RNA結(jié)合蛋白與轉(zhuǎn)錄后調(diào)控

RNA結(jié)合蛋白與轉(zhuǎn)錄后調(diào)控第1頁/共45頁RegulatoryfactorsformRNAdecayandtranslationRNAbindingproteinsmicroRNAs第2頁/共45頁RNAbindingproteins(RBPs)RNA結(jié)合蛋白種類很多，估計占細(xì)胞編碼蛋白6-8%者為RNA結(jié)合蛋白,但迄今只有為數(shù)不多的幾種RNA結(jié)合蛋白（如HuR，AUF1，TTP，TIA1，CUGBP2等）被證實(shí)可特異參與mRNA穩(wěn)定性、翻譯、或其它層面的基因調(diào)控。第3頁/共45頁HuR?Hu/ELAVRNA結(jié)合蛋白家族（包括HuA/HuR，Hel-N1，HuC，HuD）的主要成員。與其它成員僅表達(dá)于神經(jīng)細(xì)胞不同，HuR幾乎在所有組織都有表達(dá)。主要位于細(xì)胞核，但可穿梭于細(xì)胞漿/核間，且只有細(xì)胞漿HuR

可調(diào)控mRNA穩(wěn)定性（及翻譯）。核內(nèi)HuR則與mRNA成熟及export有關(guān)。?結(jié)合所有三類AREs。結(jié)合后的結(jié)局主要為穩(wěn)定目標(biāo)。因此也被稱為mRNA穩(wěn)定因子。?結(jié)合并穩(wěn)定VEGF,COX-2,p21,cyclinA,cyclinB1,c-fos,TNFα，IL-1，

MyoD，Myogenin，bcl-2等mRNA。因此可調(diào)節(jié)應(yīng)激反應(yīng)，細(xì)胞周期，腫瘤，分化，調(diào)亡等過程。?

HuR也可結(jié)合目標(biāo)并調(diào)節(jié)其翻譯。

1）調(diào)控翻譯效率，如p53，p27mRNA,c-myc。

2）調(diào)控mRNAexport,如CD83，c-fos,COX-2。第4頁/共45頁AUF1

也叫HnRNPD（heterogeneousnuclearribonucleoproteinD).主要位于核內(nèi)，但可穿梭于核/細(xì)胞漿間，結(jié)合I，II類AREs。結(jié)合目標(biāo)mRNA后使之不穩(wěn)定（易于降解），如P21，CyclinD1，也可使目標(biāo)穩(wěn)定，如TNF-alpha。第5頁/共45頁TTP與HuR及AUF1不同，TTP主要位于細(xì)胞漿，結(jié)合II類AREs。結(jié)合目標(biāo)后主要使目標(biāo)不穩(wěn)定。如：COX-2，TNF-alpha,GM-CSF,等。第6頁/共45頁

