RNA结合蛋白与转录后调控(精).ppt

RNA结合蛋白与转录后调控 王文恭 mRNAturnover Translationlevel Post translationlevel Transcriptionlevel GeneRegulation DNAandchromatinlevels Post transcriptionallevel Maturation mRNAexport RegulatoryfactorsformRNAdecayandtranslation RNAbindingproteinsmicroRNAs RNAbindingproteins RBPs RNA结合蛋白种类很多 估计占细胞编码蛋白6 8 者为RNA结合蛋白 但迄今只有为数不多的几种RNA结合蛋白 如HuR AUF1 TTP TIA1 CUGBP2等 被证实可特异参与mRNA稳定性 翻译 或其它层面的基因调控 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 TNF 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 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 Interactionofthefactorsinvolvinginpost transcriptionalregulation 1 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 BPsClassMotifExamplesI1to5个散在AUUUAc mycAUF1 HuR Hel N1 HuC HuD AUH GAPDH Hsp70c fosAUF1 HuR Hel N1 HuD KSRP AUHBeta1 ARAUF1PTHAUF1Interferon gammaGAPDH Hsp70MyoDHuRp21AUF1 HuR HuDCyclinAHuRCyclinB1HuRCyclinD1AUF1 HuRPAI 2HuRNOSII iNOSHuR KSRPIIA AUUU 5AGM CSFAUF1 HuR Hel N1 TTP BRF1 TIAR KSRP AUH GAPDH Hsp70 hnRNP A1 hnRNP CTNF alphaAUF1 HuR TTP BRF1 TIA 1 TIAR KSRPIIB AUUU 4AInterferon alphahnRNP A1 hnRNP A2 hnRNP A3IIC A U AUUU 3A A U Cox 2AUF1 HuR HuD TTP TIA 1 TIAR hnRNP A1 hnRNP A2 hnRNP A3 CUDBP2IL 2AUF1 HuR HuD TTP Hsp70 Hsp110 hnRNP A1IL 3HuR BEF1 AUH Nucleolin TINObcl 2AUF1 HuR VEGFHuRHuC HuD PAIP2IIINoAUUUA c junTIAR CUGBP1U richregionGLUT1Hel N1 hnRNP A2 hnRNP Lp53HuR IdHel N1hsp70HuRMyogeninHuRNF MHel N1GAP 43HuDAmatrixpresentationhasbeenusedtorepresenttheidentifiedassociationsbetweenARE BPsandARE containingmRNAs ThemRNAscontainingidentifiedfunctionalAREsarelistedverticallyandaregroupedaccordingtotheclassificationsproposedby 24 and 26 TheARE BPsaredisplayedhorizontally WhereappropriatethedifferentnamesusedtodenotethesameproteinormRNAaregiven ThelistsofARE containingmRNAsandofARE BPsarenotexhaustiveandonlydirectinteractionshavebeenconsidered Wheretheexperimentalmethodsusedidentifiedendogenousinteractions theseareindicatedbyanasterisk DataontheexperimentalmethodsarepresentedintheSupplementarydata Numberscorrespondtolistedreferences InteractionbetweenAREandRBPs RNA 蛋白质相互作用 结合 的特点 可在细胞核 也可在细胞浆发生在核与胞浆的结合功能截然不同 如在胞浆与翻译及mRNA稳定性有关 在核可能与拼接或成熟有关 RNA结合蛋白对目标的序列要求不如DNA结合蛋白般严格 结合部位大多在3 UTR 少数在5 UTR 绝少见于CDS mRNA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 systemisactivatedinthepresenceofdoxycycline theDNAbindingdomainoftheTet onregulator rTetR containsmutations repressorthatonlybindsDNAintheabsenceofligandisconvertedtoaligand dependentDNAbindingprotein RNA pol Tetracyclinecontrolledtransactivator tTA TET OFFindetails ManfredGossenandHermannBujard www biochem arizona edu The Tet off systemisrepressedinthepresenceofthedoxycycline TET VPproducingvector Geneofinterestexpressingvector VP RNApolinteractingpart TetR tetbindingpart TET OFFsystem Tetracyclinecontrolledtransactivator tTA 如 EGFP interesttargetchimeric mRNA翻译研究特用技术新生蛋白分析 Polysome分离 PolysomalRNA Polysomal蛋白质分离 其它经典技术 Reference Barreau etal NucleicAcidsResearch 33 22 7138 7150 20052 3 Wang etal MCB 20 760 769 20004 Lal etal EMBOJ 23 3092 3102 2004 www gmu edu departments mmb baranova pages ppt biotech lec5 ppt HuRRegulatesp21mRNAStabilizationbyUVLightWang 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 Example UVC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 proteincomplexinthecytoplasm A 复合物形成在UVC辐射半小时后明显被诱导 与P21被诱导相吻合 B 竞争抑制试验说明复合物的特异性 Cold探针可竞争抑制复合物形成 C EMSA后 UV交联 SDS PAGE分离复合物 发现复合物中一条大约40Kd的复合物 单一蛋白与大约10个碱基的短片断转录物形成 