rna结合蛋白与转录后调控

1、rna结合蛋白与转录后调控rna结合蛋白与转录后调控第1页mRNA turnover Translation level Post-translation levelTranscription levelGene RegulationDNA and chromatin levelsPost-transcriptional levelMaturation mRNA export rna结合蛋白与转录后调控第2页Regulatory factors for mRNA decay and translation RNA binding proteins microRNAs rna结合蛋白与转录后调控

2、第3页RNA binding proteins (RBPs) RNA结合蛋白种类很多，预计占细胞编码蛋白6-8%者为RNA结合蛋白, 但迄今只有为数不多几个RNA结合蛋白（如HuR，AUF1，TTP，TIA1，CUGBP2等）被证实可特异参加mRNA稳定性、翻译、或其它层面基因调控。 rna结合蛋白与转录后调控第4页HuR Hu /ELAV RNA 结合蛋白家族（包含HuA/HuR，Hel-N1，HuC，HuD） 主要组员。与其它组员仅表示于神经细胞不一样，HuR几乎在全部组织 都有表示。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

3、mRNA成熟及export相关。 结合全部三类AREs。结合后结局主要为稳定目标。所以也被 称为mRNA 稳定因子。 结合并稳定VEGF, COX-2, p21, cyclin A, cyclin B1, c-fos, TNF ，IL-1， MyoD，Myogenin，bcl-2等mRNA。所以可调整应激反应，细胞周期，肿瘤， 分化，调亡等过程。 HuR也可结合目标并调整其翻译。 1）调控翻译效率， 如 p53，p27 mRNA, c-myc。 2）调控mRNA export, 如 CD83，c-fos, COX-2。rna结合蛋白与转录后调控第5页AUF1 也叫HnRNPD（heteroge

4、neous 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），如P21， CyclinD1，也可使目标稳定，如TNF-alpha。rna结合蛋白与转录后调控第6页TTP与HuR及AUF1不一样，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。rna结合蛋白与转录后调控第7页 Effect of ARE-BPs on the stability and translation of ARE-containin

5、g mRNAsARE-BPs mRNA stability Protein expression Translational efficiency Abundance Increase 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 (

6、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/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)

7、 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 (18,81,8385,91) TNF-alpha (80) TNF-alpha (18,81,8386,89,90) IL-2 (82) Cox-2 (87) IL-3 (88) IL-2 (

8、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 Cox-2 (150) Cox-2 (150) Cox-2 (150) Nucleolin b

9、cl-2 (175)TINO bcl-2 (176) PAIP2 VEGF (177) VEGF (177)rna结合蛋白与转录后调控第8页Interaction of the factors involving in post-transcriptional regulation1. 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，且功效相反。2. RNA结合蛋白与microRNA间相互作用。如， HuR与let-7, miR-122, TTP与miR-16 。rna结合蛋白与转录后调控第9页AU-rich Elements (

10、AREs)1.主要指位于3-非翻译区富含AU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结构基础。4. 依组成份为三类1）含1-5个分散AUUUA。2）最少有两个Overlapping UUAUUUA(U/A)(U/A). 依AUUUA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二级结构也与RNA-蛋白质相互作用亲密相关。 如，约70-80% HuR或AUF1结合序列含有相同二级结构rna结合蛋白与转录后调控第10页mRNAs ARE-BPsClass Motif Exam

11、ples 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， KSRPIIA (AUUU)5A GMCSF AUF1， HuR，

12、Hel-N1， TTP， BRF1， TIAR，KSRP，AUH，GAPDH，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， Hsp1

13、10， hnRNP-A1 IL-3 HuR， BEF1， AUH，Nucleolin，TINO bcl-2 AUF1， HuR， 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 associa

