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Title: | uORFchop 對轉譯調控之探討 Study of the uORFchop-driven translation |
Authors: | Yi-Jiun Chen 陳怡君 |
Advisor: | 呂勝春(Sheng-Chung Lee) |
Keyword: | uORF,CHOP,轉譯調控,anisomycin, uORF,CHOP,translation regulation,anisomycin, |
Publication Year : | 2009 |
Degree: | 博士 |
Abstract: | 細胞面臨逆境時,會藉由啟動轉錄、後轉錄、轉譯和後轉譯等機制來進行基因的調控,並引導著細胞走向存活或是凋亡的命運。CHOP 蛋白為 stress 誘導基因表現的一員,其功能之一與細胞凋亡有關。CHOP除了受到上游轉錄因子 ATF4 的轉錄調控外,其蛋白表現亦受到mRNA 5'非轉譯區 uORF 的轉譯控制。在低濃度 anisomycin 處理的情況下,細胞能透過轉錄和轉譯兩層面來誘導 CHOP 的表現。為了能夠專一的研究 CHOP 的 uORF 如何透過轉譯作用來調控 CHOP 的表現,並釐清它在轉譯起始的調控機制,我們建立了由 CMV 啟動子的uORF 調控報導蛋白表現平台的 mRNA。利用這樣的平台我們發現:anisomycin 所誘導的CHOP 表現與轉譯起始因子eIF4E/S209 和 eIF2α/S51 的磷酸化有著密切的關連,而 thapsigargin 的誘導表現主要則是受 eIF2α/S51 磷酸化所影響。此外,我們也利用了不同的 kinase 抑制劑及變性的 kinase,確立了p38MAPK-Mnk-eIF4E 和 mTOR 此二訊息傳遞途徑共同參與著anisomycin 的誘導表現;同樣地,受 mTOR 所控制的 4E-BP1 (eIF4E 的結合蛋白) 亦為此調控機制中的一個重要環節。RNA-IP 與 polysome profile 的分析更確立了uORF 在轉譯作用上的角色,並透過 eIF4E/S209 的磷酸化來參與anisomycin 的誘導表現。另一個有趣的發現是:在缺少 eIF2α/S51 磷酸化的情況下,雖然胰島素能活化 ERK-Mnk-eIF4E 及 mTOR 訊息傳遞途徑,卻無法促進 CHOP 的表現,這意味著 uORFchop 在不同逆境環境下對 CHOP 的表現具有決定性的影響力。 Cells respond to environmental stress by inducing translation of a subset of mRNAs important for survival or apoptosis. CHOP, a downstream transcriptional target of stress-induced ATF4, is also regulated translationally in a uORF-dependent manner under stress. Low concentration of anisomycin induces CHOP expression at both transcriptional and translational levels. To study specifically the translational aspect of CHOP expression, and further clarify the regulatory mechanisms underlying stress-induced translation initiation, we developed a CMV promoter-regulated, uORFchop-driven reporter platform. Here we show that anisomycin-induced CHOP expression depends on phosphorylated eIF4E/S209 and eIF2α/S51. Contrary to phospho-eIF2α/S51, phospho-eIF4E/S209 is not involved in thapsigargin-induced CHOP expression. Studies using various kinase inhibitors and mutants uncovered that both the p38MAPK-Mnk-eIF4E and mTOR signaling pathways contribute to stress-responsive reporter and CHOP expression. We also demonstrated that anisomycin-induced translation is tightly regulated by partner binding preference of eIF4E. Furthermore, mutating the uORF sequence abolished the anisomycin-induced association of chop mRNA with phospho-eIF4E and polysomes, thus demonstrating the significance of this cis-regulatory element in conferring on the transcript a stress-responsive translational inducibility. Strikingly, although insulin treatment activated ERK-Mnk and mTOR pathways, and consequently eIF4E/S209 phosphorylation, it failed to induce phospho-eIF2α/S51 and reporter translation, thus pinpointing a crucial determinant in stress-responsive translation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45428 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 分子醫學研究所 |
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ntu-98-1.pdf Restricted Access | 2.28 MB | Adobe PDF |
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