EffectofARE-BPsonthestabilityandtranslationofARE-containingmRNAsARE-BPsmRNAstabilityProteinexpressionTranslationalefficiencyAbundanceIncreaseDecreaseIncreaseDecreaseUpregulatedDownregulatedAUF1c-myc(42)c-myc(46)GM–CSF(55)c-fos(42,67)c-fos(53)IL-3(55)PTH(56)p21(48)GM–CSF(42)CyclinD1(48)TNF-alpha(42)GM–CSF(53,54)IL-3(55)HuRc-fos(59,63,67)p53(99,137)TNF-alpha(139)p21(69)TNF-alpha(139)MyoD(68)Cox-2(139)CyclinA(70)p21(48,68,69)CyclinB1(70)CyclinA(70)NOSII/iNOS(64)CyclinB1(70)GM–CSF(55)CyclinD1(48)Cox-2(71,173)NOSII/iNOS(64)IL-3(55)GM–CSF(59)VEGF(173)TNF-alpha(65,74,139)p53(99,137)Cox-2(71,139)IL-3(55,66)VEGF(62)Myogenin(68)Hel-N1TNF-alpha(74)NF-M(73)NF-M(73)GLUT1(72)GLUT1(72)GLUT1(72)HuDGAP-43(75–77)GAP-43(75,76)TTPc-fos(90)GM–CSF(81)GM–CSF(18,81,83–85,91)TNF-alpha(80)TNF-alpha(18,81,83–86,89,90)IL-2(82)Cox-2(87)IL-3(88)IL-2(82,90)IL-3(18,66,83,84,88)BRF1TNF-alpha(89,93)GM–CSF(55)IL-3(55,92,93)IL-3(55)TIA-1TNF-alpha(120)TNF-alpha(120)Cox-2(121)Cox-2(121)KSRPc-fos(90,93)NOSII/iNOS(102)NOSII/iNOS(102)TNF-alpha(90,93)IL-2(90,93)c-jun(93)CUG-BP2Cox-2(150)Cox-2(150)Cox-2(150)Nucleolinbcl-2(175)TINObcl-2(176)PAIP2VEGF(177)VEGF(177)第7頁/共45頁Interactionofthefactorsinvolvinginpost-transcriptionalregulation1.RNA結(jié)合蛋白相互作用。如，HuR與AUF1均可結(jié)合于p21與cyclinD13’UTR，但二者有競爭，且功能相反。2.RNA結(jié)合蛋白與microRNA間相互作用。如，HuR與let-7,miR-122,TTP與miR-16。第8頁/共45頁AU-richElements(AREs)1.主要指位于3‘-非翻譯區(qū)的富含AU的序列。2.被RNA結(jié)合蛋白識別，結(jié)合。3.主要為mRNA不穩(wěn)定元件，是mRNA在完成使命后快速降解的結(jié)構(gòu)基礎(chǔ)。4.依構(gòu)成分為三類1）含1-5個分散的AUUUA。2）至少有兩個OverlappingUUAUUUA(U/A)(U/A).

依AUUUA的重復(fù)方式分為5類，IIA，IIB，IIC，等。3）富含U，但無AUUUA。注：除一級結(jié)構(gòu)外，mRNA的二級結(jié)構(gòu)也與RNA-蛋白質(zhì)相互作用密切相關(guān)。如，約70-80%HuR或AUF1結(jié)合序列具有相似的二級結(jié)構(gòu)第9頁/共45頁mRNAsARE-BPs

ClassMotifExamples

I1to5個散在

AUUUAc-mycAUF1,HuR，Hel-N1，HuC，HuD，AUH，GAPDH，Hsp70

c-fosAUF1，HuR，Hel-N1，HuD，KSRP，AUH

Beta1-ARAUF1

PTHAUF1

Interferon-gammaGAPDH，Hsp70

MyoDHuR

p21AUF1，HuR，HuD

CyclinAHuR

CyclinB1HuR

CyclinD1AUF1，HuR

PAI-2HuR

NOSII/iNOSHuR，KSRP

IIA

(AUUU)5AGM–CSFAUF1，HuR，Hel-N1，TTP，BRF1，TIAR，KSRP，AUH，GAPDH，Hsp70，hnRNP-A1，hnRNP-C

TNF-alphaAUF1，HuR，TTP，BRF1，TIA-1，TIAR，KSRP

IIB

(AUUU)4AInterferon-alphahnRNP-A1，hnRNP-A2,hnRNP-A3

IIC

(A/U)(AUUU)3A(A/U)

