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1、RNA结合蛋白与转录后调控,王文恭,mRNA turnover,Translation level,Post-translation level,Transcription level,Gene Regulation,DNA and chromatin levels,Post-transcriptional level,Maturation,mRNA export,Regulatory factors for mRNA decay and translation,RNA binding proteins microRNAs,RNA binding proteins(RBPs),RNA结合蛋白种
2、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/ELAV RNA 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
3、子。结合并稳定VEGF,COX-2,p21,cyclin A,cyclin B1,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，p27 mRNA,c-myc。2）调控mRNA export,如 CD83，c-fos,COX-2。,AUF1,也叫HnRNPD（heterogeneous nuclear ribonucleoprotein D).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），
4、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。,Effect of ARE-BPs on the stability and translation of ARE-containing mRNAsARE-BPs mRNA stability Protein expression Translational efficiency Abundance Increase Decrease Increase Decrease
5、Up regulated Down regulated AUF1 c-myc(42)c-myc(46)GMCSF(55)c-fos(42,67)c-fos(53)IL-3(55)PTH(56)p21(48)GMCSF(42)Cyclin D1(48)TNF-alpha(42)GMCSF(53,54)IL-3(55)HuR c-fos(59,63,67)p53(99,137)TNF-alpha(139)p21(69)TNF-alpha(139)MyoD(68)Cox-2(139)Cyclin A(70)p21(48,68,69)Cyclin B1(70)Cyclin A(70)NOS II/iN
6、OS(64)Cyclin B1(70)GMCSF(55)Cyclin D1(48)Cox-2(71,173)NOS II/iNOS(64)IL-3(55)GMCSF(59)VEGF(173)TNF-alpha(65,74,139)p53(99,137)Cox-2(71,139)IL-3(55,66)VEGF(62)Myogenin(68)Hel-N1 TNF-alpha(74)NF-M(73)NF-M(73)GLUT1(72)GLUT1(72)GLUT1(72)HuD GAP-43(7577)GAP-43(75,76)TTP c-fos(90)GMCSF(81)GMCSF(18,81,8385
7、,91)TNF-alpha(80)TNF-alpha(18,81,8386,89,90)IL-2(82)Cox-2(87)IL-3(88)IL-2(82,90)IL-3(18,66,83,84,88)BRF1 TNF-alpha(89,93)GMCSF(55)IL-3(55,92,93)IL-3(55)TIA-1 TNF-alpha(120)TNF-alpha(120)Cox-2(121)Cox-2(121)KSRP c-fos(90,93)NOS II/iNOS(102)NOS II/iNOS(102)TNF-alpha(90,93)IL-2(90,93)c-jun(93)CUG-BP2 C
8、ox-2(150)Cox-2(150)Cox-2(150)Nucleolin bcl-2(175)TINO bcl-2(176)PAIP2 VEGF(177)VEGF(177),Interaction of the factors involving in post-transcriptional regulation,1.RNA结合蛋白相互作用。如，HuR与AUF1均可结合于p21 与cyclin D1 3UTR，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-rich Elements(AREs
9、),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Overlapping UUAUUUA(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,mRNAs ARE-BPsClass Motif Examples I 1 to 5个散在 AUU
10、UA c-myc AUF1,HuR，Hel-N1，HuC，HuD，AUH，GAPDH，Hsp70 c-fos AUF1，HuR，Hel-N1，HuD，KSRP，AUH Beta1-AR AUF1 PTH AUF1 Interferon-gamma GAPDH，Hsp70 MyoD HuR p21 AUF1，HuR，HuD Cyclin A HuR Cyclin B1 HuR Cyclin D1 AUF1，HuR PAI-2 HuR NOS II/iNOS HuR，KSRPIIA(AUUU)5A GMCSF AUF1，HuR，Hel-N1，TTP，BRF1，TIAR，KSRP，AUH，GAPDH
11、，Hsp70，hnRNP-A1，hnRNP-C TNF-alpha AUF1，HuR，TTP，BRF1，TIA-1，TIAR，KSRPIIB(AUUU)4A Interferon-alpha hnRNP-A1，hnRNP-A2,hnRNP-A3IIC(A/U)(AUUU)3A(A/U)Cox-2 AUF1,HuR,HuD,TTP,TIA-1,TIAR,hnRNP-A1，hnRNP-A2,hnRNP-A3，CUDBP2 IL-2 AUF1，HuR，HuD，TTP。Hsp70，Hsp110，hnRNP-A1 IL-3 HuR，BEF1，AUH，Nucleolin，TINO bcl-2 AUF1，H
12、uR，VEGF HuR HuC，HuD，PAIP2IIINo AUUUA,c-jun TIAR，CUGBP1 U-rich region GLUT1 Hel-N1，hnRNP-A2，hnRNP-L p53 HuR，Id Hel-N1 hsp70 HuR Myogenin HuR NF-M Hel-N1 GAP-43 HuD A matrix presentation has been used to represent the identified associations between ARE-BPs and ARE-containing mRNAs.The mRNAs containin
13、g identified functional AREs are listed vertically and are grouped according to the classifications proposed by(24)and(26).The ARE-BPs are displayed horizontally.Where appropriate the different names used to denote the same protein or mRNA are given.The lists of ARE-containing mRNAs and of ARE-BPs a
