RNA結(jié)合蛋白與轉(zhuǎn)錄后調(diào)控.ppt

1、RNA結(jié)合蛋白與轉(zhuǎn)錄后調(diào)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結(jié)合蛋白

2、種類很多，估計占細胞編碼蛋白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）被證實可特異參與mRNA穩(wěn)定性、翻譯、或其它層面的基因調(diào)控。,HuR, Hu /ELAV RNA 結(jié)合蛋白家族（包括HuA/HuR，Hel-N1，HuC，HuD） 的主要成員。與其它成員僅表達于神經(jīng)細胞不同，HuR幾乎在所有組織 都有表達。主要位于細胞核，但可穿梭于細胞漿/核間，且只有細胞漿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。因此也被 稱為mR

3、NA 穩(wěn)定因子。 結(jié)合并穩(wěn)定VEGF, COX-2, p21, cyclin A, cyclin B1, c-fos, TNF ，IL-1， MyoD，Myogenin，bcl-2等mRNA。因此可調(diào)節(jié)應(yīng)激反應(yīng)，細胞周期，腫瘤， 分化，調(diào)亡等過程。 HuR也可結(jié)合目標并調(diào)節(jié)其翻譯。 1）調(diào)控翻譯效率， 如 p53，p27 mRNA, c-myc。 2）調(diào)控mRNA export, 如 CD83，c-fos, COX-2。,AUF1,也叫HnRNPD（heterogeneous nuclear ribonucleoprotein D). 主要位于核內(nèi)，但可穿梭于核/細胞漿間，結(jié)合I，II類ARE

4、s。 結(jié)合目標mRNA 后使之不穩(wěn)定（易于降解），如P21， CyclinD1，也可使目標穩(wěn)定，如TNF-alpha。,TTP,與HuR及AUF1不同，TTP主要位于細胞漿，結(jié)合II類AREs。 結(jié)合目標后主要使目標不穩(wěn)定。如：COX-2，TNF-alpha,GM-CSF,等。,Effect of ARE-BPs on the stability and translation of ARE-containing mRNAs ARE-BPs mRNA stability Protein expression Translational efficiency Abundance Increase

5、 Decrease Increase Decrease 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

6、 (139) Cyclin A (70) p21 (48,68,69) Cyclin B1 (70) Cyclin A (70) NOS II/iNOS (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

7、 (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 (18,81,8385,91) 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)

8、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 Cox-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 inv

9、olving in post-transcriptional regulation,1. RNA結(jié)合蛋白相互作用。 如， HuR與AUF1均可結(jié)合于p21 與cyclin D1 3UTR， 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-rich Elements (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AUU

10、UA。 2）至少有兩個Overlapping UUAUUUA(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),mRNAs ARE-BPsClass Motif Examples I 1 to 5個散在 AUUUA c-myc AUF1 , HuR ，Hel-N1， HuC， HuD， AUH，GAPDH，Hsp70 c-fos AUF1，HuR， Hel-N1， HuD ，KSRP，AUH

11、 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，Hsp70，hnRNP-A1， hnRNP-C TNF-alpha AUF1，HuR， TTP， BRF1，TIA-1， TIAR，KSRPIIB

12、(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， HuR， VEGF HuR HuC， HuD，PAIP2IIINo AUUUA, c-jun TIAR，

13、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 containing identified functional AREs are listed verti

14、cally 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 are not exhaustive and only direct inter

15、actions 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 between ARE and RBPs,RNA-蛋白質(zhì)相互作用(結(jié)合

16、)的特點,可在細胞核, 也可在細胞漿 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)合蛋白對目標的序列要求不如DNA 結(jié)合蛋白般嚴格. 結(jié)合部位大多在3-UTR, 少數(shù)在5-UTR,絕少見于CDS.,mRNA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Transcript 的體外轉(zhuǎn)錄與標記 2）細胞漿抽提物的制備。 3) EMSA (gel-shift, supershift) 4) rChip, pull-down assays (using paramagnetic streptavidin d

17、ynabeads, biotinyl-labeled transcripts) 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) 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,repressor that o

18、nly 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 p

19、roducing vector,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 翻譯研究特用技術(shù) 新生蛋白分析。 Polysome 分離， Polysomal RNA, Polysomal 蛋白質(zhì)分離。 其它經(jīng)典技術(shù)。,Reference,Barreau, etal; Nucle

20、ic Acids Research, 33(22): 7138-7150, 2005 2) 3) Wang, et al; MCB, 20:760-769, 2000 4) Lal, et al; EMBO J., 23:3092-3102, 2004,/departments/mmb/baranova/pages/ppt/biotech-lec5.ppt,HuR Regulates p21 mRNA Stabilization by UV LightWang, et al; Mol Cell Biol. 2000， 20(3): 760769.,細胞暴露于多種應(yīng)激（St

21、resses）如short-wavelength UV light (UVC)時, cyclin-dependent kinase inhibitor p21的表達明顯被誘導(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)，細胞暴露于UVC后，p21 mRNA 穩(wěn)定性增加。 問題： UVC誘導(dǎo)P21表達（穩(wěn)定性增加）的機制如何？ 與HuR有關(guān)否？,Background:,Example,UVC 輻射誘導(dǎo)蛋白-P21 RNA復(fù)合物形成。 復(fù)合物形成為P53不依賴性的，因為無論RKO細胞是否有野生型P53，復(fù)合物的

