分子生物學(xué)Chapter5講義

分子生物學(xué)Chapter5講義Chapter5mRNAModificationsinEukaryotes

第5章真核生物mRNA的修飾Inprokaryotes,transcriptionproducesanearlyexactmRNAcopyoftheDNA,andthetranscriptisimmediatelytranslatedintoprotein.Ineukaryotes,aseriesofmodificationsoccurtomRNAduringandaftertranscription.在原核生物中，轉(zhuǎn)錄產(chǎn)生的mRNA幾乎是DNA的準(zhǔn)確拷貝，并且這一轉(zhuǎn)錄產(chǎn)物會(huì)立即被轉(zhuǎn)譯成蛋白質(zhì)。在真核生物中，轉(zhuǎn)錄時(shí)以及轉(zhuǎn)錄后會(huì)對(duì)mRNA進(jìn)行一系列修飾。mRNAModificationsinEukaryotes5.1Capping5.2Polyadenylation5.3Splicing5.4mRNAEditing5.5Experiments5.1加帽5.2聚腺苷酸化5.3剪接5.4mRNA編輯5.5實(shí)驗(yàn)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(鳥(niǎo)苷轉(zhuǎn)移酶).Structureofthecap/帽的結(jié)構(gòu)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

幫助防止降解2.Helpstransportintocytoplasm

幫助轉(zhuǎn)運(yùn)到細(xì)胞質(zhì)中3.Enhancestranslation/增強(qiáng)轉(zhuǎn)譯4.Helpsremovethefirstintron

幫助去除第一個(gè)內(nèi)含子RNaseRNase5’-5’triphosphatebond5’-3’phosphodiesterbond5’-3’phosphodiesterbond1.HelpspreventdegradationRNAsinthecellcanberapidlydegradedbyribonucleases.However,theseenzymesaregenerallynotcapableofdegradingthetriphosphatebondinthecapstructure.2.HelpstransportintocytoplasmCapstructurehelpstheRNAtranscripttopassthroughselectiveporesofthenuclearmembraneandintothecytoplasm.TranscriptionoccursinthenucleusTranslationoccursinthecytoplasmCap-bindingproteinNotranslationoccurs.3.Enhancestranslation/增強(qiáng)轉(zhuǎn)譯Inordertobindtheribosome,mRNArequireshelpfromcap-bindingprotein.thisproteinrequiresthepresenceofthecapstructure.ribosomeRNApolymeraseIIRNADNAThefirstintron4.HelpsremovethefirstintronCapisrequiredformRNAsplicing,tooccurcompletly.Specifically,itspresenceisrequiredforsplicingofthefirstintron.AAAAAAA------AAAA5.2Polyadenylation/聚腺苷酸化Poly(A)tailPolyadenylationPre-mRNAPolyadenylationisamodificationprocessinwhichastringofabout250adeninenucleotidesisaddedtothe3’endofthetranscript.Polyadenylationsignal/聚腺苷酸化信號(hào)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（切割與聚腺苷酸化特異因子）,CstF（切割激活因子）,CFI,CFII（切割因子I和II）,poly(A)polymerase,andRNApolymeraseII(inparticular,theCTDofRPb2).Thecleavagecomplex/切割復(fù)合體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，成為剪接過(guò)程重要的識(shí)別序列。人類(lèi)許多重要疾病的病因

Junctionseq.mut.→異常剪接→病癥地中海貧血癥：

junctionsequencemutationofglobingene干擾mRNA成熟Junctionsequence5.3.1TheBasicSplicingReaction

基本的剪接反應(yīng)5’AG/GUAUGU…bodyofintron…UACUAAC-YAG/3’SplicesitesinyeastSplicesites:Sequencesthatmarkthebeginningandendsofintrons.AlmostallintronshaveGUon5’endandhaveAGon3’end.Thistypicalstructureformsthesplicesitesforsplicingreaction.5’splicingsiteordonorsite3’splicingsiteoracceptorsite核內(nèi)前體mRNAsplicingGroupⅢintronsobservedbaseattacksiteLariatinronGroupⅢsplicingmodelHydroxylgrouponthe‘A’attacksthe5’splicesite.ThebondbetweenGoftheexonandtheGof

intronattheboundary

isbroken.The-OHoftheexonterminalGattacksthe3’splicesite.Removaloftheintroniscomplete.2’-OH2次轉(zhuǎn)酯反應(yīng)套索內(nèi)含子ProteinsinvolvedinSplicing