Cox-2AUF1,HuR,HuD,TTP,TIA-1,TIAR,hnRNP-A1，hnRNP-A2,hnRNP-A3，CUDBP2

IL-2AUF1，HuR，HuD，TTP。Hsp70，Hsp110，hnRNP-A1

IL-3HuR，BEF1，AUH，Nucleolin，TINO

bcl-2AUF1，HuR，

VEGFHuRHuC，HuD，PAIP2

III

NoAUUUA,c-junTIAR，CUGBP1

U-richregionGLUT1Hel-N1，hnRNP-A2，hnRNP-L

p53HuR，

IdHel-N1

hsp70HuR

MyogeninHuR

NF-MHel-N1

GAP-43HuD

AmatrixpresentationhasbeenusedtorepresenttheidentifiedassociationsbetweenARE-BPsandARE-containingmRNAs.ThemRNAscontainingidentifiedfunctionalAREsarelistedverticallyandaregroupedaccordingtotheclassificationsproposedby(24)and(26).TheARE-BPsaredisplayedhorizontally.WhereappropriatethedifferentnamesusedtodenotethesameproteinormRNAaregiven.ThelistsofARE-containingmRNAsandofARE-BPsarenotexhaustiveandonlydirectinteractionshavebeenconsidered.Wheretheexperimentalmethodsusedidentifiedendogenousinteractions,theseareindicatedbyanasterisk.DataontheexperimentalmethodsarepresentedintheSupplementarydata.Numberscorrespondtolistedreferences.InteractionbetweenAREandRBPs第10頁/共45頁RNA-蛋白質(zhì)相互作用(結(jié)合)的特點(diǎn)可在細(xì)胞核,也可在細(xì)胞漿發(fā)生在核與胞漿的結(jié)合功能截然不同,如在胞漿與翻譯及mRNA穩(wěn)定性有關(guān),在核可能與拼接或成熟有關(guān).RNA結(jié)合蛋白對目標(biāo)的序列要求不如DNA結(jié)合蛋白般嚴(yán)格.結(jié)合部位大多在3’-UTR,少數(shù)在5’-UTR,絕少見于CDS.第11頁/共45頁mRNA穩(wěn)定性（turnover）研究的特殊技術(shù)蛋白質(zhì)-RNA結(jié)合試驗(yàn)：

1）目標(biāo)Transcript的體外轉(zhuǎn)錄與標(biāo)記

2）細(xì)胞漿抽提物的制備。

3)EMSA(gel-shift,supershift)4)rChip,pull-downassays(usingparamagneticstreptavidindynabeads,biotinyl-labeledtranscripts)mRNA半衰期測定

基本思路:終止轉(zhuǎn)錄后,收取不同時間點(diǎn)之RNA,定量分析RNA降解速率.1）用ActinomycinD終止轉(zhuǎn)錄

2）Tet-off/on（或類似）報告基因系統(tǒng)

3)invitroRNA降解分析第12頁/共45頁"Tet-on"systemisactivatedinthepresenceofdoxycyclinetheDNAbindingdomainoftheTet-onregulator(rTetR)containsmutations

repressorthatonlybindsDNAintheabsenceofligand

isconvertedtoaligand-dependentDNAbindingprotein.RNA-polTetracyclinecontrolledtransactivator(tTA)第13頁/共45頁TET-OFFindetailsManfredGossenandHermannBujard

The"Tet-off"systemisrepressed

inthepresenceofthedoxycyclineTET-VPproducingvectorGeneofinterestexpressingvectorVP–RNApolinteractingpartTetR-tetbindingpartTET-OFFsystemTetracyclinecontrolledtransactivator(tTA)如：EGFP-interesttargetchimeric…第14頁/共45頁mRNA翻譯研究特用技術(shù)新生蛋白分析。Polysome分離，PolysomalRNA,Polysomal蛋白質(zhì)分離。其它經(jīng)典技術(shù)。第15頁/共45頁ReferenceBarreau,etal;NucleicAcidsResearch,33(22):7138-7150,20052)3)Wang,etal;MCB,20:760-769,20004)Lal,etal;EMBOJ.,23:3092-3102,2004/departments/mmb/baranova/pages/ppt/biotech-lec5.ppt第16頁/共45頁HuRRegulatesp21mRNAStabilizationbyUVLight

Wang,etal;MolCellBiol.2000，20(3):760–769.

