RNA結(jié)合蛋白與轉(zhuǎn)錄后調(diào)控課件

RNA結(jié)合蛋白與轉(zhuǎn)錄后調(diào)控mRNAturnoverTranslationlevelPost-translationlevelTranscriptionlevelGeneRegulationDNAandchromatinlevelsPost-transcriptionallevelMaturationmRNAexportRegulatoryfactorsformRNAdecayandtranslation

RNAbindingproteins

microRNAs

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。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。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。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。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)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.InteractionbetweenAREandRBPsmRNA穩(wěn)定性（turnover）研究的特殊技術(shù)蛋白質(zhì)-RNA結(jié)合試驗：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"Tet-on"systemisactivatedinthepresenceofdoxycyclinetheDNAbindingdomainoftheTet-onregulator(rTetR)containsmutations

repressorthatonlybindsDNAintheabsenceofligand

isconvertedtoaligand-dependentDNAbindingprotein.RNA-polTetracyclinecontrolledtransactivator(tTA)TET-OFFindetailsManfredGossenandHermannBujard

The"Tet-off"systemisrepressed

inthepresenceofthedoxycyclineTET-VPproducingvectorGeneofinterestexpressingvectorVP–RNApolinteractingpartTetR-tetbindingpartTET-OFFsystemTetracyclinecontrolledtransactivator(tTA)如：EGFP-interesttargetchimeric…ReferenceBarreau,etal;NucleicAcidsResearch,33(22):7138-7150,20052)3)Wang,etal;MCB,20:760-769,20004)Lal,etal;EMBOJ.,23:3092-3102,2004

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）的機制如何？與HuR有關(guān)否？Background:ExampleⅠUVC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，因為無論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-proteincomplexinthecytoplasmHuR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’UTRofp21mRNAHuR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-lifeofp21mRNAUVC輻射下，B2（含HuR結(jié)合位點）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-regulationofHuRSubcellularlocalizationofHuR.GFP-HuRwasvisualizedbyfluorescencemicroscopyintransientlytransfectedRKOcellsthatwereeitherleftuntreatedortreatedwith20JofUVC/m2(4hearlier).DAPIstainingservedtovisualizethenucleus.NotethedistinctoverlapofDAPIandGFP-HuRsignalsinuntreatedcells;whileUVC-irradiatedcellsalsoexhibitabundantnuclearGFP-HuR,thetreatmentcausesasubstantialincreaseinthecytoplasmicGFP-HuRsignal,notseeninuntreatedcells.UVCinducescytoplasmicHuRIncreasedcytoplasmicHuRandp21RNAbindingafterexposuretostresses.(A)WesternblotanalysistomonitorHuRexpressionincytoplasmicandnuclearfractionsaftertreatmentwiththeindicatedagents.Sampleswerecollected2hafteradditionofactinomycin(Act.)D(1μg/ml)or4hafterexposureto100μMH2O2,MMS(100μg/ml),48μMPGA2,orUVC(20J/m2).HybridizationsusingantibodiesagainstactinandBAF57cwerecarriedouttoassessuniformityinloadingandtransferamongcytoplasmicandnuclearsamples,respectively.(B)B2bindingactivityincytoplasmiclysatesofcellstreatedasforpanelandsupershiftanalysisofcomplexesformingafterexposuretosuchstresses.InductionofcytoplasmicHuRanditsbindingtop21mRNAbyUVCBackgroundCDKinhibitorp16INK4isinducedwithreplicativesenescence.Althoughtranscriptionalregulationofp16hasbeenintensivelystudied,regulationbypost-transcriptionalmechanismhasnotbeenreported.RNAbindingproteinAUF1isexpressedasafamilyoffourproteinisoforms(p37,p40,p42andp45)arisingthroughalternativesplicing.AUF1bindstoAU-richelements(ARE)orAUUUAmotifsinthe3’-UTRoftargetmRNAsanddestabilizesthem(differentfromHuR).Question:IsAUF1mediatedmRNAturnoverinvolvedintheregulationofp16duringcellularsenescence?

P16mRNA3’-UTRcontainsmotifsforbidingbyRNAbindingproteinsSenescenceYoungP163’-UTRisimportantfortheinstabilityofEGFP-p163’-UTR(Inyoungcells)TimeinDox(h)TimeinDox(h)

AUF1bindstop163’-UTR.BindingofAUF1top163’-UTRattenuatedwithcellularsenescence.AUF1levelsreducedwithcellularsenescence.AUF1bindstoAUrichregionofp163’-UTR.ABCP163’-UTRconfersinstabilitytochimerictranscriptsinlungcarcinomacells(H2)TimeinDox(h)Knock-downofAUF1stabilizesEGFP-p163’-UTRchimerictranscriptsinH2cells

Knock-downofAUF1increasesp16expressionandacceleratescellularsenescenceofWI-38cellsSummarymRNAturnoverisimportantforp16regulationduringcellaging.2.AUF1bindstop163’-UTRanddestabilizesp16mRNA.3.AUF1expressionreducedwithcellularsenescence.ReductionofAUF1duringcellularsenescencecanleadtop16up-regulationandacceleratecellsenescent.RNA-bindingproteinHuRenhancesp53translationinresponsetoultravioletlightirradiation.Mazan-MamczarzK,etal;2003,PNASExampleⅢBackgroundp53是重要的抗癌基因，功能廣泛。在UVC輻射下，p53被誘導(dǎo)，但機制不明。2.UVC誘導(dǎo)細(xì)胞漿HuR。Questions：

HuR是否調(diào)控p53?如何調(diào)控?Fig.1.UVCinducesp53expressionatproteinlevelUVCinducesp53expressionp53mRNAlevelsisnotinfluencedbyUVCp53mRNAhalf-lifeisnotalteredbyUVCSubcellularandpolysomalp53mRNAisnotalteredunderUVC.UVCinducesp53expressionatproteinlevelFig.2.

Westernblot