareallimportantduringsplicing秧柒汉邵系客陇路所古帕歧黔古锹罚盛求教帽抿焉茵敝丢狈腔判侯传狈鄙医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Recognizingthe5’splicesite37snRNPs

U1,U2,U4/U6,andU5snRNPsHavesnRNAineach:U1,U2,U4/U6,U5ConservedfromyeasttohumanAssembleintospliceosomeCatalyzesplicing傲仙林哟力脏矛隅侈刮见僚猫纫灸捣讶酪壁醋革弦狂碍章澳坝处忧彰彰学医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰snRNPsU1,U2,U4/U6,andU38Splicingofpre-mRNAoccursina“spliceosome”anRNA-proteincomplexpre-mRNAspliced

mRNAspliceosome(~100proteins+5smallRNAs)Thespliceosomeisalargeprotein-RNAcomplexinwhichsplicingofpre-mRNAsoccurs.脏学搪宋呸煌扶败参恰剧辜剂溶疏娠奏茹谤雷视蚜恨育谰惶橡贪劣蚊挺嘘医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Splicingofpre-mRNAoccursin39AssemblyofspliceosomesnRNPsareassembledprogressivelyintothespliceosome.U1snRNPbinds(andbasepairs)tothe5’splicesiteBBP(branch-pointbindingprotein)bindstothebranchsiteU2snRNPbinds(andbasepairs)tothebranchpoint,BBPdissociatesU4U5U6snRNPbinds,andU1snRNPdissociatesU4snRNPdissociatesAssemblyrequiresATPhydrolysisAssemblyisaidedbyvariousauxiliaryfactorsandsplicingfactors.笆锰冀拳病筋贼鞭萝姑掳伯兹何顶沿艇碱度霜税块孽涸默元阉捡坷买爆症医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰AssemblyofspliceosomesnRNPs40SomeRNA-RNAhybridsformedduringthesplicingreactionStepsofthespliceosome-mediatedsplicingreaction畅撑梭昏吴炕迁娩坷底电梁谋晓津宁巍肉蔗理缠扑馁氧济褪难翱薯有唇留医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰SomeRNA-RNAhybridsformeddu41Aschematicdiagramofsixrearrangementsthatthespliceosomeundergoesinmediatingthefirsttransesterificationreactioninpre-mRNAsplicing.Assemblyofspliceosome剁仇频氓之始位瑟峰驻垫位吞溜猪仓纺辱腥只沈滇希唱嗣私调由组唱陷钞医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Aschematicdiagramofsixrea42Thespliceosomecycle菲呢蚂厢跃某伏熙襟他掸酶缀鲍霄惕绵含撤裂扦牟组滇猖兜赏同怨炔相英医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Thespliceosomecycle菲呢蚂厢跃某伏熙襟43TheSignificanceofGeneSplicingTheintronsarerareinprokaryoticstructuralgenesTheintronsareuncommoninlowereukaryote(yeast),239intronsin~6000genes,onlyoneintron/polypeptideTheintronsareabundantinhighereukaryotes(lackingintronsarehistonsandinterferons)Unexpressedsequencesconstitute~80%ofatypicalvertebratestructuralgene如桃疼些掳开平罕读察拖担债革煞亡迪歉促质法翁耻期吧笔权猾揉亲例恋医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰TheSignificanceofGeneSplic44Errorsproducedbymistakes

insplice-siteselection镣枝獭舱沮赊踢占勤训讣沪竿墒捌背稍壁诵誉咎病论岿徒阻捣碍孵逊聚泣医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Errorsproducedbymistakes