細(xì)胞暴露于多種應(yīng)激（Stresses）如short-wavelengthUVlight(UVC)時,cyclin-dependentkinaseinhibitorp21的表達(dá)明顯被誘導(dǎo)。P21的調(diào)控，尤其P53調(diào)節(jié)的轉(zhuǎn)錄已被廣泛，深入研究。先前的研究發(fā)現(xiàn)，UVC可通過P53-不依賴的方式誘導(dǎo)P21。研究還發(fā)現(xiàn)，細(xì)胞暴露于UVC后，p21mRNA穩(wěn)定性增加。問題：UVC誘導(dǎo)P21表達(dá)（穩(wěn)定性增加）的機(jī)制如何？與HuR有關(guān)否？Background:ExampleⅠ第17頁/共45頁UVC輻射誘導(dǎo)蛋白-P21RNA復(fù)合物形成。復(fù)合物形成為P53不依賴性的，因?yàn)闊o論RKO細(xì)胞是否有野生型P53，復(fù)合物的形成無區(qū)別。復(fù)合物由蛋白質(zhì)與3‘UTR間結(jié)合而成。5’UTR及缺失ARE的3‘-UTR（A1，C5）幾乎無蛋白結(jié)合。核與細(xì)胞漿蛋白均可與P213’UTR形成復(fù)合物，但只有胞漿中的復(fù)合物形成可被UVC誘導(dǎo)。UVCinducestheformationofp213’-UTR-proteincomplexinthecytoplasm第18頁/共45頁A。復(fù)合物形成在UVC輻射半小時后明顯被誘導(dǎo)，與P21被誘導(dǎo)相吻合。B。競爭抑制試驗(yàn)說明復(fù)合物的特異性，Cold探針可競爭抑制復(fù)合物形成。C。EMSA后，UV交聯(lián)，SDS分離復(fù)合物，發(fā)現(xiàn)復(fù)合物中一條大約40Kd的復(fù)合物（單一蛋白與大約10個堿基的短片斷轉(zhuǎn)錄物形成），說明有一35-40KdRNA結(jié)合蛋白被UVC誘導(dǎo)并與P213’-UTR結(jié)合。D。UVC誘導(dǎo)該未知復(fù)合物的趨勢與P21被誘導(dǎo)一致。Elevationofp21byUVCisaccompaniedwithincreasedformationofP213’UTR-proteincomplex第19頁/共45頁HuR結(jié)合p21mRNA（invivoandinvitro）

(A)，(B)HuR抗體可特異結(jié)合細(xì)胞漿蛋白與B2形成的復(fù)合物。(C)RNaseT1SelectionAssaywascarriedoutwithB2andA1,incubatedwith10nMGSTorGST-HuR(seeMaterialsandMethods).T1,digestionswithRNaseT1alone;M,molecularweightmarkers.(D)GelretardationassaysusingB2andtheindicatedconcentrationsofeitherGSTorGST-HuR.(E)EMSA-WesternAssay:Left,cytoplasmicfractionswereeitherincubatedwithB2ornot,cross-linked,digestedwithRNaseT1,resolvedbySDS(15%gel),andtransferredontopolyvinylidenedifluoridemembranes,whichweresequentiallyexposedtoX-rayfilmfor24h(Radioactivesignal)andsubjectedtoWesternblotanalysistodetectHuR(Westernsignal);exposuretime,30s.Right,LysatesfromUVC-treatedoruntreatedcellswereincubatedwithB2andthensubjectedtoWesternblotanalysis.EstimatedsizeoftheHuR-p21complexes,37to40kDa.HuRbindstothe3’UTRofp21mRNA第20頁/共45頁HuR表達(dá)降低后，

