探討酵母菌核醣核酸蛋白Rbp1p之轉譯後修飾

Abstract

在真核細胞中，基因的表現在轉錄後會受到很多方面的調控，例如訊息核醣核酸的修飾、運送、降解，以及最後訊息核唐核酸要轉譯成蛋白質的過程，在這些調控的步驟中有許多可以和核醣核酸結合的蛋白質來協助調控的過程，這些蛋白質稱為核醣核酸結合蛋白。本實驗室在過去發現一個在酵母菌中可以與核醣核酸結合的蛋白質Rbp1p，最初它被鑑定為抑制細胞生長的負調控因子，結構上它包含三個核醣核酸識別基序RRM及兩個富含麩氨酸區域，我們已知這個Rbp1p在酵母菌生長階段的晚期、葡萄糖剝奪、滲透壓力下可以作落到細胞質中特殊的細胞質聚集顆粒P-bodies，並且經由二次元電泳的分析顯示Rbp1p分布在同一個分子量上但不同的等電點，暗示Rbp1p可能含有不同的轉譯後修飾，在本論文中再次利用二次元電泳的分析發現Rbp1p在生長階段的不同二維電泳上圖譜也會不同，我們推測外界的刺激會使Rbp1p被轉譯後修飾所調控，我們藉由質譜儀的分析找到了六個磷酸化的點，利用單點突變的技術將它們突變為丙氨酸，實驗證明同時突變其中的三個點459、462、463則Rbp1p負生長因子的能力下降，且突變型的Rbp1p在二維電泳上呈現的圖譜和野生型Rbp1p不同，但是這三個點的突變並不會影響Rbp1p坐落到P-bodies，同時我們也研究一個Rbp1p可能的磷酸激酶Ksp1p，我們發現剔除Ksp1p後Rbp1p負生長因子的能力也會下降，若挽回Ksp1的表現則Rbp1p負生長因子的能力又回復了，剔除Ksp1p後也會影響到Rbp1p在二次元電泳圖譜上的分布，但是剔除Ksp1p後依然不會影響Rbp1p坐落到P-bodies。這樣的結果說明了Ksp1p對於Rbp1p抑制生長的功能以及Rbp1p的轉譯後修飾是重要的。另外在本研究中我們藉由質譜儀的分析鑑定出Rbp1p在二次元電泳上每一個點的轉譯後修飾，並且也找到幾個可能與Rbp1p結合的蛋白質包括Dhh1p、Hrp1p、Porin1p、Psp1p以及 Pub1p。

In eukaryotic cells gene expression subjected several level of posttranscriptional regulation included mRNA processing, transport, turnover, and tanslation regulation.bove regulation process involves various of RNA-binding proteins to support. Previously our lab found the Saccharomyces cerevisiae RNA-binding protein Rbp1p was first identified as a negative growth regulator, which contains three copies of an RNA recognition motif (RRM) and two glutamine-rich stretches. We have known that Rbp1p can localize to specific cytoplasm foci called P body when cell growth to stationary phase, glucose deprivation, and osmotic stress. Rbp1p revealed mutiple spot with the same molecular weight but different isoeletric points in 2-DE analysis.his result suggested that Rbp1p contained diverse post-translational modification.n this study using 2-DE analysis found under different growth stage, Rbp1p subjected to different post-translational modification. We speculated that when cell received external stimulus Rbp1p may regulated by post-translational modification. We also found six phosphorylation sites of Rbp1p by means of mass spectrometry. Using site-directed mutagenesis technique to produce phosphorylation sites mutants of Rbp1p. Data revealed simultaneously mutation on serine 459, 462, and 463 to alanine show partial growth inhibition ability lost. Compare to wild Rbp1p the mutant form show different post-translational modification pattern in 2-DE gel. However the mutant of Rbp1p had no effect to localize to P body. In addition we also studied a putative Rbp1p kinase Ksp1p, found that Rbp1p growth inhibition ability lost in Δksp1 strain. Futhermore, in rescue experiment where ADH drove Myc-Ksp1p overexpression in Δksp1 cells, Rbp1p restored its growth inhibition ability. However Ksp1p deletion had no effect on the localization of Rbp1p to P-body. These result suggested thatKsp1p is important for the function of Rbp1p, at least in growth phenotype and post-translational modification. Besides, in this study we determined each of the multiple post-translational modification spots of Rbp1p by mass spectrometry. Moreove we also identified several Rbp1p associated protein included Dhh1p, Hrp1p, Porin1p, Psp1p, and Pub1p.