RNA结合蛋白与转录后调控课件

RNA结合蛋白与转录后调控mRNAturnoverTranslationlevelPost-translationlevelTranscriptionlevelGeneRegulationDNAandchromatinlevelsPost-transcriptionallevelMaturationmRNAexportRegulatoryfactorsformRNAdecayandtranslation

RNAbindingproteins

microRNAs

HuR•Hu/ELAVRNA结合蛋白家族（包括HuA/HuR，Hel-N1，HuC，HuD）的主要成员。与其它成员仅表达于神经细胞不同，HuR几乎在所有组织都有表达。主要位于细胞核，但可穿梭于细胞浆/核间，且只有细胞浆HuR可调控mRNA稳定性（及翻译）。核内HuR则与mRNA成熟及export有关。•结合所有三类AREs。结合后的结局主要为稳定目标。因此也被称为mRNA稳定因子。•结合并稳定VEGF,COX-2,p21,cyclinA,cyclinB1,c-fos,TNFα，IL-1，MyoD，Myogenin，bcl-2等mRNA。因此可调节应激反应，细胞周期，肿瘤，分化，调亡等过程。•

HuR也可结合目标并调节其翻译。1）调控翻译效率，如p53，p27mRNA,c-myc。2）调控mRNAexport,如CD83，c-fos,COX-2。AUF1

也叫HnRNPD（heterogeneousnuclearribonucleoproteinD).主要位于核内，但可穿梭于核/细胞浆间，结合I，II类AREs。结合目标mRNA后使之不稳定（易于降解），如P21，CyclinD1，也可使目标稳定，如TNF-alpha。TTP与HuR及AUF1不同，TTP主要位于细胞浆，结合II类AREs。结合目标后主要使目标不稳定。如：COX-2，TNF-alpha,GM-CSF,等。Interactionofthefactorsinvolvinginpost-transcriptionalregulation1.RNA结合蛋白相互作用。如，HuR与AUF1均可结合于p21与cyclinD13’UTR，但二者有竞争，且功能相反。2.RNA结合蛋白与microRNA间相互作用。如，HuR与let-7,miR-122,TTP与miR-16。AU-richElements(AREs)1.主要指位于3‘-非翻译区的富含AU的序列。2.被RNA结合蛋白识别，结合。3.主要为mRNA不稳定元件，是mRNA在完成使命后快速降解的结构基础。4.依构成分为三类1）含1-5个分散的AUUUA。2）至少有两个OverlappingUUAUUUA(U/A)(U/A).依AUUUA的重复方式分为5类，IIA，IIB，IIC，等。3）富含U，但无AUUUA。注：除一级结构外，mRNA的二级结构也与RNA-蛋白质相互作用密切相关。如，约70-80%HuR或AUF1结合序列具有相似的二级结构mRNAsARE-BPs

ClassMotifExamples

I1to5个散在

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稳定性（turnover）研究的特殊技术蛋白质-RNA结合试验：1）目标Transcript的体外转录与标记2）细胞浆抽提物的制备。3)EMSA(gel-shift,supershift)4)rChip,pull-downassays(usingparamagneticstreptavidindynabeads,biotinyl-labeledtranscripts)mRNA半衰期测定

基本思路:终止转录后,收取不同时间点之RNA,定量分析RNA降解速率.1）用ActinomycinD终止转录2）Tet-off/on（或类似）报告基因系统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.

细胞暴露于多种应激（Stresses）如short-wavelengthUVlight(UVC)时,cyclin-dependentkinaseinhibitorp21的表达明显被诱导。P21的调控，尤其P53调节的转录已被广泛，深入研究。先前的研究发现，UVC可通过P53-不依赖的方式诱导P21。研究还发现，细胞暴露于UVC后，p21mRNA稳定性增加。问题：UVC诱导P21表达（稳定性增加）的机制如何？与HuR有关否？Background:ExampleⅠUVC辐射诱导蛋白-P21RNA复合物形成。复合物形成为P53不依赖性的，因为无论RKO细胞是否有野生型P53，复合物的形成无区别。复合物由蛋白质与3‘UTR间结合而成。5’UTR及缺失ARE的3‘-UTR（A1，C5）几乎无蛋白结合。核与细胞浆蛋白均可与P213’UTR形成复合物，但只有胞浆中的复合物形成可被UVC诱导。UVCinducestheformationofp213’-UTR-proteincomplexinthecytoplasmHuR结合p21mRNA（invivoandinvitro）(A)，(B)HuR抗体可特异结合细胞浆蛋白与B2形成的复合物。(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表达降低后，

p213′UTR与细胞浆蛋白间形成的复合物（在UVC辐射后）降低，p21mRNA稳定性降低（半衰期缩短），表达降低。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辐射下，B2（含HuR结合位点）赋予P21mRNA稳定性。用反义方法降低内源性HuR表达后，由B2赋予的稳定性消失。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，功能广泛。在UVC辐射下，p53被诱导，但机制不明。2.UVC诱导细胞浆HuR。Questions：

HuR是否调控p53?如何调控?Fig.1.UVCinducesp53expressionatproteinlevelUVCinducesp53expressionp53mRNAlevelsisnotinfluencedbyUVCp53mRNAhalf-lifeisnotalteredbyUVCSubcellularandpolysomalp53mRNAisnotalteredunderUVC.UVCinducesp53expressionatproteinlevelFig.2.

Westernblot