p213′UTR與細(xì)胞漿蛋白間形成的復(fù)合物（在UVC輻射后）降低，p21mRNA穩(wěn)定性降低（半衰期縮短），表達(dá)降低。DecreasedHuRexpressionlowersbindingtothep213′UTRandreducesp21mRNAstabilityandp21inductionbyUVC.(A)WesternblotanalysisofHuRexpressioninRKOcells,eitheruntransfected(untr.)ortransfectedwithpZeoSV2(?)HuR,expressingASHuR.Chosenclonalisolatesareshown.Blotsweresequentiallystrippedandrehybridizedwithanantibodyrecognizingactin(43kDa),tovisualizedifferencesinloadingandtransfer,andwithanantibodyrecognizinghnRNPC(43kDa).(B)B5bindingactivityinlysatesfromuntransfectedandASHuR-expressingcells6hafterUVCirradiation.(C)Northernblotanalysisofp21mRNAexpressioninuntransfectedandASHuR-expressingRKOcells8haftereithernotreatment(?)orexposuretotheindicatedUVCdoses.Evennessinloadingandtransferamongsampleswasassessedafterstrippingthemembraneandrehybridizingitwithanoligomerproberecognizing18SrRNA.(D)Westernblotanalysistoassesstheexpressionofp21,c-Jun(39kDa),andactininuntransfectedandASHuR-expressingRKOcells10haftereithernotreatmentorexposureto20J/m2UVC.p-jun,phosphorylatedJun.(E)Graphsdepicttherateoflossofp21andβ-actinmRNAsincellswithdifferentHuRlevelsafteractinomycinD(2μg/ml)additionwithorwithoutUVCirradiation.Atthetimesindicated,totalRNAwasextractedandp21andβ-actinmRNAsweremonitoredbyNorthernblotting;signalswerequantitatedwithaPhosphorImager,normalizedagainst18S(notshown),andplottedonalogarithmicscale.ThemRNAhalf-lifeineachtreatmentgroupisindicatedinparentheses.Valuesrepresentmeans±standarderrorsofthemeansofthreeindependentexperiments.EctopicexpressionoftheantisenseHuRinhibitedtheinteractionofHuRwithp21mRNAandreducedthelevelsaswellasthehalf-lifeofp21mRNA第21頁/共45頁UVC輻射下，B2（含HuR結(jié)合位點(diǎn)）賦予P21mRNA穩(wěn)定性。用反義方法降低內(nèi)源性HuR表達(dá)后，由B2賦予的穩(wěn)定性消失。Effectofthefull-lengthandmutantp213′UTRonexpressionofaluciferasereporterconstruct.(Top)ExpressionvectorspGL3,pGL3-FL,andpGL3-ΔB2(seeMaterialsandMethods)weretransientlycotransfectedintoRKOparental(untransfected[Untr.]),AS.2,orAS.7cellsalongwithpSV-βgal(usedtonormalizefortransfectionefficiency);cellswereirradiatedwithUVC(20J/m2)orleftuntreated,andluciferaseandβ-galactosidaseactivitieswereexamined24hlater.(Bottom)RelativefoldincreaseinluciferaseactivityafterUVCexposure,seenwitheitherpGL3-FLorpGL3-ΔB2comparedwiththatseenwiththecontrolvectorpGL3.Valuesrepresentmeans±standarderrorsofthemeansoffiveindependentexperimentsTheB2fragmentconfersPGL3-B2reporterabilitytorespondtothedown-regulationofHuR第22頁/共45頁WesternblotanalysisofHuRexpressionandsubcellularlocalization.(A)SixhoursafterirradiationwiththeindicateddosesofUVC,whole-cell(20μg),cytoplasmic(40μg),nuclear(10μg),andcytosolic(40μg)lysateswerepreparedandsubjectedtoWesternblotanalysistomonitortheexpressionofHuR,hnRNPC,AUF1,BAF57c(57kDa),andactin.CelllysateswerecollectedatthetimesindicatedafterirradiationwithUVC(20J/m2)(B)or6hafterirradiationwiththeindicateddosesofUVC(C),andWesternblotanalysisofHuRexpressionperformedoncytoplasmic(40μg)andnuclear(10μg)fractions.(D)Indicateddosesofionomycin(Ion.;micromolar)orlithiumacetate(LiAc;millimolar)wereaddedtocells1hbeforeUVCirradiationwith20J/m2andWesternblotanalysisofcytoplasmicHuR.HybridizationusingantibodiesagainstactinandBAF57cwascarriedouttoassessuniformityinloadingandtransferamongcytoplasmicandnuclearsamples,respectively.UVCinducesthecytoplasmicpresenceofHuR第23頁/共45頁SubcellularlocalizationofHuR.GFP-HuRwasvisualizedbyfluorescencemicroscopyintransientlytransfectedRKOcellsthatwereeitherleftuntreatedortreatedwith20JofUVC/m2(4hearlier).DAPIstainingservedtovisualizethenucleus.NotethedistinctoverlapofDAPIandGFP-HuRsignalsinuntreatedcells;whileUVC-irradiatedcellsalsoexhibitabundantnuclearGFP-HuR,thetreatmentcausesasubstantialincreaseinthecytoplasmicGFP-HuRsignal,notseeninuntreatedcells.UVCinducescytoplasmicHuR第24頁/共45頁IncreasedcytoplasmicHuRandp21RNAbindingafterexposuretostresses.(A)WesternblotanalysistomonitorHuRexpressionincytoplasmicandnuclearfractionsaftertreatmentwiththeindicatedagents.Sampleswerecollected2hafteradditionofactinomycin(Act.)D(1μg/ml)or4hafterexposureto100μMH2O2,MMS(100μg/ml),48μMPGA2,orUVC(20J/m2).HybridizationsusingantibodiesagainstactinandBAF57cwerecarriedouttoassessuniformityinloadingandtransferamongcytoplasmicandnuclearsamples,respectively.(B)B2bindingactivityincytoplasmiclysatesofcellstreatedasforpanelandsupershiftanalysisofcomplexesformingafterexposuretosuchstresses.InductionofcytoplasmicHuRanditsbindingtop21mRNAbyUVC第25頁/共45頁SummaryUVC在不影響總HuR水平的條件下誘導(dǎo)細(xì)胞漿HuR水平UVC誘導(dǎo)HuR與p21mRNA的結(jié)合HuR結(jié)合P213’-UTR后使P21mRNA穩(wěn)定性增高，進(jìn)而引起P21表達(dá)增高。人為降低HuR水平可降低HuR與P213’-UTR間的相互結(jié)合，降低P21mRNA穩(wěn)定性，降低P21表達(dá)，提示HuR對UVC誘導(dǎo)的P21mRNA穩(wěn)定性是必須的。第26頁/共45頁ExampleII

