1. RNA 结合蛋白与转录后调控 王文恭

2. mRNA turnover Translation level Post-translation level Transcription level Gene Regulation DNA and chromatin levels Post-transcriptional level Maturation mRNA export

3. Regulatory factors for mRNA decay and translation 1. RNA binding proteins 2. microRNAs

4. RNA binding proteins (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。

5. 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 稳定因子。 结合并稳定 VEGF, COX-2, p21, cyclin A, cyclin B1, 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 ， p27 mRNA, c-myc 。 2 ）调控 mRNA export, 如 CD83 ， c-fos, COX-2 。

6. AUF1 1. 也叫 HnRNPD （ heterogeneous nuclear ribonucleoprotein D). 2. 主要位于核内，但可穿梭于核 / 细胞浆间， 结合 I ， II 类 AREs 。 3. 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 。

7. 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。

8. Effect of ARE-BPs on the stability and translation of ARE-containing mRNAs ARE-BPs mRNA stability Protein expression Translational efficiency Abundance Increase Decrease Increase Decrease Up regulated Down regulated AUF1 c-myc (42) c-myc (46) GM–CSF (55)424655 c-fos (42,67) c-fos (53) IL-3 (55)42675355 PTH (56) p21 (48)5648 GM–CSF (42) Cyclin D1 (48)4248 TNF-alpha (42) GM–CSF (53,54)425354 IL-3 (55)55 HuR c-fos(59,63,67) p53(99,137) TNF-alpha (139) p21 (69) TNF-alpha (139)69139 MyoD (68) Cox-2 (139) Cyclin A (70)6813970 p21 (48,68,69) Cyclin B1 (70)48686970 Cyclin A (70) NOS II/iNOS (64)7064 Cyclin B1 (70) GM–CSF (55)7055 Cyclin D1 (48) Cox-2 (71,173)4871173 NOS II/iNOS (64) IL-3 (55)6455 GM–CSF (59) VEGF (173)59173 TNF-alpha (65,74,139) p53 (99,137)657413999137 Cox-2 (71,139)71139 IL-3 (55,66)5566 VEGF (62)62 Myogenin (68)68 Hel-N1 TNF-alpha (74) NF-M (73) NF-M (73)7473 GLUT1 (72) GLUT1 (72) GLUT1 (72)72 HuD GAP-43 (75–77) GAP-43 (75,76)75777576 TTP c-fos (90) GM–CSF (81)9081 GM–CSF (18,81,83–85,91) TNF-alpha (80)188183859180 TNF-alpha (18,81,83–86,89,90) IL-2 (82)18818386899082 Cox-2 (87) IL-3 (88)8788 IL-2 (82,90)8290 IL-3 (18,66,83,84,88)1866838488 BRF1 TNF-alpha (89,93) GM–CSF (55)899355 IL-3 (55,92,93) IL-3 (55)55929355 TIA-1 TNF-alpha (120) TNF-alpha (120)120 Cox-2 (121) Cox-2 (121)121 KSRP c-fos (90,93) NOS II/iNOS (102)9093102 NOS II/iNOS (102)102 TNF-alpha (90,93)9093 IL-2 (90,93)9093 c-jun (93)93 CUG-BP2 Cox-2 (150) Cox-2 (150) Cox-2 (150)150 Nucleolin bcl-2 (175)175 TINO bcl-2 (176)176 PAIP2 VEGF (177) VEGF (177)177

9. 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 3’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 。

10. AU-rich Elements (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 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

11. mRNAs ARE-BPs Class Motif Examples I 1 to 5 个散在 AUUUA 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 ， KSRP IIA (AUUU)5A GM–CSF AUF1 ， HuR ， Hel-N1 ， TTP ， BRF1 ， TIAR ， KSRP ， AUH ， GAPDH ， Hsp70 ， hnRNP-A1 ， hnRNP-C TNF-alpha AUF1 ， HuR ， TTP ， BRF1 ， TIA-1 ， TIAR ， KSRP IIB (AUUU)4A Interferon-alpha hnRNP-A1 ， hnRNP-A2, hnRNP-A3 IIC (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 ， PAIP2 III No 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 containing 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 are 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.2426 Interaction between ARE and RBPs

12. 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.

13. 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 paramagnetic 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

14. "Tet-on" system is activated in the presence of doxycycline the DNA binding domain of the Tet-on regulator (rTetR) contains mutations repressor that only binds DNA in the absence of ligand is converted to a ligand-dependent DNA binding protein. RNA-pol Tetracycline controlled transactivator (tTA )

15. TET-OFF in details Manfred Gossen and Hermann Bujard www.biochem.arizona.edu The "Tet-off" system is repressed in the presence of the doxycycline TET-VP producing 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…

16. mRNA 翻译研究特用技术 1. 新生蛋白分析。 2.Polysome 分离， Polysomal RNA, Polysomal 蛋白质分离。 3. 其它经典技术。

17. Reference 1)Barreau, etal; Nucleic 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 www.gmu.edu/departments/mmb/baranova/pages/ppt/biotech-lec5.ppt

18. HuR Regulates p21 mRNA Stabilization by UV Light Wang, et al; Mol Cell Biol. 2000 ， 20(3): 760–769. 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，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 Ⅰ

19. 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 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 P21 3’UTR 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 formation of p213’-UTR-protein complex in the cytoplasm

