分子生物学Chapter5讲义

分子生物学Chapter5讲义Chapter5mRNAModificationsinEukaryotes

第5章真核生物mRNA的修饰Inprokaryotes,transcriptionproducesanearlyexactmRNAcopyoftheDNA,andthetranscriptisimmediatelytranslatedintoprotein.Ineukaryotes,aseriesofmodificationsoccurtomRNAduringandaftertranscription.在原核生物中，转录产生的mRNA几乎是DNA的准确拷贝，并且这一转录产物会立即被转译成蛋白质。在真核生物中，转录时以及转录后会对mRNA进行一系列修饰。mRNAModificationsinEukaryotes5.1Capping5.2Polyadenylation5.3Splicing5.4mRNAEditing5.5Experiments5.1加帽5.2聚腺苷酸化5.3剪接5.4mRNA编辑5.5实验研究Chapter5mRNAModificationsinEukaryotesconceptTheremoveofnucleotides

bybothendonucleasesandexonucleases.Theaddtionofnucleotidestothe5’-or3’-endsoftheprimarytranscriptionortheircleavageproduct.Aprimarytranscriptorpre-mRNAisanmRNAthathasbeentranscribedbutisnotyetreadyfortranslation.RNAprocessingisthecollectivetermusedtodescribethese

alterationstotheprimarytranscript.5.1Capping/加帽Cappingistheprocessofaddingaderivative(m7G)ofguaninenucleotidetothe5’endofthepre-mRNA.This

derivative

isattachedtothe

5’end

ofthepre-mRNAbya

5’-5’triphosphate

bondlinkage.Thereactioniscarriedout

byanenzymecalled

guanyltransferase(鸟苷转移酶).Structureofthecap/帽的结构5’-3’phospho-diesterbond5’-5’triphosphatebond7-methylguaninenucleotidemethylgroup5’-CapRNApolymeraseIIRNADNACappingtakesplacequiteearly.TranscriptionofanmRNAoccursinthe5’to3’direction.So,the5’endofthemRNA

comesoutfirst.Cappingtakesplacequiteearly,beforetherest

ofthegeneistranscribed.CappingprocessInvertedguaninenucleotideRNApolymeraseIIDNAMethyltransferaseGuanylyltransferaseRNAtriphosphataseCTD:C-terminaldomainThesethreeenzymesstaytogetheraroundtheC-terminaldomainoftheRpb2subunitofRNApolymeraseII.Theyacttogethertocompletethecappingprocess.Functionsofthecapstructure1.Helpspreventdegradation

帮助防止降解2.Helpstransportintocytoplasm

帮助转运到细胞质中3.Enhancestranslation/增强转译4.Helpsremovethefirstintron

帮助去除第一个内含子RNaseRNase5’-5’triphosphatebond5’-3’phosphodiesterbond5’-3’phosphodiesterbond1.HelpspreventdegradationRNAsinthecellcanberapidlydegradedbyribonucleases.However,theseenzymesaregenerallynotcapableofdegradingthetriphosphatebondinthecapstructure.2.HelpstransportintocytoplasmCapstructurehelpstheRNAtranscripttopassthroughselectiveporesofthenuclearmembraneandintothecytoplasm.TranscriptionoccursinthenucleusTranslationoccursinthecytoplasmCap-bindingproteinNotranslationoccurs.3.Enhancestranslation/增强转译Inordertobindtheribosome,mRNArequireshelpfromcap-bindingprotein.thisproteinrequiresthepresenceofthecapstructure.ribosomeRNApolymeraseIIRNADNAThefirstintron4.HelpsremovethefirstintronCapisrequiredformRNAsplicing,tooccurcompletly.Specifically,itspresenceisrequiredforsplicingofthefirstintron.AAAAAAA------AAAA5.2Polyadenylation/聚腺苷酸化Poly(A)tailPolyadenylationPre-mRNAPolyadenylationisamodificationprocessinwhichastringofabout250adeninenucleotidesisaddedtothe3’endofthetranscript.Polyadenylationsignal/聚腺苷酸化信号AAAAAAA------AAAAPoly(A)tailPolyadenylationPre-mRNAPolyadenylationsignalhasatypicaltypicalsequenceofAAUAAA.Thissequencegivesaninstructionas“tocutthemRNAabout20nucleotidesdownstream,nearaGU-richsequence”.AAUAAAGUAAUAAAmotifbyitselfnotsufficientforpolyadenylation.Ifitwere,thenpolyadenylationwouldoccurdownstreamofthemanyAAUAAAsequencefoundinintrons,butitdoesnot.PolyadenylationSignals:50-250poly(A)Cleavageandpolyadenylationofapre-mRNApolyadenylationinvolvesboth

