BIOCHEMICAL AND GENETIC CHARACTERIZATION OF YRA1P IN BUDDING YEAST

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Yra1p及其脊椎动物同源物与mRNA输出因子Mex67p/TAP结合，并被认为在体内mRNA输出中发挥作用。为了进一步表征Yra1p，我们使用免疫亲和层析纯化内源性Yra1p复合物。这些实验表明，两个输入β同源物（Kap123p和Pse1p）和聚A尾部结合蛋白Pab1p和Nab2p与Yra1p相关。与Yra1p相关的其他主要蛋白质包括参与mRNA和rRNA加工的蛋白质以及与Yra1p相关的蛋白质Yra2p。进一步的生化和遗传学实验表明，Yra1p和Yra2p之间存在密切的功能关系。我们生成了一个YRA1的温度敏感等位基因，并用它来证明缺乏Yra1p和Yra2p功能的细胞能够退出G0阻滞，并在阻滞前经历几轮细胞分裂。我们还发现了yra1-2温度敏感生长缺陷的高拷贝抑制子。其中包括SUB2，一个在mRNA输出中重要的剪接因子，ULP1，一个定位于核孔并参与Smt3p/SUMO加工的核半胱氨酸蛋白酶，以及YRA2。综上所述，这些结果表明Yra1p除了在mRNA输出中起作用外，还在多种RNA加工事件中发挥作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Yra1p and its vertebrate homologues bind to the mRNA export factor Mex67p/TAP and are thought to play a role in mRNA export in vivo. To further characterize Yra1p, we used immunoaffinity chromatography to purify endogenous Yra1p complexes. These experiments demonstrated that two importin beta homologues (Kap123p and Pse1p) and the poly A tail-binding proteins Pab1p and Nab2p associate with Yra1p. The other major proteins that associate with Yra1p include proteins involved in mRNA and rRNA processing and the Yra1p-related protein Yra2p. Additional biochemical and genetic experiments suggest a close functional relationship between Yra1p and Yra2p. We generated a temperature-sensitive allele of YRA1 and used it to demonstrate that cells which lack the function of both Yra1p and Yra2p are able to exit a G0 arrest and go through several rounds of cell division before arresting. We also identified high-copy suppressors of the yra1-2 temperature-sensitive growth defect. These include SUB2, a splicing factor important in mRNA export, ULP1, a nuclear cysteine protease localized to the nuclear pore and involved in Smt3p/SUMO processing, and YRA2. Taken together, these results suggest that Yra1p has roles in diverse RNA processing events in addition to a role in mRNA export.