20. 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 accompanied with increased formation of P21 3’UTR-protein complex

21. 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 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 either 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 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 size of the HuR-p21 complexes, 37 to 40 kDa. HuR binds to the 3’UTR of p21 mRNA

22. 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 expression 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 recognizing 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. Evenness 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 treatment or exposure to 20 J/m2 UVC. p-jun, phosphorylated Jun. (E) Graphs depict the rate of loss of p21 and β-actin mRNAs in cells with different HuR levels after actinomycin D (2 μg/ml) addition with or without UVC irradiation. At the times indicated, total RNA was extracted and p21 and β- actin mRNAs were monitored 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 experiments. 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

23. 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 construct. (Top) Expression vectors pGL3, pGL3-FL, and pGL3-ΔB2 (see Materials and Methods) were transiently cotransfected into RKO parental (untransfected [Untr.]), AS.2, or AS.7 cells along with pSV-βgal (used to normalize for transfection efficiency); cells were irradiated with UVC (20 J/m2) or left untreated, and luciferase and β- galactosidase activities were examined 24 h later. (Bottom) Relative fold increase in luciferase activity after UVC exposure, seen with either pGL3-FL or pGL3-ΔB2 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 down-regulation of HuR

24. Western blot analysis of HuR expression and subcellular localization. (A) Six hours after irradiation with the indicated doses of UVC, whole-cell (20 μg), cytoplasmic (40 μg), nuclear (10 μg), and cytosolic (40 μg) 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 analysis of HuR expression performed on cytoplasmic (40 μg) and nuclear (10 μg) 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 to assess uniformity in loading and transfer among cytoplasmic and nuclear samples, respectively. UVC induces the cytoplasmic presence of HuR

25. Subcellular localization of HuR. GFP-HuR was visualized by fluorescence microscopy in transiently transfected RKO cells 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 increase in the cytoplasmic GFP- HuR signal, not seen in untreated cells. UVC induces cytoplasmic HuR

26. 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 after addition of actinomycin (Act.) D (1 μg/ml) or 4 h after exposure to 100 μM H2O2, MMS (100 μg/ml), 48 μM 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) 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

27. 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 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。

28. Example II AUF1 Regulates Replicative Senescence through Mediating p16 mRNA Turnover Wang, et al; EMBO Rep., 6 : 158-164, 2005.

29. Background CDK inhibitor p16 INK4 is induced with replicative 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 AU-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?

30. P16 mRNA 3’-UTR contains motifs for biding by RNA binding proteins

31. SenescenceYoung P16 3’-UTR is important for the instability of EGFP-p16 3’-UTR (In young cells) Time in Dox (h)

32. AUF1 binds to p16 3’-UTR. Binding of AUF1 to p16 3’-UTR attenuated with cellular senescence.

33. AUF1 levels reduced with cellular senescence. AUF1 binds to AU rich region of p16 3’-UTR. A B C

34. P16 3’-UTR confers instability to chimeric transcripts in lung carcinoma cells (H2) Time in Dox (h)

35. Knock-down of AUF1 stabilizes EGFP-p16 3’-UTR chimeric transcripts in H2 cells

36. Knock-down of AUF1 increases p16 expression and accelerates cellular senescence of WI-38 cells

37. Summary 1.mRNA turnover is important for 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.

38. RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation. Mazan-Mamczarz K, et al; 2003, PNAS Example Ⅲ

39. Background 1.p53 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 ?

40. Fig. 1. UVC induces p53 expression at protein level (A)UVC induces p53 expression (B)p53 mRNA levels is not influenced by UVC (C)p53 mRNA half-life is not altered by UVC (D)Subcellular and polysomal p53 mRNA is not altered under UVC. UVC induces p53 expression at protein level

41. Fig. 2. (A)Western blot analysis of p53 expression in RKO whole-cell lysates prepared at the times indicated after treatment with lactacystin (5 μM), UVC (15 J/m2), or a combination of both. Signals were quantitated by densitometry (graph). (B) Newly translated p53 is increased by UVC. UVC induced p53 is not associated with proteasome turnover

42. 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 with either 15 or 30 J/m2 UVC. (B) Plasmids pGL3-Luc and pGL3-Luc-p53(3′UTR) were used in transient transfections; 24 h after transfection, cells were either irradiated (15 J/m2) or left untreated, and the relative luciferase activity was calculated 6, 12, and 24 h later.11 Binding of cytoplasmic proteins to the p53 3′ UTR is linked to p53 translational up-regulation.

43. Fig. 4. (A)(REMSA) HuR binds to the p53 3’UTR (B)(pull-down assays) HuR binds to the p53 3’UTR. (C) (rCHip assays) HuR binds to the p53 3’UTR. (D)UVC induces the presence of HuR in the cytoplasm and polysome. HuR binds to the p53 3′ UTR in a UVC-dependent manner

44. Fig. 5. (A)Western blot analysis of HuR and p53 in HuR-over- expressing (S11) cells under UVC treatment. (B)Western blot analysis of HuR and p53 in HuR- silencing cells under UVC treatment. (C)Analysis of newly translated p53 in HuR- silenced cells. Modulation of HuR levels affects p53 translation and steady-state levels.

45. Conclussion UVC induces p53 translation. HuR binds to the 3’UTR of p53. HuR enhances p53 translation under UVC treatment.