14、re not exhaustive and only direct interactions have been considered.Where the experimental methods used identified endogenous interactions,these are indicated by an asterisk.Data on the experimental methods are presented in the Supplementary data.Numbers correspond to listed references.,Interaction
15、between ARE and RBPs,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-down assays(using paramagn
16、etic streptavidin dynabeads,biotinyl-labeled transcripts)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)in vitro RNA降解分析,Tet-on system is activated in the presence of doxycycline,the DNA binding domain of the Tet-on regulator(rTetR)contains mutations,repre
17、ssor that only binds DNA in the absence of ligand is converted to a ligand-dependent DNA binding protein.,RNA-pol,Tetracycline controlled transactivator(tTA),TET-OFF in details,Manfred Gossen and Hermann Bujard,The Tet-off system is repressed in the presence of the doxycycline,TET-VP producing vecto
18、r,Gene of interest expressing vector,VP RNA pol interacting part,TetR-tet binding part,TET-OFF system,Tetracycline controlled transactivator(tTA),如：EGFP-interest target chimeric,mRNA 翻译研究特用技术新生蛋白分析。Polysome 分离，Polysomal RNA,Polysomal 蛋白质分离。其它经典技术。,Reference,Barreau,etal;Nucleic Acids Research,33(22)
19、:7138-7150,20052)3)Wang,et al;MCB,20:760-769,20004)Lal,et al;EMBO J.,23:3092-3102,2004,HuR Regulates p21 mRNA Stabilization by UV LightWang,et al;Mol Cell Biol.2000，20(3):760769.,细胞暴露于多种应激（Stresses）如short-wavelength UV light(UVC)时,cyclin-dependent kinase inhibitor p21的表达明显被诱导。P21 的调控，尤其P53调节的转录已被广泛，
20、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，p21 mRNA 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-P21 RNA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3UTR间结合而成。5UTR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P21 3UTR 形成复合物，但只有胞浆中的复合物形成可被UVC诱导。,UVC induces the
21、formation of p213-UTR-protein complex in the cytoplasm,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），说明有一35-40 Kd RNA结合蛋白被UVC诱导并与P21 3-UTR 结合。D。UVC 诱导该未知复合物的趋势与P21被诱导一致。,Elevation of p21 by UVC is accom
22、panied with increased formation of P21 3UTR-protein complex,HuR 结合p21 mRNA（in vivo and in vitro）(A)，(B)HuR抗体可特异结合细胞浆蛋白与B2形成的复合物。(C)RNase T1 Selection Assay was carried out with B2 and A1,incubated with 10 nM GST or GST-HuR(see Materials and Methods).T1,digestions with RNase T1 alone;M,molecular weig
23、ht markers.(D)Gel retardation assays using B2 and the indicated concentrations of either GST or GST-HuR.(E)EMSA-Western Assay:Left,cytoplasmic fractions were either incubated with B2 or not,cross-linked,digested with RNase T1,resolved by SDS-PAGE(15%gel),and transferred onto polyvinylidene difluorid
24、e membranes,which were sequentially exposed to X-ray film for 24 h(Radioactive signal)and subjected to Western blot analysis to detect HuR(Western signal);exposure time,30 s.Right,Lysates from UVC-treated or untreated cells were incubated with B2 and then subjected to Western blot analysis.Estimated
25、 size of the HuR-p21 complexes,37 to 40 kDa.,HuR binds to the 3UTR of p21 mRNA,HuR表达降低后，p21 3 UTR 与细胞浆蛋白间形成的复合物（在UVC辐射后）降低，p21 mRNA 稳定性降低（半衰期缩短），表达降低。Decreased HuR expression lowers binding to the p21 3 UTR and reduces p21 mRNA stability and p21 induction by UVC.(A)Western blot analysis of HuR expre
26、ssion in RKO cells,either untransfected(untr.)or transfected with pZeoSV2()HuR,expressing AS HuR.Chosen clonal isolates are shown.Blots were sequentially stripped and rehybridized with an antibody recognizing actin(43 kDa),to visualize differences in loading and transfer,and with an antibody recogni