cleavageofthepre-RNAand

polyadenylation

atthecleavagesite.Requiresseveralproteins:CPSF（切割与聚腺苷酸化特异因子）,CstF（切割激活因子）,CFI,CFII（切割因子I和II）,poly(A)polymerase,andRNApolymeraseII(inparticular,theCTDofRPb2).Thecleavagecomplex/切割复合体CleavagecomplexCPSF(cleavageandpolyadenylationspecificityfactor)CstF(cleavagestimulationfactor)CFI(cleavagefactorI)CFII(cleavagefactorsII)Poly(A)-BindingProteinControlthelengthoftail

AAUAAAsignalhasbeentranscribed,CPSFbindstotheAAUAAAsequence.CstFbindstotheG-UrichsequencePBPcanhelppoly(A)polymerasetoworkmoreefficiently.Promotionpoly(A)elongationCTD:C-terminaldomainProteinsforcappingatCTDProteinsfortailingatCTDaftercappingisperformed,CTDwillbephosphorylatedGraduallyandthecappingproteinsareremoved.Functionsofthepoly(A)tail

poly(A)尾的功能AAAAAAA------AAAAAAAAAAA---AAAAAAThemainfunctionofpoly(A)tailistoprotectthemRNAfromdegradationbyribonucleases.ItdependsonthelongstringofAsthatseparatethecodingregionfromtheendofthemRNAAAAAAAA------AAAAThereisalsosomeevidencethatthepoly-Atailisinvolvedin

splicingandenhancestranslationofmRNAs.5.3Splicing/剪接Exons:Partsofagenethatareexpressedasprotein.Introns:Sequencesthatdonotcodeforproteinandinterruptthecodingregions.Splicing:Theprocessofremovingintronsandrejointheexonfromapre-mRNA.InterruptgeneJunctionsequenceofintronlinkingwithexon高度保守，成为剪接过程重要的识别序列。人类许多重要疾病的病因

Junctionseq.mut.→异常剪接→病症地中海贫血症：

junctionsequencemutationofglobingene干扰mRNA成熟Junctionsequence5.3.1TheBasicSplicingReaction

基本的剪接反应5’AG/GUAUGU…bodyofintron…UACUAAC-YAG/3’SplicesitesinyeastSplicesites:Sequencesthatmarkthebeginningandendsofintrons.AlmostallintronshaveGUon5’endandhaveAGon3’end.Thistypicalstructureformsthesplicesitesforsplicingreaction.5’splicingsiteordonorsite3’splicingsiteoracceptorsite核内前体mRNAsplicingGroupⅢintronsobservedbaseattacksiteLariatinronGroupⅢsplicingmodelHydroxylgrouponthe‘A’attacksthe5’splicesite.ThebondbetweenGoftheexonandtheGof

intronattheboundary

isbroken.The-OHoftheexonterminalGattacksthe3’splicesite.Removaloftheintroniscomplete.2’-OH2次转酯反应套索内含子ProteinsinvolvedinSplicing

