Characterization of post-translational modification and protein-protein interaction of RNA-binding protein 1, Rbp1p, in Saccharomyces cerevisiae
Date Issued
2007
Date
2007
Author(s)
Fang, Chiung-Ying
DOI
en-US
Abstract
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.
Subjects
酵母菌
核醣核酸蛋白
後轉譯修釋作用
蛋白交互作用
Saccharomyces cerevisiae
RNA-binding protein
post-translational modification
protein-protein interaction
Type
other
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