27、zing hnRNP C(43 kDa).(B)B5 binding activity in lysates from untransfected and AS HuR-expressing cells 6 h after UVC irradiation.(C)Northern blot analysis of p21 mRNA expression in untransfected and AS HuR-expressing RKO cells 8 h after either no treatment()or exposure to the indicated UVC doses.Even
28、ness in loading and transfer among samples was assessed after stripping the membrane and rehybridizing it with an oligomer probe recognizing 18S rRNA.(D)Western blot analysis to assess the expression of p21,c-Jun(39 kDa),and actin in untransfected and AS HuR-expressing RKO cells 10 h after either no
29、 treatment or exposure to 20 J/m2 UVC.p-jun,phosphorylated Jun.(E)Graphs depict the rate of loss of p21 and-actin mRNAs in cells with different HuR levels after actinomycin D(2 g/ml)addition with or without UVC irradiation.At the times indicated,total RNA was extracted and p21 and-actin mRNAs were m
30、onitored by Northern blotting;signals were quantitated with a PhosphorImager,normalized against 18S(not shown),and plotted on a logarithmic scale.The mRNA half-life in each treatment group is indicated in parentheses.Values represent means standard errors of the means of three independent experiment
31、s.,Ectopic expression of the antisense HuR inhibited the interaction of HuR with p21 mRNA and reduced the levels as well as the half-life of p21 mRNA,UVC 辐射下，B2（含HuR结合位点）赋予P21 mRNA 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。Effect of the full-length and mutant p21 3 UTR on expression of a luciferase reporter
32、 construct.(Top)Expression vectors pGL3,pGL3-FL,and pGL3-B2(see Materials and Methods)were transiently cotransfected into RKO parental(untransfected Untr.),AS.2,or AS.7 cells along with pSV-gal(used to normalize for transfection efficiency);cells were irradiated with UVC(20 J/m2)or left untreated,an
33、d luciferase and-galactosidase activities were examined 24 h later.(Bottom)Relative fold increase in luciferase activity after UVC exposure,seen with either pGL3-FL or pGL3-B2 compared with that seen with the control vector pGL3.Values represent means standard errors of the means of five independent
34、 experiments,The B2 fragment confers PGL3-B2 reporter ability to respond to the down-regulation of HuR,Western blot analysis of HuR expression and subcellular localization.(A)Six hours after irradiation with the indicated doses of UVC,whole-cell(20 g),cytoplasmic(40 g),nuclear(10 g),and cytosolic(40
35、 g)lysates were prepared and subjected to Western blot analysis to monitor the expression of HuR,hnRNP C,AUF1,BAF57c(57 kDa),and actin.Cell lysates were collected at the times indicated after irradiation with UVC(20 J/m2)(B)or 6 h after irradiation with the indicated doses of UVC(C),and Western blot
36、 analysis of HuR expression performed on cytoplasmic(40 g)and nuclear(10 g)fractions.(D)Indicated doses of ionomycin(Ion.;micromolar)or lithium acetate(LiAc;millimolar)were added to cells 1 h before UVC irradiation with 20 J/m2 and Western blot analysis of cytoplasmic HuR.Hybridization using antibod
37、ies against actin and BAF57c was carried out to assess uniformity in loading and transfer among cytoplasmic and nuclear samples,respectively.,UVC induces the cytoplasmic presence of HuR,Subcellular localization of HuR.GFP-HuR was visualized by fluorescence microscopy in transiently transfected RKO c
38、ells that were either left untreated or treated with 20 J of UVC/m2(4 h earlier).DAPI staining served to visualize the nucleus.Note the distinct overlap of DAPI and GFP-HuR signals in untreated cells;while UVC-irradiated cells also exhibit abundant nuclear GFP-HuR,the treatment causes a substantial