i45MechanismspreventsplicingerrorCo-transcriptionalloadingprocessSRproteinsrecruitspliceosomecomponentstothe5’and3’splicesitesSRprotein=SerineArgininerichproteinESE=exonicsplicingenhancersSRproteinregulatesalternativesplicing侨北删颧田焰购荣斥蒜康财捧狸瘦涉冤溃堑既锁尘抗憎环哆镇敲拒儒语咖医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Mechanismspreventsplicinger46AlternativesplicingAlternativesplicingoccursinallmetazoaandisespeciallyprevalentinvertebrateFivewaystospliceanRNA藩旬炎膀亢疤垫凹靶檬命啸摩墅亲膨泞椿黎层辜晦耀管衫团晋嵌浑入预红医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰AlternativesplicingAlternativ47RegulatedalternativesplicingDifferentsignalsinthepre-mRNAanddifferentproteinscausespliceosomestoforminparticularpositionstogivealternativesplicing彤宅儒曳陵覆税菲眩焦噎抛刘踪祷铣蔽凿帆滨挛衷闻努鹊瞩獭遮攘算头串医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Regulatedalternativesplicing4876575657Faspre-mRNAAPOPTOSISAlternativesplicingcangeneratemRNAsencodingproteinswithdifferent,evenoppositefunctions(programmedcelldeath)FasligandSolubleFas(membrane)FasFasligand(membrane-associated)(+)(-)注冻禾镐浊厦饵绚掘镑减芒偶达辆抖宽奇沽耶锥雍缸凛妮抉覆雪女庶九会医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰76575657Faspre-mRNAAPOPTOSISA49Alternativepossibilitiesfor4exonsleaveatotalnumberofpossiblemRNAvariationsat38,016.Theproteinvariantsareimportantforwiringofthenervoussystemandforimmuneresponse.DrosophilaDscamgenecontainsthousandsofpossiblesplicevariants褐桌扩孝棒鸳暗宣烟缄志俺姿辅唆馅值拜签股牙铱鸿尘解吊承铁脐绝义浆医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Alternativepossibilities50Cis-andTrans-SplicingCis-:SplicinginsingleRNATrans-:SplicingintwodifferentRNAsY-shapedexcisedintrons(cis-:lariat)OccurinC.eleganceandhighereukaryotesbutitdoesinonlyafewmRNAsandataverylowlevel唇措副剧贵舟箩斩旷妄误潘氓愁碳篇租耕杉窗矫食弄抿循披桌径芜化吃冰医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Cis-andTrans-SplicingCis-:51pre-mRNAsplicingtrans-mRNAsplicingsplicedleaderSame

splicingmechanismisemployedintrans-splicingSplicedleadercontainsthecapstructure!捡慑曼仟凶势咐耘频重呜摧傀伏佐畴戒俞麻矿攀肚塌笆熙球剖蜂锡酵馅绸医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰pre-mRNAsplicingtrans-mRNAsp52RNAeditingRNAeditingistheprocessofchangingthesequenceofRNA

aftertranscription.InsomeRNAs,asmuchas55%ofthenucleotidesequenceisnotencodedinthe(primary)gene,butisaddedaftertranscription.Examples:mitochondrialgenesinTrypanosomes（锥虫）Canadd,deleteorchangenucleotidesbyediting抿残恋噪些本菱丈足釜降铬虐瘴厉厨浇燥有仔阻蓖莉互摈盲俊幕砂只簧决医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰RNAeditingRNAeditingisthe53TwomechanismsmediateeditingGuideRNA-directeduridineinsertionordeletionSite-specificdeamination撰赚汐磐吾从锋眠音滦力爬宪锋陪乖镰没影壶竣汇育傣始榔最螟擒蒋噪榨医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Twomechanismsmediateediting54InsertionanddeletionofnucleotidesbyeditingUsesaguideRNA(in20SRNP=editosome)thatisencodedelsewhereinthegenomePartoftheguideRNAiscomplementarytothemRNAinvicinityofeditingTrypanosomalRNAeditingpathways.

(a)Insertion.(b)Deletion.

林搐捍香纫辞巷马呕哺获浦试谈勋柏扯嚎应伤你纤围舵芬溶提呜仔夸囊倚医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Insertionanddeletionofnucl55MammalianexampleofeditingTheCisconvertedtoUinintestinebyaspecificdeaminatingenzyme,notbyaguideRNA.难秸骗胀哨拓搏胸葵步媚遗锣剂锁铸押斩拓鼎钳转直聂屉接轮爱戍初胸香医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰MammalianexampleofeditingTh56CuttingandTrimmingRNACanuseendonucleasestocutatspecificsiteswithinalongerprecursorRNACanuseexonucleasestotrimbackfromthenewendstomakethematureproductThisgeneralprocessisseeninprokaryotesandeukaryotesforalltypesofRNA淑戊蹲译息汤胯两渡若热暮闺请幕哈炯碌楞坍找嗡砚昔年湍嘱捶刮颇爪恍医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰CuttingandTrimmingRNACanus57Theposttranscriptionalprocessingof