在剪接中发挥作用的蛋白质Spliceosome:Thecollectionoffactors,especiallysnRNPs,thathelpwiththesplicingofintrons.Theseproteinsformspliceosome.snRNPs－核内小核糖核蛋白剪接体snRNPs:smallnuclear

ribonucleoproteinssnRNPsaresmallparticlesfoundinthenucleusandcontainbothproteinandRNA（SnRNA）.snRNPsfunctioninginsplicing:U1,U2,U4,U5andU6.SnRNAcanformspecific

basepairswiththepre-mRNA.U1bindsatthe5’splicesite.Next,U2bindsattheattackingA.SplicingprocessofspliceosomeU4andU6jointhe5’splicesitewhileU5holdsthe5’and3’splicesitestogether.Finally,U6andU2worktogethertocarryoutthemajorreactionsofsplicing.U4andU6bindtoeachother.Next,U4

unbindsU6.ThisactivatesU6,anditremovesU1fromthe5’splicesite.5.3.2

Self-Splicing/自我剪接Tetrahymenathermophilia嗜热四膜虫

SplicingthatoccurswithoutthehelpofproteinsorsnRNPsiscalledself-splicing.低等真核生物rRNA，线粒体和叶绿体基因内含子。Intron含有多个保守的CentralCoreSeqence（中部核心结构）它们反向平行，序列互补，形成分子内二级结构。Internalguidesequence(IGS):

与5’donor，3’acceptor序列互补，使U与G靠近。Self-splicingofgroupIintronIGSoftetrahymenarRNAIGS：内含子内存在的一段序列，可以与5’供点或3’受点边界序列互补。IGS保守序列：GGAGGGCCSReactionofSelf-splicing5’splicesite

isbrokenbyattackfromaG.Thefreeendoftheexonattacksthe3’splicesite,displacingtheintronandforminganewbondwiththenextexon.二次转酯反应413ntintron15ntRibozyme(核酶)：象35srRNA具有酶活性，能自我催化完成剪接的RNA。IGSSelf-splicingofgroupIIintron核mRNA，tRNA攻击位点牛仔套马索5’-2’磷酸二酯键twotransesterification5.3.4Trans-Splicing/反式剪接被剪接的前导序列cis-splicing小外显子SL：含内含子5’供点GULS：受点3’AG-OHattackthe5’splicesitemRNA前体含有LeadingsequenceY-内含子5.3.5Alternativesplicing/可变剪接Alternativesplicing:akindofsplicingthatcanproducevariousproteins((isoformprotein)fromonegene.constitutive同源异型蛋白Bychoosingvariouscombinationsof17exonsfroma8,000differentproteinscanbeproducedfromthisonegene.DrosophilaDscamgeneExon3Exon54.14.24.34.44.54.64.74.84.94.104.114.12Exon4Exon6Exon9AlternativeSplicingGoesMainstream可变剪接成为主流TheScientist,Volume17(24):28,Dec.15,2003contains116exonsstickantibodyinthemembraneAlternativesplicinginimmunecellstransmembraneregionsecretedantibody5.4mRNAEditing/mRNA编辑RNA编辑是指在mRNA水平上改变遗传信息的过程。指基因转录产生的mRNA分子中，由于核苷酸的缺失，插入或置换，基因转录物的序列不与基因编码序列互补，使翻译生成的蛋白质的氨基酸组成，不同于基因序列中的编码信息现象。mRNA编辑通过编辑，可以给mRNA前体添加新的遗传信息。Sleepingsicknessisoneofthemostneglectedhumandiseases,andmainlyaffectsthemostdeprivedpeoplesofsomeAfricancountries.Trypanosome锥虫TrypanosomesareprotozoathatcauseAfricansleepingsickness.EditingmechanismWhatdetermineswheretheeditingsystemshouleaddUMP?GuideRNAisasmallRNAwhosepartialsequenceiscomplementarytothesequenceofanRNAthatwillbeedited.Insertionediting---向导RNA介导的编辑：指导RNA：一类小RNA分子，其部分序列可与被编辑地RNA序列互补。Atthe5’-end,anchorsequencedirectsthegRNAtotheregionofthemRNAitwilledit.apolipoproteinB(APOB)（载脂蛋白）TheapolipoproteinB(APOB)carriescholesterolinthebody.Intheliver,alargeversion