酵母菌核糖核酸結合蛋白之作用蛋白-Psp1p之功能特性探討

Abstract

真核細胞中，訊息核糖核酸的表現過程必須經過一連串的轉錄後調控，包括在五端加上覆蓋、疊接、以及在三端加上多個腺嘌伶。在這個過程中，核糖核酸結合蛋白扮演著重要的角色。過去實驗室發現酵母菌中一個核糖核酸結合蛋白Rbp1p可以促進粒腺體外膜孔蛋白porin訊息核糖核酸降解，同時，Rbp1p在環境壓力之下，譬如說葡萄糖剝奪、滲透壓力改變、或是熱刺激的時候，會坐落到細胞質中聚集的顆粒P-bodies。在這裡我們研究另一個與Rbp1p有相互作用的蛋白質：Psp1p。Psp1p轉譯出一個有著八百四十一個胺基酸的蛋白質，上面有三個富含天冬醯胺(N)區域，一個富含麩胺醯胺(Q)區域，以及一個Psp1p保留區域。我們發現Psp1p大量表現的時候抑制細胞生長，也會抑制轉譯作用的進行。酵母菌雙雜合實驗發現Psp1p能與P-bodies的成員之一，Dhh1p有交互作用，並且Psp1p大量表現時抑制細胞生長的現象在dhh1突變菌株會消失，而抑制轉錄功能能力不受影響。另外，酵母菌雙雜合實驗指出Rbp1p利用碳端NMP區域與Psp1p的55-216個胺基酸區域相互作用。雖然大量表現Rbp1p與Psp1p都會抑制生長，但這個現象並不需要依賴對方達成。我們也發現Psp1p在葡萄糖剝奪和熱休克下會形成細胞質聚集顆粒，與Rbp1p聚集的顆粒有相同的分佈，且可能扮演著幫助Rbp1p在葡萄糖剝奪的時候聚集到P-bodies的角色。除此之外，我們也發現RBP1和PSP1在維持酵母菌細胞壁的功能上有交互作用，且RBP1在細胞壁維持的功能上可能在PSP1的上游。

In eukaryote cells, messenger RNA (mRNA) processing involves post-transcriptional modification process, 5’-capping, splicing, and 3’ polyadenylation. mRNA binding proteins play important roles in these processes. Previously, we found that a yeast RNA-binding protein, Rbp1p, enhances degradation of mitochondria outer membrane porin mRNA. Under environmental stress conditions, such as glucose deprivation, osmotic stress and heat shock, Rbp1p localizes into cytoplasmic granules, referred to P-bodies. In this thesis, we focus on an interacting protein of Rbp1p, Psp1p. Our current knowledge of Psp1p in Saccharomyces cerevisiae is limit. Structurally, Psp1p comprises three asparagines-rich regions, a glutamine-rich region, and a Psp1-conserved region in its C-terminal. In this work, we found that overexpression of Psp1p not only caused growth inhibition, but also repressed translation. Two-hybrid analysis indicated that Psp1p interacts with a P-body component, the decapping activator Dhh1p. In dhh1△, Psp1p lost its growth inhibition ability, but translation repression property still remained. Moreover, by two-hybrid analysis showed that the C-terminal NMP region of Rbp1p interacted to amino acid 55-216 of Psp1p. Psp1p translocated into cytoplasmic compartments upon glucose deprivation and heat shock, and co-localized with Rbp1p. Psp1p may plays a role in recruitment of Rbp1p recruited to P-bodies under glucose deficient conditions. Finally, Psp1p and Rbp1p may play a role in cell wall maintenance, and Rbp1p probably acted upstream of Psp1p in regulating cell wall integrity.