核糖核酸聚合酶第四次單元 RPB4 在哺乳類動物細胞之功能

Abstract

基因的表現受到許多階段的調控，從染色體轉錄出 mRNA 之後到轉譯成蛋白質，每階段的調控都可以影響到最終蛋白質的表現，但每個階段之間的連接還不是那麼清楚。然而在酵母菌中 Rpb4 蛋白為核糖核酸聚合酶的次單元之一，已被報導參與 mRNA 外送出細胞核、轉譯、轉錄、mRNA 降解等過程，暗示著 Rpb4 在調控基因表現上佔有重要的地位，同時也顯現出轉譯、轉錄和 mRNA 降解之間是具有連結的。目前的研究主要集中在酵母菌，較少探討其在哺乳類動物細胞中的功能，所以本篇研究主要放在 RPB4 在哺乳類動物細胞中的功能。首先我們利用 GFP-RPB4 發現 RPB4 有部分分布在細胞質，並且在 HeLa 細胞中會與 P-body 有交互作用，接著利用 MS2繫留分析 (tethering assay) 將 RPB4 帶到 reporter luciferase 的 mRNA 上，我們發現 RPB4 在 HEK293T 細胞中會使得 reporter gene 活性下降，然而在 HeLa 細胞卻沒有這樣的現象，之後觀察其 mRNA 含量，發現 mRNA 並沒有明顯減少，再則我們分離出多核糖體在40S的部分發現到有 RPB4 的存在，之後再從多核糖體層分離出 RNA，發現 reporter mRNA 含量明顯下降，暗示著 reporter mRNA 沒有進入到轉譯程序，最後我們還發現到當 reporter 帶有 MS2 接合序列，會使得 Flag-MS2-RPB4 在細胞核出現。綜合上述，我們發現 RPB4 降低 reporter 表現量的機制可能是：一.影響轉譯初始階段，使 mRNA 無法進入到多核糖體，二.RPB4 透過 MS2 結合序列結合到 reporter mRNA 上，並使 mRNA 留在細胞核。

Gene expression is a complex process that involves several distinct stages, but it is little known about crosstalk among these stages. The Rpb4 (ScRpb4, Rpb4) is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II) which is composed of 12 subunits. The major function of Pol II is responsible for mRNA synthesis (transcription) in the nucleus. However, Rpb4 and Rpb7 can form a heterodimer and depart from the nucleus to the cytoplasm. In cytoplasm, Rpb4 has been found to act in mRNA decay pathway and mRNA translation. It raises the possibility that Rpb4 links between the nuclear and cytoplasmic processes. Rpb4 plays a coordinator, which integrates the various stages to determine the gene expression. In our study, we aim to explore the function of Homo Sapiens RPB4 (HsRPB4,RPB4) in mammalian cell. Firstly, we found RPB4 localizes partially in cytosol and colocalizes with P-bodies in HeLa cell by transfecting GFP-RPB4. Secondly, in HEK293T cell overexpression of RPB4 down-regulates the activity and protein expression level of reporter luciferase by using MS2-tethering system where Flag-MS2-RPB4 fusion protein binds luciferase mRNA containing MS2 binding site; however, RPB4 does not decrease the luciferase mRNA level compared to the control. Furthermore, we performed sucrose gradients to fractionate polysomes and examined the distribution of Flag-MS2-RPB4 and luciferase mRNA. We found RPB4 is located in 40S ribosome fraction and the amount of luciferase mRNA in polysome was declined in the presence of Flag-MS2-RPB4. Subsequent subcellular analysis showed that more Flag-MS2-RPB4 was detected in nuclei when cotranstected with MS2-binding elements containing luciferase reporter than the control luciferase reporter. Taken together, these findings suggest the possible mechanism of RPB4 mediated reporter down-regulation is by retaining reporter mRNA in nucleus to reduce protein translation.