E.colirRNA.武蜘嫉铀掏乍失涝贬诲膨碧返左道刁狗谁袜羌曰氏郑前并浇握瞩诵冤国寄医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Theposttranscriptionalproces58RNaseIIIcutsinstemsofstem-loops16SrRNA23SrRNARNaseIIINoapparentprimarysequencespecificity-perhapsRNaseIIIrecognizesaparticularstemstructure.涧讶考崖楚泄敢寞深噎纂脆魂恋涡铲趾遏矾详俘戈外税扮缕潮课织蒂亿琴医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰RNaseIIIcutsinstemsofste59EukaryoticrRNAProcessingTheprimaryrRNAtranscript(~7500nt,45SRNA)contains18S,5.8Sand28SMethylation occurmostlyinrRNAsequence 80%:O2-methylribose,20%:bases(AorG)peudouridine 95UinrRNAinhumanareconvertedtoY’s maycontributerRNAtertiarystability泽基聚治报铡煽兆澈福边异俞豫氨兆磊因梨信消劲鼓逸钢淖吭乒跋侧琐抿医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰EukaryoticrRNAProcessingThe60TransferRNAProcessingCloverleafstructureCCA:aminoacidbindingsiteAnticodon~60tRNAgenesinE.coliAschematicdiagramofthetRNAcloverleafsecondarystructure.睹娶搂堵骂龚硷菩谢咽肆否操痒爽梭孟荫物樊蜡粗继植栽胰偿讽风竹垢柿医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰TransferRNAProcessingCloverl61Endo-andexonucleasestogenerateendsoftRNAEndonucleaseRNasePcleavestogeneratethe5’end.EndonucleaseRNaseFcleaves3nucleotidespastthemature3’end.ExonucleaseRNaseDtrims3’to5’,leavingthemature3’end.得软奏妄翅币观咆兽爱汉鲜讥盛涌辕描龙噎复畜计斑侵该缸挂瞅瓢镍变抽医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Endo-andexonucleasestogene62Splicingofpre-tRNAIntronsinpre-tRNAareveryshort(about10-20nucleotides)Haveno

consensussequencesAreremovedbyaseriesofenzymaticsteps:CleavagebyanendonucleasePhosphodiesterasetoopenacyclicintermediateandprovidea3’OHActivationofoneendbyakinase(withATPhydrolysis)Ligationoftheends(withATPhydrolysis)Phosphatasetoremovetheextraphosphateonthe2’OH(remainingafterphosphodiesterase)误碧坟脱领狡峙凛陋幸蠢喳苏裤苫定卧森全拒舍凳丰员万蛮宰墒吱宪福盈医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Splicingofpre-tRNAIntronsin63Stepsinsplicingofpre-tRNAPOH5’2’,3’cyclicphosphateExcisedintronIntronof10-20nucleotides1.Endo-nuclease2.Phospho-diesterase3.Kinase(ATP)4.Ligase(ATP)5.Phosphatase++SplicedtRNA筛绕渭泵桶由吠稀沙骤训冻圆队芥磊淆哮惭朗雪哥说瓜奸住箔忌橙米迪炳医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Stepsinsplicingofpre-tRNAP64CCAat3’endoftRNAsAlltRNAsendinthesequenceCCA.AminoacidsareaddedtotheCCAendduring“charging”oftRNAsfortranslation.FormosteukaryotictRNAs,theCCAisaddedaftertranscription,inareactioncatalyzedbytRNAnucleotidyltransferase.突纱署宅弦早扒尼者拐腿七誓演帮悸泊扒扦刑羊敢钢泪造商何嚎忘沤敖代医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰CCAat3’endoftRNAsAlltRNA65AllofthefourbasesintRNAcanbemodified刚遂廓傣验腆福俩织痰佛持粥速靡乱喂歼于豁隋任闻点盘今囚文卢彦佬揽医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰AllofthefourbasesintRNA66Pathologiesresulting

fromaberrantsplicing

can

begroupedintwomajorcategoriesMutationsaffectingproteinsthatareinvolvedinsplicing

Examples:

SpinalMuscularAtrophy RetinitisPigmentosa MyotonicDystrophyMutationsaffectingaspecificmessengerRNAanddisturbingitsnormalsplicingpattern

Examples:

β-Thalassemia DuchenneMuscularDystrophy CysticFibrosis FrasierSyndrome FrontotemporalDementiaandParkinsonism疾殆剃噶阴盘岗兄琵常坟煮镊歌握啦技吨翰足骸纵窘恶蓟飞疗靛苟岭撬平医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Pathologiesresultingfromabe67IntronAdvantage?OnebenefitofgeneswithintronsisaphenomenoncalledalternativesplicingApre-mRNAwithmultipleintronscanbesplicedindifferentwaysThiswillgeneratematuremRNAswithdifferentcombinationsofexonsThisvariationinsplicingcanoccurindifferentcelltypesorduringdifferentstagesofdevelopment昧而练宦捉伪呛拇瘟廖奎析凳饯纬缝菱发刀困组莲甥蝶谭倍皑砸欠稀怨褥医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰IntronAdvantage?Onebenefito68IntronAdvantage?Thebiologicaladvantageofalternativesplicingisthattwo(ormore)polypeptidescanbederivedfromasinglegeneThisallowsanorganismtocarryfewergenesinitsgenome蝶析诚灿彰闹泡徊济佳耳辛陷瘤疗跺篙诌傅业肺卉抢谤仲擞诈沙肚此帛从医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰IntronAdvantage?Thebiologica69Doyoubelieve?搁爵僳蜘莫洁时问晌震誊汐拌渺橙叮挎领脂豪荣憋嘻毗舌凶腹阎追喻幢专医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Doyoubelieve?搁爵僳蜘莫洁时问晌震誊汐拌渺橙70RNAinterferenceorRNAiisaremarkableprocesswherebysmallnoncodingRNAsilencespecificgenes.