AUF1RegulatesReplicativeSenescencethroughMediatingp16mRNATurnover

Wang,etal;EMBORep.,6:158-164,2005.

第27頁/共45頁BackgroundCDKinhibitorp16INK4isinducedwithreplicativesenescence.Althoughtranscriptionalregulationofp16hasbeenintensivelystudied,regulationbypost-transcriptionalmechanismhasnotbeenreported.RNAbindingproteinAUF1isexpressedasafamilyoffourproteinisoforms(p37,p40,p42andp45)arisingthroughalternativesplicing.AUF1bindstoAU-richelements(ARE)orAUUUAmotifsinthe3’-UTRoftargetmRNAsanddestabilizesthem(differentfromHuR).Question:IsAUF1mediatedmRNAturnoverinvolvedintheregulationofp16duringcellularsenescence?

第28頁/共45頁P(yáng)16mRNA3’-UTRcontainsmotifsforbidingbyRNAbindingproteins第29頁/共45頁SenescenceYoungP163’-UTRisimportantfortheinstabilityofEGFP-p163’-UTR(Inyoungcells)TimeinDox(h)TimeinDox(h)第30頁/共45頁

AUF1bindstop163’-UTR.BindingofAUF1top163’-UTRattenuatedwithcellularsenescence.第31頁/共45頁AUF1levelsreducedwithcellularsenescence.AUF1bindstoAUrichregionofp163’-UTR.ABC第32頁/共45頁P(yáng)163’-UTRconfersinstabilitytochimerictranscriptsinlungcarcinomacells(H2)TimeinDox(h)第33頁/共45頁Knock-downofAUF1stabilizesEGFP-p163’-UTRchimerictranscriptsinH2cells第34頁/共45頁

Knock-downofAUF1increasesp16expressionandacceleratescellularsenescenceofWI-38cells第35頁/共45頁SummarymRNAturnoverisimportantforp16regulationduringcellaging.2.AUF1bindstop163