14、tions 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 prot

15、ein 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

16、the Supplementary data. Numbers correspond to listed references.Interaction between ARE and RBPsrna结合蛋白与转录后调控第11页RNA-蛋白质相互作用(结合)特点可在细胞核, 也可在细胞浆发生在核与胞浆结合功效截然不一样, 如在胞浆与翻译及mRNA稳定性相关, 在核可能与拼接或成熟相关.RNA结合蛋白对目标序列要求不如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.rna结合蛋白与转录后调控第12页mRNA稳定性（turnover）研究特殊技术蛋白质-RNA结

17、合试验： 1）目标Transcript 体外转录与标识 2）细胞浆抽提物制备。 3) EMSA (gel-shift, supershift) 4) rChip, pull-down assays (using paramagnetic streptavidin dynabeads, biotinyl-labeled transcripts)mRNA半衰期测定 基础思绪:终止转录后,收取不一样时间点之RNA, 定量分析RNA降解速率. 1）用ActinomycinD终止转录 2）Tet-off/on （或类似）汇报基因系统 3) in vitro RNA降解分析rna结合蛋白与转录后调控第13

18、页Tet-on system is activated in the presence of doxycyclinethe 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-polTetracycline controlled transactivator (tTA)rna结合蛋白

19、与转录后调控第14页TET-OFF in detailsManfred Gossen and Hermann Bujard The Tet-off system is repressed in the presence of the doxycyclineTET-VP producing vectorGene of interest expressing vectorVP RNA pol interacting partTetR - tet binding partTET-OFF systemTetracycline controlled transactivator (tTA)如：EGFP-

20、interest target chimericrna结合蛋白与转录后调控第15页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。 rna结合蛋白与转录后调控第16页Reference Barreau, etal; Nucleic Acids Research,33(22): 7138-7150, 2) 3) Wang, et al; MCB, 20:760-769, 4) Lal, et al; EMBO J., 23:3092-3102, /departments/mmb/baranova/pag

21、es/ppt/biotech-lec5.ppt rna结合蛋白与转录后调控第17页HuR Regulates p21 mRNA Stabilization by UV LightWang, et al; Mol Cell Biol. ， 20(3): 760769. 细胞暴露于各种应激（Stresses）如short-wavelength UV light (UVC)时, cyclin-dependent kinase inhibitor p21表示显著被诱导。P21 调控，尤其P53调整转录已被广泛，深入研究。先前研究发觉，UVC可经过P53-不依赖方式诱导P21。研究还发觉，细胞暴露于UV

22、C后，p21 mRNA 稳定性增加。问题：UVC诱导P21表示（稳定性增加）机制怎样？ 与HuR相关否？ Background:Example rna结合蛋白与转录后调控第18页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，因为不论RKO细胞是否有野生型P53，复合物形成无区分。复合物由蛋白质与3UTR间结合而成。5UTR及缺失ARE3-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，但只有胞浆中复合物形成可被UVC诱导。UVC induces the formation of p213-UTR-protein complex

23、 in the cytoplasmrna结合蛋白与转录后调控第19页A。复合物形成在UVC 辐射半小时后显著被诱导，与P21 被诱导相吻合。B。 竞争抑制试验说明复合物特异性，Cold 探针可竞争抑制复合物形成。C。 EMSA 后，UV交联，SDS分离复合物，发觉复合物中一条大约40Kd 复合物（单一蛋白与大约10个碱基短片断转录物形成），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 诱导该未知复合物趋势与P21被诱导一致。Elevation of p21 by UVC is accompanied with increased format

24、ion of P21 3UTR-protein complexrna结合蛋白与转录后调控第20页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

25、 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(15% gel), and transferred onto polyvinylidene diflu

26、oride 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 analys

27、is. Estimated size of the HuR-p21 complexes, 37 to 40 kDa.HuR binds to the 3UTR of p21 mRNArna结合蛋白与转录后调控第21页HuR表示降低后， p21 3 UTR 与细胞浆蛋白间形成复合物（在UVC辐射后）降低， p21 mRNA 稳定性降低（半衰期缩短），表示降低。Decreased HuR expression lowers binding to the p21 3 UTR and reduces p21 mRNA stability and p21 induction by UVC. (A) We