说明有一35 40KdRNA结合蛋白被UVC诱导并与P213 UTR结合 D UVC诱导该未知复合物的趋势与P21被诱导一致 Elevationofp21byUVCisaccompaniedwithincreasedformationofP213 UTR proteincomplex HuR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 PAGE 15 gel andtransferredontopolyvinylidenedifluoridemembranes whichweresequentiallyexposedtoX rayfilmfor24h Radioactivesignal andsubjectedtoWesternblotanalysistodetectHuR Westernsignal exposuretime 30s Right LysatesfromUVC treatedoruntreatedcellswereincubatedwithB2andthensubjectedtoWesternblotanalysis EstimatedsizeoftheHuR p21complexes 37to40kDa HuRbindstothe3 UTRofp21mRNA HuR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 Graphsdepicttherateoflossofp21and actinmRNAsincellswithdifferentHuRlevelsafteractinomycinD 2 g ml additionwithorwithoutUVCirradiation Atthetimesindicated totalRNAwasextractedandp21and actinmRNAsweremonitoredbyNorthernblotting signalswerequantitatedwithaPhosphorImager normalizedagainst18S notshown andplottedonalogarithmicscale ThemRNAhalf lifeineachtreatmentgroupisindicatedinparentheses Valuesrepresentmeans standarderrorsofthemeansofthreeindependentexperiments EctopicexpressionoftheantisenseHuRinhibitedtheinteractionofHuRwithp21mRNAandreducedthelevelsaswellasthehalf lifeofp21mRNA UVC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 B2 seeMaterialsandMethods weretransientlycotransfectedintoRKOparental untransfected Untr AS 2 orAS 7cellsalongwithpSV gal usedtonormalizefortransfectionefficiency cellswereirradiatedwithUVC 20J m2 orleftuntreated andluciferaseand galactosidaseactivitieswereexamined24hlater Bottom RelativefoldincreaseinluciferaseactivityafterUVCexposure seenwitheitherpGL3 FLorpGL3 B2comparedwiththatseenwiththecontrolvectorpGL3 Valuesrepresentmeans standarderrorsofthemeansoffiveindependentexperiments TheB2fragmentconfersPGL3 B2reporterabilitytorespondtothedown regulationofHuR WesternblotanalysisofHuRexpressionandsubcellularlocalization A SixhoursafterirradiationwiththeindicateddosesofUVC whole cell 20 g cytoplasmic 40 g nuclear 10 g andcytosolic 40 g lysateswerepreparedandsubjectedtoWesternblotanalysistomonitortheexpressionofHuR hnRNPC AUF1 BAF57c 57kDa andactin CelllysateswerecollectedatthetimesindicatedafterirradiationwithUVC 20J m2 B or6hafterirradiationwiththeindicateddosesofUVC C andWesternblotanalysisofHuRexpressionperformedoncytoplasmic 40 g andnuclear 10 g fractions D Indicateddosesofionomycin Ion micromolar orlithiumacetate LiAc millimolar wereaddedtocells1hbeforeUVCirradiationwith20J m2andWesternblotanalysisofcytoplasmicHuR HybridizationusingantibodiesagainstactinandBAF57cwascarriedouttoassessuniformityinloadingandtransferamongcytoplasmicandnuclearsamples respectively UVCinducesthecytoplasmicpresenceofHuR SubcellularlocalizationofHuR GFP HuRwasvisualizedbyfluorescencemicroscopyintransientlytransfectedRKOcellsthatwereeitherleftuntreatedortreatedwith20JofUVC m2 4hearlier DAPIstainingservedtovisualizethenucleus NotethedistinctoverlapofDAPIandGFP HuRsignalsinuntreatedcells whileUVC irradiatedcellsalsoexhibitabundantnuclearGFP HuR thetreatmentcausesasubstantialincreaseinthecytoplasmicGFP HuRsignal notseeninuntreatedcells UVCinducescytoplasmicHuR IncreasedcytoplasmicHuRandp21RNAbindingafterexposuretostresses A WesternblotanalysistomonitorHuRexpressionincytoplasmicandnuclearfractionsaftertreatmentwiththeindicatedagents Sampleswerecollected2hafteradditionofactinomycin Act D 1 g ml or4hafterexposureto100 MH2O2 MMS 100 g ml 48 MPGA2 