39、increase in the cytoplasmic GFP-HuR signal,not seen in untreated cells.,UVC induces cytoplasmic HuR,Increased cytoplasmic HuR and p21 RNA binding after exposure to stresses.(A)Western blot analysis to monitor HuR expression in cytoplasmic and nuclear fractions after treatment with the indicated agen
40、ts.Samples were collected 2 h after addition of actinomycin(Act.)D(1 g/ml)or 4 h after exposure to 100 M H2O2,MMS(100 g/ml),48 M PGA2,or UVC(20 J/m2).Hybridizations using antibodies against actin and BAF57c were carried out to assess uniformity in loading and transfer among cytoplasmic and nuclear s
41、amples,respectively.(B)B2 binding activity in cytoplasmic lysates of cells treated as for panel and supershift analysis of complexes forming after exposure to such stresses.,Induction of cytoplasmic HuR and its binding to p21 mRNA by UVC,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p21 mRNA 的结合HuR 结合
42、P21 3-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P21 3-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P21 mRNA 稳定性是必须的。,Example II AUF1 Regulates Replicative Senescence through Mediating p16 mRNA TurnoverWang,et al;EMBO Rep.,6:158-164,2005.,Background,CDK inhibitor p16INK4 is induced with replicati
43、ve senescence.Although transcriptional regulation of p16 has been intensively studied,regulation by post-transcriptional mechanism has not been reported.RNA binding protein AUF1 is expressed as a family of four protein isoforms(p37,p40,p42 and p45)arising through alternative splicing.AUF1 binds to A
44、U-rich elements(ARE)or AUUUA motifs in the 3-UTR of target mRNAs and destabilizes them(different from HuR).Question:Is AUF1 mediated mRNA turnover involved in the regulation of p16 during cellular senescence?,P16 mRNA 3-UTR contains motifs for biding by RNA binding proteins,Senescence,Young,P16 3-UT
45、R is important for the instability of EGFP-p16 3-UTR(In young cells),Time in Dox(h),Time in Dox(h),AUF1 binds to p16 3-UTR.Binding of AUF1 to p16 3-UTR attenuated with cellular senescence.,AUF1 levels reduced with cellular senescence.AUF1 binds to AU rich region of p16 3-UTR.,A,B,C,P16 3-UTR confers
46、 instability to chimeric transcripts in lung carcinoma cells(H2),Time in Dox(h),Knock-down of AUF1 stabilizes EGFP-p16 3-UTR chimeric transcripts in H2 cells,Knock-down of AUF1 increases p16 expression and accelerates cellular senescence of WI-38 cells,SummarymRNA turnover is important for p16 regul
47、ation during cell aging.2.AUF1 binds to p16 3-UTR and destabilizes p16 mRNA.3.AUF1 expression reduced with cellular senescence.Reduction of AUF1 during cellular senescence can lead to p16 up-regulation and accelerate cell senescent.,RNA-binding protein HuR enhances p53 translation in response to ult
48、raviolet light irradiation.Mazan-Mamczarz K,et al;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.UVC induces p53 expression at protein levelUVC induces p53 expressionp53 mRNA levels is not influenced by UVCp53 mRNA half-life is not a
49、ltered by UVCSubcellular and polysomal p53 mRNA is not altered under UVC.,UVC induces p53 expression at protein level,Fig.2.Western blot analysis of p53 expression in RKO whole-cell lysates prepared at the times indicated after treatment with lactacystin(5 M),UVC(15 J/m2),or a combination of both.Si
50、gnals were quantitated by densitometry(graph).(B)Newly translated p53 is increased by UVC.,UVC induced p53 is not associated with proteasome turnover,Fig.3.(A)REMSA(11)by using either CR or 3 UTR p53 radiolabeled transcripts and cytoplasmic lysates prepared 4 and 6 h after treatment of RKO cells wit
