Identification and functional analysis of RNA-binding sites in proteins involved in nuclear mRNP assembly in S. cerevisiae

基因表达的Hermes是信使RNA，它将编码蛋白质的基因的信息携带到核糖体中。在核mRNP生物发生过程中，许多蛋白质与mRNA结合，加工并包装成信使核糖核蛋白颗粒(MRNP)。MRNP的这些RNA结合蛋白(RBPs)不仅在结合时发挥作用，而且经常影响基因表达的后期步骤。因此，mRNP在细胞核内的组装过程是必不可少的，同时也是令人望而生畏的复杂过程。在这里提出的项目中，我们结合质谱学和生化方法来揭示核内mRNP的组装过程和所涉及的限制性商业惯例的功能。在过去的三年中，我们确定了RNA结合位点，开发了鉴定蛋白质-RNA交联链的新技术，并对第一个核RBP NPL3的RNA结合突变体进行了功能鉴定。我们将继续通过“经典”光交联法和化学交联法结合高分辨质谱学，系统地鉴定酿酒酵母限制性商业惯例中的RNA结合位点。利用所确定的RNA结合位点上的突变，我们将评估每个RBP在mRNP组装、核mRNA输出和mRNA稳定性中的功能。我们期望这一结果将阐明核内mRNP的组装过程以及mRNP的组成对细胞内mRNA生命周期的影响。

The Hermes of gene expression is the messenger RNA (mRNA), which carries the information of a protein-coding gene to the ribosome. During nuclear mRNP biogenesis many proteins bind to the mRNA, processing and packaging it into a messenger ribonucleoprotein particle (mRNP). These RNA-binding proteins (RBPs) of the mRNP not only function at the time of their binding, but often also affect later steps of gene expression. Consequently, the process of mRNP assembly in the nucleus is essential and at the same time dauntingly complex.In the project proposed here, we combine mass-spectrometric and biochemical approaches to unravel the process of nuclear mRNP assembly and the functions of the RBPs involved. During the last three years we identified RNA-binding sites, developed new techniques for the identification of protein-RNA cross-links and functionally characterized RNA-binding mutants of the first nuclear RBP, Npl3. We will continue to systematically identify the RNA-binding sites in these RBPs in S. cerevisiae by “classical” photo-cross-linking and additionally by chemical cross-linking combined with high-resolution mass spectrometry. Using mutants in the RNA-binding sites thus identified, we will assess each RBP's function in mRNP assembly, nuclear mRNA export and mRNA stability. We expect that the results will elucidate the process of nuclear mRNP assembly and the influence of mRNP composition on the life cycle of mRNA in the cell.