-RNAiwasfirstobservedinplantinimmuneresponsetoviralpathgens.-MicroRNAregulategeneexpressioninorganismsfromnematodetoman.RNAInterferenceandInterferenceRNANobelPrizeinPhysiologyorMedicine2006,Andrew.FireandCraig.Mello站击旺乱冠斧躯兢剐度戎协择彦估杆鳃由授衡富矩变症语僳爸履茂棒电货医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰RNAinterferenceorRNAi71RNAInterference:AMechanismforSilencingGene

Expression1. SmalldsRNAfragmentscansilencetheexpressionofamatchinggene.ThisisRNAinterference(RNAi),recentlydiscoveredinC.elegans.a. InjectingdsRNAintoadultwormsresultsinspecificlossofthecorrespondingmRNAinthewormanditsprogeny.b. RNAialsooccursinmanyotherorganisms,whereitprotectsagainstviralinfectionandregulatesdevelopmentalprocesses.2. RNAiishighlyspecificandsensitive,withonlyafewmoleculesofdsRNAneeded,makingitanexcellentresearchtool.篱妮厨聚禾缚付吵禹哑韶沥眯昭敲铺八饭缚怔搂菊警豆差捉泽红酵吨嘴濒医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰RNAInterference:AMechanism72岳燥略件豆嘴夕履垫乳田见庆派哀姿俘哈耍吠婉迹承盲磺阐箍定冰罐棒知医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰岳燥略件豆嘴夕履垫乳田见庆派哀姿俘哈耍吠婉迹承盲磺阐箍定冰罐73时拙暮冠豪元痊怯鹊售符匆涤鸡特芥侨连嫉轨藻骨慢犬甘昂煎秀菜更直余医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰时拙暮冠豪元痊怯鹊售符匆涤鸡特芥侨连嫉轨藻骨慢犬甘昂煎秀菜更74ComparisonofsiRNAandmiRNAPrecursorStructureFunctionTargetmRNABiologicalsiRNAEndogenousorexogenousdsRNAdsRNAmRNAcleavageperfectcomplimentarityInhibittransponandvirusinfectionmiRNAEndogenoustranscriptssRNATranslationinhibitionandmRNAcleavageImperfectcomplimentaritydevelopment鞍某颂蛊嘴掏啼钞毡鸦匹榴儒拆肮焰阎兰虑络杠惶袄拙喀标劫锄瓦屈颗惧医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰ComparisonofsiRNAandmiRNAs75ForeignDNAandTransgeneForeignDNAandTransgeneAberrantsenseRNAdsRNAsiRNAsHeterochromatinformationandTranscriptionalsilencingRdRPDicerNature2004,Vol431,Sept.16:343予艰憋滓音照跪丝洁麻痉片放枣睹舟蓖守亚付焉垄这仆巷咕抗霓赋妻苑老医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰ForeignDNAandTransgeneForei76Mitrons:Shortintronichairpinspri-miRNApre-miRNAsMicroRNAs(MiRNAs)~22NTsRNAPloymeraseIIorIIIDicerSelfsplicingmiRNARISCNoneedofDroshaSplicingmachineryLariatdebranchingenzymeCell130,July132007:89-100站溺俯鸣露狰显废惺挚递戈帧寇阎假柄耻讽衡郊野阶肄哀淹花净虫铣蓑丑医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰Mitrons:Shortintronichairpi77lncRNAfunctions盒熟刑态女彬呕药枷囤挨跪寻宙嫌埋哟呜砚诡眺廓患歧朝叫吐扯弘州名健医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰lncRNAfunctions盒熟刑态女彬呕药枷囤挨跪寻宙78SomethingImaynotcare,butyouhaveto.胞莎缄艘酞躇嘻鸦湾蔓抚婪率齐盐绢赫娄田柏狙捕寞疯呀惯郧镍破蓑隔朔医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰SomethingImaynotcare,bu79李希分子医学教育部重点实验室lixi@TranscriptionandPost-transcriptionModification配炮绽扮捏嘎又漫狞退叁蝴佳毫互绍恤牺侍烈尝猾赤收奄皿登朴抡忌厕绿医用分子遗传学转录和转录后修饰医用分子遗传学转录和转录后修饰李希TranscriptionandPost-t80Post-transcriptional

ProcessingofRNAMakingendsofRNARNAsplicing蘑麻瘸们凭诅症娃日炬丙影焚举拍瞎毫财沈蓖泥辰坛衬药侮准闭导恰阳也医用分子遗传学转录