探討酵母菌核醣核酸蛋白Rbp1p及粒線體膜孔蛋白porin 之調控

Abstract

真核細胞中，訊息核醣核酸在調控基因表現中扮演重要的角色。細胞中的訊息核醣核酸需與不同的核醣核酸結合蛋白結合才得以進行核醣核酸新陳代謝，包括訊息核醣核酸的修飾、運輸、降解、及轉譯成蛋白質的過程。過去實驗室發現酵母菌中結合蛋白Rbp1p為一生長調控因子。目前已知在細胞中大量表現Rbp1p會抑制酵母菌的生長。Rbp1p也能夠促進粒線體外膜孔蛋白porin訊息核醣核酸之降解。我們發現當細胞缺少Rbp1p時會增加細胞生長速率，然而在野生型或缺少Rbp1p的突變株中大量表現Rbp1p蛋白質則會相似的減緩生長速率。因此，我們猜測Rbp1p可能有自我調節表現的機制。進一步的，利用報導者分析發現報導者接上RBP1訊息核醣核酸的3'非轉譯區後表現量會受到Rbp1p的抑制。我們找到RBP1訊息核醣核酸的3'非轉譯區中含有一些保守序列可能參與Rbp1p的自我調控機制。同時，我們證實Rbp1p的自我調控機制需要其蛋白質上的三個訊息核醣核酸結合區。當細胞缺少參與訊息核醣核酸脫帽反應的蛋白質Dhh1p或是Lsm1p-7p-Pat1複合物時，Rbp1p無法自我調控表現。 在酵母菌雙雜合系統中觀察到Rbp1和porin的結合，我們假設這兩者的結合可能調控由Rb1p1所調節的porin訊息核醣核酸降解。然而，實驗結果顯示無論在野生型或是缺少RBP1的突變株中大量表現外生性porin都會抑制內生性porin訊息核醣核酸的表現量。Rbp1p和porin結合的功能仍需未來深入研究。

Regulation of eukaryotic messenger RNAs (mRNAs) is an important factor in controlling gene expression. Cellular mRNAs associate to RNA binding proteins to go through RNA metabolism, including processing, export, turnover, localization, and translation of mRNA. Rbp1p, an RNA-binding protein, was previously characterized as a negative growth regulator in Saccharomyces cerevisiae. Overexpression of Rbp1p, but not the N-terminally truncated form, decreases the porin mRNA expression by enhancing degradation. In vitro binding assay indicates Rbp1p binds the (C/G)U-rich element of the porin mRNA 3’-untranslated region (3’UTR). Our lab concluded that Rbp1p is involved in the post-transcriptional regulation of porin mRNA. Plating result showed the growth rate is increased in yeast lacking Rbp1p; however, the growth rate of yeast was roughly the same whether expression of exogenous Rbp1p in wild-type or rbpl△ yeast. We suspected that Rbp1p might regulate its own expression. We showed that overexpression of exogenous Rbp1p decreased endogenous Rbp1p mRNA and protein level. Using reporter assay, we demonstrated that Rbp1p decreased the expression of reporter carrying 3’UTR of RBP1. We further determined the length of RBP1 mRNA 3’UTR and searched for several conserved regions, including putative AU-rich element and polyuridine-rich sequences were located in RBP1 mRNA 3’UTR. We demonstrated that RNA regulate its own expression. We showed that overexpression of exogenous Rbp1p recognition ability of Rbp1p is needed for its autoregulation. Moreover, our results indicated autoregulation of Rbp1p depends on decapping factors, Dhh1p and Lsm1p-7p-Pat1 complex. Theis observation suggests autoregulation of Rbp1p through 5’ to 3’ mRNA decay mechanism. Previous yeast two-hybrid result in our lab showed the Rbp1p interacts with porin. We speculated that the interaction between porin and Rbp1p might involve in Rbp1-mediated porin mRNA degradation. However, we observed that amount of porin was very similar after epichromosomal (gene in a 2μ plasmid controlled by an ADH1 promoter), and chromosomal expression (endogenous gene controlled by the endogenous promoter). On the other hand, we showed exogenous porin decrease endogenous POR1 mRNA both in wild-type and rbp1