22、形成無區(qū)別。復(fù)合物由蛋白質(zhì)與3UTR間結(jié)合而成。5UTR及缺失ARE的3-UTR（A1，C5）幾乎無蛋白結(jié)合。核與細胞漿蛋白均可與P21 3UTR 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)。,UVC induces the formation of p213-UTR-protein complex in the cytoplasm,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-PAGE 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

23、與大約10個堿基的短片斷轉(zhuǎn)錄物形成），說明有一35-40 Kd RNA結(jié)合蛋白被UVC誘導(dǎo)并與P21 3-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。,Elevation of p21 by UVC is accompanied with increased formation of P21 3UTR-protein complex,HuR 結(jié)合p21 mRNA（ in vivo and in vitro） (A)， (B) HuR抗體可特異結(jié)合細胞漿蛋白與B2形成的復(fù)合物。 (C) RNase T1 Selection Assay was carried out

24、with B2 and A1, incubated with 10 nM GST or GST-HuR (see Materials and Methods). T1, digestions with RNase T1 alone; M, molecular weight 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 ei

25、ther incubated with B2 or not, cross-linked, digested with RNase T1, resolved by SDS-PAGE (15% gel), and transferred onto polyvinylidene difluoride membranes, which were sequentially exposed to X-ray film for 24 h (Radioactive signal) and subjected to Western blot analysis to detect HuR (Western sig

26、nal); exposure time, 30 s. Right, Lysates from UVC-treated or untreated cells were incubated with B2 and then subjected to Western blot analysis. Estimated size of the HuR-p21 complexes, 37 to 40 kDa.,HuR binds to the 3UTR of p21 mRNA,HuR表達降低后， p21 3 UTR 與細胞漿蛋白間形成的復(fù)合物（在UVC輻射后）降低， p21 mRNA 穩(wěn)定性降低（半衰期縮

27、短），表達降低。 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 expression in RKO cells, either untransfected (untr.) or transfected with pZeoSV2()HuR, expressing AS HuR. Chosen clonal isolates are shown. Blo

28、ts were sequentially stripped and rehybridized with an antibody recognizing actin (43 kDa), to visualize differences in loading and transfer, and with an antibody recognizing hnRNP C (43 kDa). (B) B5 binding activity in lysates from untransfected and AS HuR-expressing cells 6 h after UVC irradiation

29、. (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. Evenness in loading and transfer among samples was assessed after stripping the membrane and rehybridizing it with an oligomer

30、 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 treatment or exposure to 20 J/m2 UVC. p-jun, phosphorylated Jun. (E) Graphs depict the rate of loss of p21 and -acti

31、n 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 monitored by Northern blotting; signals were quantitated with a PhosphorImager, normalized against 18S (not

32、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 experiments.,Ectopic expression of the antisense HuR inhibited the interaction of HuR with p21 mRNA and reduced

33、 the levels as well as the half-life of p21 mRNA,UVC 輻射下，B2 （含HuR結(jié)合位點）賦予P21 mRNA 穩(wěn)定性。用反義方法降低內(nèi)源性HuR 表達后，由B2賦予的穩(wěn)定性消失。 Effect of the full-length and mutant p21 3 UTR on expression of a luciferase reporter construct. (Top) Expression vectors pGL3, pGL3-FL, and pGL3-B2 (see Materials and Methods) were tr

34、ansiently 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, and luciferase and -galactosidase activities were examined 24 h later. (Bottom) Relativ

35、e 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 experiments,The B2 fragment confers PGL3-B2 reporter ability to respond to the

36、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 g) lysates were prepared and subjected to Western blot analysis to mo

37、nitor 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 analysis of HuR expression performed on cytoplasmic (40 g

38、) 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 antibodies against actin and BAF57c was carried out

39、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 cells that were either left untreated or tre

40、ated 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 increase in the cytoplasmic GFP-HuR si

41、gnal, 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 agents. Samples were collected 2 h afte

42、r 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 samples, respectively. (B

43、) 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在不影響總HuR水平的條件下誘導(dǎo)細胞漿HuR水平 UVC誘導(dǎo)HuR與p21 mRNA 的結(jié)合 HuR 結(jié)合P21 3-UTR后使P21mRNA

44、穩(wěn)定性增高，進而引起P21表達增高。 人為降低HuR水平可降低HuR與P21 3-UTR間的相互結(jié)合，降低P21mRNA 穩(wěn)定性，降低P21表達，提示HuR對UVC誘導(dǎo)的P21 mRNA 穩(wěn)定性是必須的。,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 replicative senescenc

45、e. 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 AU-ri

46、ch 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-U

47、TR 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 c

48、onfers 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,Summary mRNA turnover is important for

49、p16 regulation 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 r

50、esponse to ultraviolet light irradiation. Mazan-Mamczarz K, et al; 2003, PNAS,Example ,Background p53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)細胞漿HuR。 Questions： HuR是否調(diào)控p53? 如何調(diào)控?,Fig. 1. UVC induces p53 expression at protein level UVC induces p53 expression p53 mRNA levels is not influenced by UVC p53 mRNA half-lif