探討酵母菌核醣核酸蛋白Rbp1p面對環境壓力之後轉譯修飾

Abstract

在真核細胞中，核醣核酸結合蛋白調控著信使核醣核酸(mRNA)的轉錄和轉錄後事件(post-transcriptional events)，例如剪接(splicing)、編輯(editing)、運輸出核(export)、穩定性(stability)、位置(localization)和轉譯(translation)。之前實驗室發現酵母菌核醣核酸結合蛋白Rbp1p，分布在細胞質中，為具有抑制酵母菌生長的負調控因子。此外，在葡萄糖剝奪(glucose depletion)、熱壓力(heat shock)等環境壓力下，Rbp1p分別會座落到細胞質中的細胞質處理小體(P-bodies)及壓力體(stress granules; SGs)。利用二維電泳分析，發現在這兩種環境壓力下Rbp1p等電點圖譜皆有往酸性端移動之現象。推測在處在環境壓力下Rbp1p含有不同的轉譯後修飾。藉由質譜儀分析Rbp1p轉譯後修飾，發現了八個磷酸化的位置，並且利用定點突變(site-directed mutagenesis)的技術將它們突變為丙氨酸，觀察到這些突變型的Rbp1p不會影響抑制酵母菌生長的能力。另外，也發現在環境壓力下，這些突變型的Rbp1p依然可以座落到細胞質處理小體。同時在酵母菌雙雜交實驗發現S646A和S649A突變型的Rbp1p和野生型的Rbp1p結合的能力會減弱。藉著共同免疫沉澱實驗，我們發現了Ssa2p為Rbp1p的結合蛋白，並且在熱壓力的環境下結合的能力會下降。此外，在ssa2突變株中大量表現Rbp1p，Rbp1p抑制酵母菌生長的能力增強了。

In eukaryotic cells, RNA binding proteins regulate mRNA transcription and post-transcriptional events, such as splicing, editing, export, stability, localization, and translation. Our laboratory has demonstrated that RNA binding protein1, Rbp1p, appears punctate in cytoplasm and as a negative growth regulator. In addition, Rbp1p localizes to P-bodies and stress granules in cytoplasm upon glucose deprivation and heat shock, respectively. Using two-dimensional electrophoresis analysis, the iso-electric point pattern of Rbp1p shows spots toward acidic pore after glucose deprivation and heat shock treating. The results imply that Rbp1p contains different post-translational modifications in stress conditions. By mass spectrometry analysis, we identify eight phosphorylation sites of Rbp1p (Ser459, Ser462, Ser463, Ser524, Ser526, Thr637, Ser646, and Ser649), and using site-directed mutagenesis approach to substitute alanine for serine/threonine. S646A and S649A mutants of Rbp1p decrease the ability of interaction with Rbp1p by yeast two-hybrid assay. Under glucose deprivation, however, phosphorylation mutants of Rbp1p can still localize to P-bodies, and these mutants cannot affect ability of growth inhibition. In co-immunoprecipitation experiments, I identify that Ssa2p is one of associated proteins with Rbp1p, and the interaction ability diminishes at heat shock. Besides, overexpression of Rbp1p in ssa2Δ strain enhances the growth-inhibition ability. It implies that Ssa2p can inhibit Rbp1p negative regulator growth.