李玉梅臺灣大學：生化科學研究所翁瑞霞Weng, Jui-HsiaJui-HsiaWeng2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52735細胞中透過不同的調節機制以期達到基因表現的控制，而部分調 控機制所影響的層面即在核醣核酸的後轉錄時期。此類調控對於半生 期短、易分解的核醣核酸尤為重要，例如：原癌基因、細胞素等。而 在相關研究中發現，此種易分解的核醣核酸之3 端非轉譯區中具有特 定的排列分子重複出現，其稱之為AU-rich element 簡稱ARE； ARE 透過其鍵結蛋白的調控，而達穩定或加速分解之效。 當細胞遭遇環境變化或藥物刺激，例如熱休克或氧化性藥物影響 時，細胞將啟動防禦機制，一方面調控熱休克性蛋白等之表現另一方 面爲降低細胞損傷而暫時終止一般蛋白的轉錄，將訊息核醣核酸存放 於壓力小體中 (stress granules)，待環境危機解除才釋出訊息核醣 核酸回復其原有表現。 在本篇報告中，針對BC1 (Brain Co-regulater 1) 蛋白在細胞 遭遇環境變異前後所擔負的不同功能角色提出實驗探討。初步結果顯 示，BC1 透過其C 端區域而具有與ARE 鍵結的能力，但此鍵結的現象 並未對該核醣核酸的半生期並無顯著影響，而具有增強其轉譯的傾 向。此外，當使細胞受到氧化性藥物刺激時，透過免疫染色法證實 BC1 參與在壓力小體中。未來爲進一步釐清BC1 在形成壓力小體或對 其內核醣核酸所占調控角色，則仍須更進一步實驗。AU-rich element (ARE) is found in the 3’UTR of many short-lived mRNAs such as cytokines, and oncogenes. Many RNA-binding proteins that selectively recognize and bind to this ARE sequence are called AU-rich binding protein (AUBP) and can modulate stability and/or translation of ARE-containing mRNAs. Eukaryotic cells shut down protein synthesis and reprogram their translational machinery in response to environmental stress for conserving anabolic energy to repair of the stress-induced damage. In stressed-cells, mRNA is in a dynamic equilibrium between polysomes and stress granules (SGs). SGs are cytoplasmic foci at which stalled translation initiation complexes accumulate. Many RNA-binding protein such as TIA-1,TIAR, and HuR localized at stress granules and it has been proposed the carboxyl terminus of TIA1, the prion-related domain PRD, mediates the formation of SGs. In this study, we investigated the binding ability of BC1 to homo-polynucleotides. Our data demonstrated that BC1 is an RNA-binding protein and possesses strong binding activity toword the distinct sequence. The YTH domain of BC1, a putative RNA-binding domain, harbors ARE-binding activity by using the electrophoretic mobility shift assay (EMSA). RT-PCR and Northern blot analysis showed that BC1 does not alter the stability of its binding target RNAs, but we also prove that BC1 have tendency to promote gene expression at translational level. When cells encounter stress, BC1 is colocalized with TIA-1 and HuR in SGs. We also found that BC1 interacts with TIA-1 under stress in GST-pull down experiment. In deletion analysis, we found that the PRD domain of TIA-1 and the Extensin-like domain of BC1 are responsible for SG formation. Collectively, we report a novel RNA-binding protein BC1 which may exert its role in promoting translation of ARE-containing mRNAs and is involved in formation of SGs.Contents English Abstract…………………………………………………..……..4 Chinese Abstract…………………………………………………..…….5 Abbreviation………………………………………………………..……6 1. Introduction 1.1 Turnover of mRNA…………………………………………………………..…....7 1.1.1 Regulating mRNA decay by cis-acting element – AU-rich element………...…..8 1.1.2 AREs mediate effecter mechanisms……………………………………….…...10 1.1.3 AU-rich element binding protein.........................................................................11 1.2 Cells encountering stress……………………………….……………………..….12 1.2.1 Stress sensor…………………………………………………………………....12 1.2.2 Formation of stress granules (SGs)…………………………………………….13 1.2.3 TIA proteins and SGs assembly………………………………………………..15 1.2.4 Other proteins regulating SGs formation………………………………………16 1.2.5 Components of SGs - accumulation of stalled translation initiation complex....18 1.2.6 Components of SGs – specific RNA-binding proteins…………………….......18 1.3 The Brain Co-regulator 1……………………………………………………..