28、stern 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 a

29、nd 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 treatmen

30、t () 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 untransfecte

31、d 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 -actin mRNAs in cells with different HuR levels after actinomycin D (2 g/ml) addition with or without UVC irradiation. At the times in

32、dicated, total RNA was extracted and p21 and -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 repres

33、ent 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 mRNArna结合蛋白与转录后调控第22页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 表示后，由B2赋予稳定性消失。

34、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 transiently cotransfected into RKO parental (untransfected Untr.), AS.2, or AS.7 cells along with pSV-gal (used to nor

35、malize 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) Relative fold increase in luciferase activity after UVC exposure, seen with either pGL3-FL or pGL3-B2 compared with that se

36、en 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 rna结合蛋白与转录后调控第23页Western blot analysis of HuR expression and subcellular localization. (A) Si

37、x 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 monitor the expression of HuR, hnRNP C, AUF1, BAF57c (57 kDa), and actin. Cell lysates were collected

38、 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) and nuclear (10 g) fractions. (D) Indicated doses of ionomycin (Ion.; micromolar) or lithium acet

39、ate (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.UV

40、C induces the cytoplasmic presence of HuRrna结合蛋白与转录后调控第24页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 nucleu

41、s. 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 HuRrna结合蛋白与转录后调控第25页Increa

42、sed 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 g/ml) or 4 h after exposure to

43、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) B2 binding activity in cytoplasmic lysates of cells treated as for

44、panel and supershift analysis of complexes forming after exposure to such stresses.Induction of cytoplasmic HuR and its binding to p21 mRNA by UVCrna结合蛋白与转录后调控第26页SummaryUVC在不影响总HuR水平条件下诱导细胞浆HuR水平UVC诱导HuR与p21 mRNA 结合HuR 结合P21 3-UTR后使P21mRNA 稳定性增高，进而引发P21表示增高。人为降低HuR水平可降低HuR与P21 3-UTR间相互结合，降低P21mRNA

45、稳定性，降低P21表示，提醒HuR对UVC诱导P21 mRNA 稳定性是必须。rna结合蛋白与转录后调控第27页Example II AUF1 Regulates Replicative Senescence through Mediating p16 mRNA TurnoverWang, et al; EMBO Rep., 6 : 158-164, . rna结合蛋白与转录后调控第28页BackgroundCDK inhibitor p16INK4 is induced with replicative senescence. Although transcriptional regulat

46、ion 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

47、 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? rna结合蛋白与转录后调控第29页P16 mRNA 3-UTR contains motifs for biding by RNA binding proteinsrna结合蛋白与转录后调控第30页SenescenceYoungP16 3-UTR is

48、important for the instability of EGFP-p16 3-UTR (In young cells)Time in Dox (h)Time in Dox (h)rna结合蛋白与转录后调控第31页 AUF1 binds to p16 3-UTR. Binding of AUF1 to p16 3-UTR attenuated with cellular senescence.rna结合蛋白与转录后调控第32页AUF1 levels reduced with cellular senescence. AUF1 binds to AU rich region of p16

49、 3-UTR.ABCrna结合蛋白与转录后调控第33页P16 3-UTR confers instability to chimeric transcripts in lung carcinoma cells (H2)Time in Dox (h)rna结合蛋白与转录后调控第34页Knock-down of AUF1 stabilizes EGFP-p16 3-UTR chimeric transcripts in H2 cells rna结合蛋白与转录后调控第35页 Knock-down of AUF1 increases p16 expression and accelerates cel

50、lular senescence of WI-38 cellsrna结合蛋白与转录后调控第36页SummarymRNA 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

51、and accelerate cell senescent.rna结合蛋白与转录后调控第37页RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation.Mazan-Mamczarz K, et al; , PNASExample rna结合蛋白与转录后调控第38页Backgroundp53是主要抗癌基因， 功效广泛。 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? 怎样调控? rna结合蛋白与转录后调控第39页Fig. 1.UVC induces p53 expression at protein le