張瀞仁臺灣大學：生化科學研究所黃雅聆Huang, Ya-LingYa-LingHuang2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52760訊息RNA的穩定性是真核細胞調控基因表現的一個主要機制，因而影響細胞的生長和分化。AU-rich elements (AREs)位在許多原致癌基因、細胞激素以及一些生長因子的mRNA之3’untranslated regions當中，可能是使它們自己的mRNAs快速降解的目標序列。HuR是RNA結合蛋白當中ELAV family的一個成員，它的表現非常廣泛；在短暫轉殖的細胞，HuR會結合到AREs上，並且使含有ARE的mRNAs變的穩定。Tristetraproline（TTP）是一個immediate-early gene，它可以結合到AREs引起RNA的不穩定。為了要研究ARE-containing mRNAs穩定性的調控機制，因此我們在LPS刺激的巨噬細胞中，完成了TNFα和IL-1β mRNAs半衰期的表現和調控機制的實驗。泳動阻滯電泳分析（EMSA）顯示由LPS誘導的去穩定因子TTP可以以較高的親和力結合到TNFα ARE上，而對IL-1β ARE的親和力則較低。HuR被發現可以和TNFα ARE相互作用來穩定它的RNA。有趣的是，由LPS誘導的IL-1β mRNA穩定性是p38 signaling pathway-dependent，而TNFα mRNA的穩定性則是p38 signaling pathway-independent。p38 pathway的活化導致TTP磷酸化並且降低它和RNA結合的活性。相對於TNFα的ARE，p38的訊息可以取消TTP在IL-1β ARE上的抑制作用。我們的結果指出TTP可以反應p38的訊號來調節IL-1β等ARE-containing mRNA的表現。Messenger RNA stability is one of the key mechanisms that eukaryotic cells regulate gene expression and influence cell growth and differentiation. AU-rich elements (AREs) present in the 3’ untranslated regions of mRNAs from many protooncogenes, cytokines, and growth factors may be targets for rapid degradation. HuR, a ubiquitous expressed member of the ELAV family of RNA binding proteins, selectively binds to AREs and stabililizes ARE-containing mRNAs in transiently transfected cells. Tristetraproline (TTP) is an immediate-early gene that could bind to AREs and trigger RNA destabilization. To investigate the regulation of stability of ARE-containing mRNAs, we performed experiments on the expression and regulation of half-life of TNFα and IL-1β mRNAs in LPS-stimulated macrophages. Electrophoretic mobility shift assays showed that the LPS-induced destabilization factor TTP could bind to TNFα ARE much better than that of IL-1β ARE. HuR was found to interact with TNFα ARE to stabilize its RNA. Interestingly, LPS-induced stability of IL-1β mRNA was p38 signaling pathway-dependent while that of TNFα mRNA was p38 signaling pathway-independent. Activation of p38 pathway resulted in the phosphorylation of TTP and decrease of its RNA-binding activity. Contrary to ARE of TNFα, p38 signal could reverse the inhibitory activity of TTP on IL-1β ARE. Our results indicate that TTP could respond to p38 signal to modulate the expression of specific ARE-containing mRNAs such as IL-1β.目錄 中文摘要............................................................................................................1 Abstract.............................................................................................................2 第一章：前言.....................................................................................................3 1. AU-rich element-containing mRNAs..................................................................3 2. RNA-binding protein......................................................................................6 2.1 Hu-antigen R（HuR）...................................................................................8 2.2 Tristetraprolin（TTP）.................................................................................10 3. p38 Mitogen-activated protein kinase（MAPK） pathway.................................13 第二章: 材料與方法.........................................................................................17 1. 質體建構....................................................................................................17 1.1 HuR cDNA和TTP cDNA...............................................................................17 1.2 TNFα cDNA和IL-1β cDNA............................................................................17 1.3 TNFα ARE和IL-1β ARE...............................................................................18 1.4 其他的質體.................................................................................................18 2. 細胞培養......................................................................................................18 3. Total RNA Isolation......................................................................................19 4. Reverse Transcription-Polymerase Chain Reaction..........................................19 4.1 Reverse Transcription.................................................................................20 4.2 聚合脢鏈鎖反應.........................................................................................20 5. 細胞質及細胞核萃取物抽取............................................................................23 6. SDS 聚丙烯醯胺膠體電泳.............................................................................23 7. SDS-PAGE染色法........................................................................................24 8. 西方墨點分析................................................................................................24 9. RNA泳動阻滯電泳分析..................................................................................25 9.1 RNA探針製備.............................................................................................25 9.2 RNA泳動阻滯電泳分析.................................................................................27 10. 