在剪接中發(fā)揮作用的蛋白質(zhì)Spliceosome:Thecollectionoffactors,especiallysnRNPs,thathelpwiththesplicingofintrons.Theseproteinsformspliceosome.snRNPs－核內(nèi)小核糖核蛋白剪接體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嗜熱四膜蟲(chóng)

SplicingthatoccurswithoutthehelpofproteinsorsnRNPsiscalledself-splicing.低等真核生物rRNA，線粒體和葉綠體基因內(nèi)含子。Intron含有多個(gè)保守的CentralCoreSeqence（中部核心結(jié)構(gòu)）它們反向平行，序列互補(bǔ)，形成分子內(nèi)二級(jí)結(jié)構(gòu)。Internalguidesequence(IGS):

與5’donor，3’acceptor序列互補(bǔ)，使U與G靠近。Self-splicingofgroupIintronIGSoftetrahymenarRNAIGS：內(nèi)含子內(nèi)存在的一段序列，可以與5’供點(diǎn)或3’受點(diǎn)邊界序列互補(bǔ)。IGS保守序列：GGAGGGCCSReactionofSelf-splicing5’splicesite

isbrokenbyattackfromaG.Thefreeendoftheexonattacksthe3’splicesite,displacingtheintronandforminganewbondwiththenextexon.二次轉(zhuǎn)酯反應(yīng)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攻擊位點(diǎn)牛仔套馬索5’-2’磷酸二酯鍵twotransesterification5.3.4Trans-Splicing/反式剪接被剪接的前導(dǎo)序列cis-splicing小外顯子SL：含內(nèi)含子5’供點(diǎn)GULS：受點(diǎn)3’AG-OHattackthe5’splicesitemRNA前體含有LeadingsequenceY-內(nèi)含子5.3.5Alternativesplicing/可變剪接Alternativesplicing:akindofsplicingthatcanproducevariousproteins((isoformprotein)fromonegene.constitutive同源異型蛋白Bychoosingvariouscombinationsof17exonsfroma8,000differentproteinscanbeproducedfromthisonegene.DrosophilaDscamgeneExon3Exon54.14.24.34.44.54.64.74.84.94.104.114.12Exon4Exon6Exon9AlternativeSplicingGoesMainstream可變剪接成為主流TheScientist,Volume17(24):28,Dec.15,2003contains116exonsstickantibodyinthemembraneAlternativesplicinginimmunecellstransmembraneregionsecretedantibody5.4mRNAEditing/mRNA編輯RNA編輯是指在mRNA水平上改變遺傳信息的過(guò)程。指基因轉(zhuǎn)錄產(chǎn)生的mRNA分子中，由于核苷酸的缺失，插入或置換，基因轉(zhuǎn)錄物的序列不與基因編碼序列互補(bǔ)，使翻譯生成的蛋白質(zhì)的氨基酸組成，不同于基因序列中的編碼信息現(xiàn)象。mRNA編輯通過(guò)編輯，可以給mRNA前體添加新的遺傳信息。Sleepingsicknessisoneofthemostneglectedhumandiseases,andmainlyaffectsthemostdeprivedpeoplesofsomeAfricancountries.Trypanosome錐蟲(chóng)TrypanosomesareprotozoathatcauseAfricansleepingsickness.EditingmechanismWhatdetermineswheretheeditingsystemshouleaddUMP?GuideRNAisasmallRNAwhosepartialsequenceiscomplementarytothesequenceofanRNAthatwillbeedited.Insertionediting---向?qū)NA介導(dǎo)的編輯：指導(dǎo)RNA：一類(lèi)小RNA分子，其部分序列可與被編輯地RNA序列互補(bǔ)。Atthe5’-end,anchorsequencedirectsthegRNAtotheregionofthemRNAitwilledit.apolipoproteinB(APOB)（載脂蛋白）TheapolipoproteinB(APOB)carriescholesterolinthebody.Intheliver,alargeversion