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  • 學位論文

探討酵母菌核醣核酸蛋白Rbp1p之後轉譯修飾及蛋白交互作用

Characterization of post-translational modification and protein-protein interaction of RNA-binding protein 1, Rbp1p, in Saccharomyces cerevisiae

指導教授 : 李芳仁
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摘要


真核細胞中,基因表現在訊息核醣核酸轉錄後必須經過一連串的調控,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。調控的步驟中包含了許多核醣核酸結合蛋白和核醣核酸共同組成不同的複合體,稱為核醣核蛋白。過去實驗室發現酵母菌中一個包含三個核醣核酸識別基序RRM及兩個富含麩氨酸區域的核醣核酸結合蛋白Rbp1p。目前已經知道在細胞內大量表現Rbp1p會抑制細胞生長,Rbp1p也能夠促進粒腺體外膜孔蛋白porin訊息核醣核酸降解。Rbp1p在Δxrn1的細胞突變株中,會坐落到細胞質中特殊的細胞質聚集顆粒,稱做P-bodies;在野生型細胞株中,經由葡萄糖剝奪、滲透壓力和生長階段晚期的壓力之下,Rbp1p也會坐落在P-bodies的位置,這些位置的改變暗示著Rbp1p會被外界的訊息所調控。在這篇論文中我們證明了在細胞中Rbp1p是一個磷酸化蛋白。我們以單點突變的方式將Rbp1p五個被推測可能會被磷酸化的位置突變為丙胺酸。實驗結果顯示這些僅包含一個單點磷酸化位置突變的Rbp1p仍然具有其負調控細胞生長的功能,但若將五個磷酸化位置突變則可產生功能喪失的Rbp1p。另外我們也研究了Rbp1p可能的激酶Ksp1p及Ykl171w和Rbp1p之間的關連性,我們發現在Δrbp1Δksp1的突變株中大量表現Rbp1p並不會抑制細胞生長,暗示缺乏Ksp1p的情況下Rbp1p可能喪失部分的功能。此外,於論文中也界定了Rbp1p的三個交互作用蛋白Rbp1p本身、核酸解螺旋酵素Dhh1p、及粒線體外膜蛋白Por1p的最小交互作用區域。酵母菌雙雜交實驗顯示,Rbp1p是利用其碳端和交互作用蛋白進行交互作用,而Dhh1p是利用碳端富含麩氨酸區域和Rbp1p進行交互作用。我們也發現無法坐落到P-bodies的Rbp1p突變種也無法和野生型Rbp1p進行交互作用,更進一步確認Rbp1p的自我結合對於坐落到P-bodies是重要的。

並列摘要


Posttranscriptional control of eukaryotic gene expression is comprised of several levels of regulation including processing, export, turnover, localization, and translation of mRNAs. Each regulation step involves various combinations of RNA-binding proteins that form dynamic messenger ribonucleoproteins with the transcript. In Saccharomyces cerevisiae, the 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. Our lab has demonstrated that Rbp1p decreases the porin mRNA level by enhancing degradation. In a Δxrn1 strain, Rbp1p localized to a specific cytoplasmic foci known as a P-body. In wild-type cells, Rbp1p localized to P-bodies under glucose deprivation or treatment with KCl, suggesting that outside signaling regulates Rbp1p localization. Here we showed that Rbp1p is a phosphorylated protein in vivo. We generated several putative phosphorylation mutants by site-direct mutagenesis and characterized these mutants by functional analysis. Although all single phosphorylated mutations of Rbp1p are functional, combinations of several mutations can generate a loss-of-function mutant. Ptacek et al. used large-scale screening of kinase-substrate interaction and found two putative kinases of Rbp1p, Ksp1p and Ykl171wp (Ptacek et al., 2005). We demonstrated that overexpression of Rbp1p, but not N-terminal-truncated mutant, resulted in slow growth of the Δrbp1 strain; however, this phenomenon disappeared in the Δrbp1Δksp1 strain, suggesting that Rbp1p loss part of its function in absence of Ksp1p. The 2-D electrophoresis further confirmed that protein pattern of Rbp1p in Δrbp1Δksp1 strain shifts to lower pI compared with in the Δrbp1 strain. In the second part of this thesis, we mapped the interaction region of Rbp1p with three interaction proteins: Rbp1p itself, a DEAD-box helicase, Dhh1p, and a mitochondrial pore protein, Por1p. We found that all three interactions required the C-terminus of Rbp1p. We also found Rbp1p mutants which could not localize to P-bodies and could not interact with wild-type Rbp1p; further confirming that the oligomerization of Rbp1p is required for P-body localization.

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