轉接載體....................................................................................................28 11. DH5α轉型..................................................................................................28 12. GST 融合蛋白質.........................................................................................29 12.1 GST融合蛋白質誘導表現............................................................................29 12.2純化GST融合蛋白質...................................................................................29 13. 抗體..........................................................................................................30 14. 質體抽取....................................................................................................30 14.1小量質體抽取.............................................................................................30 14.2大量質體抽取.............................................................................................31 15. 細胞轉殖.....................................................................................................31 15.1.磷酸鈣細胞轉殖.........................................................................................31 15.2以Lipofectamine 2000進行細胞轉殖..............................................................32 16. 冷光酵素活性分析.......................................................................................33 16.1 β-gal for normalize....................................................................................33 16.2 Luciferase assay.......................................................................................34 第三章: 實驗結果..............................................................................................35 實驗安排.........................................................................................................35 1. TNFα mRNA及IL-1β mRNA在受LPS刺激的巨噬細胞內表現的情況.....................36 2. 3’UTR對TNFα和IL-1β mRNA表現的影響.......................................................37 3. TTP蛋白質及HuR蛋白質在受LPS刺激的巨噬細胞內表現的情況.........................38 4. HuR及TTP和TNFα ARE的連結能力................................................................38 5. HuR及TTP和IL-1β ARE的連結能力.................................................................39 6. 重組TTP蛋白質和ARE的結合能力..................................................................40 7. 重組HuR蛋白質的各個RNA recognition motif和ARE的結合能力.........................41 8. HuR和TTP對TNFα及IL-1β表現的調控機制.......................................................42 9. p38 MAPK pathway對LPS誘導的RNA穩定性及TTP表現的影響.........................43 10.p38 MAPK pathway對TTP、HuR表現及Luc-ARE活性的影響............................44 第四章：討論...................................................................................................47 參考文獻.........................................................................................................54 圖表與附錄......................................................................................................67 圖1 TNFα mRNA及IL-1β mRNA在受LPS刺激的巨嗜細胞內表現的情況.................68 圖2 3’UTR對TNFα和IL-1β mRNA表現的影響....................................................70 圖3 TTP蛋白質及HuR蛋白質在受LPS刺激的巨嗜細胞內表現的情況......................72 圖4 HuR及TTP和TNFα ARE的連結能力.............................................................73 圖5 HuR及TTP和IL-1β ARE的連結能力..............................................................74 圖6 重組TTP蛋白質和ARE的結合能力...............................................................75 圖7 重組HuR蛋白質的各個RNA recognition motif和ARE的結合能力......................76 圖8 HuR和TTP對TNFα及IL-1β表現的調控機制.....................................................77 圖9 p38 MAPK pathway對LPS誘導的RNA穩定性及TTP表現的影響......................79 圖10 p38 MAPK pathway對TTP、HuR表現及Luc-ARE活性的影響........................80 附錄1 各種mRNA 3’UTR中ARE的特色比較.......................................................83 附錄2 三類ARE的序列及功能特性.......................................................................83 附錄3 ARE-mRNAs的生物多樣性.......................................................................84 附錄4 mRNA降解方式.......................................................................................85 附錄5 各種ARE-BPs的特色................................................................................86 附錄6 HuR的胺基酸序列...................................................................................87 附錄7 TTP的胺基酸序列.....................................................................................87 附錄8 p38 MAPK抑制劑：SB203580..................................................................88 附錄9 Mitogen-activated protein kinase signaling pathways...................................88 縮寫表.............................................................................................................891846292 bytesapplication/pdfen-US細胞激素穩定性RNA結合蛋白cytokinestabilityRNA binding proteinotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52760/1/ntu-94-R91242001-1.pdf