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頁籤選單縮合
題名 | Analysis of ARE-binding Proteins Associated with 3' Untranslated Region of MKP-1 mRNA=去磷酸酶MKP-1信使核糖核酸的三端非轉譯區結合蛋白之分析 |
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作者姓名(中文) | 林念儀; 林子揚; 張瀞仁; 林中天; | 書刊名 | 臺灣獸醫學雜誌 |
卷期 | 35:3 2009.09[民98.09] |
頁次 | 頁174-184 |
分類號 | 437.24 |
關鍵詞 | 去磷酸酶; 核糖核酸; 轉錄後調控; 三端非轉譯區; 多腺嘌呤-尿嘧啶序列; MKP-1; RNA; Posttranscriptional regulation; 3' untranslated region; AU-rich element; |
語文 | 英文(English) |
中文摘要 | Mitogen-activated protein kinases (MAPKs)在調節細胞增生、分化、死亡以及胚胎發育和免疫反應中扮演重要角色,MAPK phosphatase-1(MKP-1)可藉由催化去磷酸的作用而抑制MAPK訊息之傳遞。MKP-1表現的異常與多種癌症的發生有關,且可能造成腫瘤細胞產生化療藥物耐受性(chemoresistance)。MKP-1的信使核糖核酸(mRNA)非常不穩定且半生期短,其三端非轉譯區(3'untranslated region, 3'UTR)具有特殊的多腺嘌呤-尿嘧啶序列(AU-rich element, ARE),推測可影響其轉錄後調控機制。本篇研究將不同長度的MKP-1之3'UTR片段融合於報導基因的三端,觀察其對報導基因表現之影響,結果發現MKP-1之3'UTR可有效降低報導基因的表現,且與已知可加速mRNA降解之蛋白質tristetraprolin (TTP)共同表現時,其表現更加受到抑制。為進一步了解MKP-1轉錄後調控的分子機制,設計RNA pull-down及RNA免疫沉澱(immunoprecipitation)等實驗,並證實此二設計可運用於RNA結合蛋白的分離鑑定,且便利未來對特定ARE序列的特性分析。 |
英文摘要 | Mitogen-activated protein kinases (MAPKs) play major roles in regulating important physiological processes. MAPK phosphatases (MKPs) dephosphorylate and inhibit MAPKs activity, thereby negatively regulating MAPKs signaling. Altered expression of MKP-1, the most studied member of the MKP family, has been implicated in the development of various kinds of cancers and in the acquisition of chemoresistance. MKP-1 mRNA is labile and has specific AU-rich elements (AREs) in its 3' untranslated region (3'UTR) that direct posttranscriptional gene regulation. In this study, activities of reporters comprised of luciferase coding region and various lengths of MKP-1 3'UTR were analyzed. The results indicated that transcripts bearing complete MKP-1 3'UTR or ARE fragments had a significant lower expression of the reporter gene. And co-expression of tristetraprolin (TTP) further inhibited the expression of reporters. An RNA pull-down assay was established for the identification of MKP-1 RNA-interacting proteins. And RNA-immunoprecipitation assay (RNA-IP) was performed and reporter mRNAs containing MKP-1 AREs were found to be associated with RNA-binding proteins such as TTP and Hu antigen R (HuR). In this paper, we proved that 3'UTR of MKP-1 mRNA contributed largely to the regulation of MKP-1 expression and we provided two practical biochemical assays for identifying ARE-binding proteins associated with MKP-1 mRNA. |
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