orUVC 20J m2 HybridizationsusingantibodiesagainstactinandBAF57cwerecarriedouttoassessuniformityinloadingandtransferamongcytoplasmicandnuclearsamples respectively B B2bindingactivityincytoplasmiclysatesofcellstreatedasforpanelandsupershiftanalysisofcomplexesformingafterexposuretosuchstresses InductionofcytoplasmicHuRanditsbindingtop21mRNAbyUVC Summary UVC在不影响总HuR水平的条件下诱导细胞浆HuR水平UVC诱导HuR与p21mRNA的结合HuR结合P213 UTR后使P21mRNA稳定性增高 进而引起P21表达增高 人为降低HuR水平可降低HuR与P213 UTR间的相互结合 降低P21mRNA稳定性 降低P21表达 提示HuR对UVC诱导的P21mRNA稳定性是必须的 ExampleIIAUF1RegulatesReplicativeSenescencethroughMediatingp16mRNATurnoverWang etal EMBORep 6 158 164 2005 Background CDKinhibitorp16INK4isinducedwithreplicativesenescence Althoughtranscriptionalregulationofp16hasbeenintensivelystudied regulationbypost transcriptionalmechanismhasnotbeenreported RNAbindingproteinAUF1isexpressedasafamilyoffourproteinisoforms p37 p40 p42andp45 arisingthroughalternativesplicing AUF1bindstoAU richelements ARE orAUUUAmotifsinthe3 UTRoftargetmRNAsanddestabilizesthem differentfromHuR Question IsAUF1mediatedmRNAturnoverinvolvedintheregulationofp16duringcellularsenescence P16mRNA3 UTRcontainsmotifsforbidingbyRNAbindingproteins Senescence Young P163 UTRisimportantfortheinstabilityofEGFP p163 UTR Inyoungcells TimeinDox h TimeinDox h AUF1bindstop163 UTR BindingofAUF1top163 UTRattenuatedwithcellularsenescence AUF1levelsreducedwithcellularsenescence AUF1bindstoAUrichregionofp163 UTR A B C P163 UTRconfersinstabilitytochimerictranscriptsinlungcarcinomacells H2 TimeinDox h Knock downofAUF1stabilizesEGFP p163 UTRchimerictranscriptsinH2cells Knock downofAUF1increasesp16expressionandacceleratescellularsenescenceofWI 38cells SummarymRNAturnoverisimportantforp16regulationduringcellaging 2 AUF1bindstop163 UTRanddestabilizesp16mRNA 3 AUF1expressionreducedwithcellularsenescence ReductionofAUF1duringcellularsenescencecanleadtop16up regulationandacceleratecellsenescent RNA bindingproteinHuRenhancesp53translationinresponsetoultravioletlightirradiation Mazan MamczarzK etal 2003 PNAS Example Backgroundp53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 UVCinducesp53expressionatproteinlevel Fig 2 Westernblotanalysisofp53expressioninRKOwhole celllysatespreparedatthetimesindicatedaftertreatmentwithlactacystin 5 M UVC 15J m2 oracombinationofboth Signalswerequantitatedbydensitometry graph B Newlytranslatedp53isincreasedbyUVC UVCinducedp53isnotassociatedwithproteasometurnover Fig 3 A REMSA 11 byusingeitherCRor3 UTRp53radiolabeledtranscriptsandcytoplasmiclysatesprepared4and6haftertreatmentofRKOcellswitheither15or30J m2UVC B PlasmidspGL3 LucandpGL3 Luc p53 3 UTR wereusedintransienttransfections 24haftertransfection cellswereeitherirradiated 15J m2 orleftuntreated andtherelativeluciferaseactivitywascalculated6 12 and24hlater Bindingofcytoplasmicproteinstothep533 UTRislinkedtop53translationalup regulation Fig 4 REMSA HuRbindstothep533 UTR pull downassays HuRbindstothep533 UTR rCHipassays HuRbindstothep533 UTR UVCinducesthepresenceofHuRinthecytoplasmandpolysome HuRbindstothep533 UTRinaUVC dependentmanner Fig 5 WesternblotanalysisofHuRandp53inHuR over expressing S11 cellsunderUVCtreatment WesternblotanalysisofHuRandp53inHuR silencingcellsunderUVCtreatment Analysisofnewlytranslatedp53inHuR silencedcells ModulationofHuRlevelsaffectsp53translationandsteady statelevels Conclussion UVCinducesp53translation HuRbindstothe3 UTRofp53 HuRenhancesp53translationunderUVCtreatment