…..19 2. Materials and Methods 2.1 Plasmid constructs………………………...………………………………….......21 2.2 Cell culture and transfection…………………...…………………………………22 2.3 In vitro translation and in vitro transcription…………………...………………...22 2.4 RNA binding assay………………………………………...……………………..23 2.5 RNA electrophoretic mobility shift assay (EMSA)…………………………...….23 2.6 RT-PCR………………………………………………………………………...…23 2.7 Northern blot analysis………………………………………………………..…..24 2.8 Luciferase assay……………………………………………………………….....24 2.9 RNA labeling by ethidium bromide……………………………………...............24 2.10 Immunofluorescence analysis…………………………………………………..25 2.11 In situ hybridization……………………………………………………………..26 2.12 GST-pull down assay…………………………………………………................263. Result 3.1 The RNA binding ability of BC1…………………………………………………28 3.2 BC1 binds AU-rich RNA in vitro…………………………………………...……28 3.3 The YTH domain is sufficient for RAN binding…………………………………30 3.4 BC1 seems neither to stabilize nor to degrade RNA ………………………...…..30 3.5 BC1 promotes protein translation in vitro………………………………………..31 3.6 BC1 form cytoplasmic foci under stress………………………………...……….32 3.7 Co-localization of BC1 with TIA-1, the marker of SGs……………………...….33 3.8 BC1, a new marker for SGs………………………………………………………34 3.9 RNA-independent interaction of BC1 and TIA-1…………………………...…...35 3.10 The essential region of BC1 for being recruited into SG……………...………..36 4. Discussion 4.1 RNA-binding ability of BC1……………………………………………………..38 4.2 DNA-binding ability of BC1……………………………………………………..38 4.3 Is BC1 involved in regulating RNA stability?........................................................39 4.4 BC1 selectively promotes the translation…………………………….………..…40 4.5 BC1 as a novel component of stress granules…………………………….……...41 4.6 Different stresses might cause different effects on BC1……...………………….41 4.7 The possible mechanisms of recruiting BC1 into SGs…………..……………….42 4.8 One view in the program of evolution……………………………………………43 5. Reference……………………………………………….……………44 6. Figure…………………………………………………….…………..53 FIG. 1. The in vitro RNA binding ability of BC1. FIG. 2. Sequences of the 3’-UTR AU-rich elements of the various RNA probes used in this study. FIG. 3. AU-rich binding assay by in vitro translated BC1 proteins. FIG. 4. RNA stability was not altered by BC1. FIG. 5. BC1 can promote the ARE-containing reporters expression. FIG. 6. BC1 forms cytosolic foci under oxidative condition. FIG. 7. Overexpression of BC1 is co-localized with TIA-1. FIG. 8. Endogenous BC1 is associated with SGs. FIG. 9. BC1 is a maker of SGs. FIG. 10. BC1 directly interacts with TIA-1FIG. 11. The PRD domain of TIA-1 and the Extensin-like domain of BC1 are essential for SGs formation. FIG. 12. Amino acid 200-300 is efficient for BC1 to associate with SGs 7. Appendix……………………………………………………….…….67 Appendix. 1. Deadenylation-dependent mRNA decay. Appendix. 2. Examples of decay determinants and their interacting proteins regulating the turnover of a few mRANs. Appendix. 3. AU-rich elements and their associated factors Appendix. 4. Model for how the ARE mediates stability and instability. Appendix. 5. Translational initiation in the absence or presence of stress Appendix. 6. The guanine nucleotide exchange activity of eIF2B Appendix. 7. The schema of TIA-1 and TIAR Appendix. 8. Amino acid sequence of BC1.1741392 bytesapplication/pdfen-US壓力小體RNA穩定性BC1AREstress granulesotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52735/1/ntu-94-R91242012-1.pdf