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

基因表达的爱马仕是信使RNA（mRNA），它将蛋白质编码基因的信息携带到核糖体。在核mRNP生物发生过程中，许多蛋白质与mRNA结合，将其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（RBP）不仅在其结合时起作用，而且通常还影响基因表达的后续步骤。因此，mRNP在细胞核中的组装过程是必不可少的，同时也是令人生畏的复杂性。在这里提出的项目中，我们结合联合收割机质谱和生物化学方法来解开核mRNP组装过程和所涉及的RBP的功能。在过去的三年中，我们确定了RNA结合位点，开发了用于鉴定蛋白质-RNA交联的新技术，并功能性地表征了第一个核RBP Np 13的RNA结合突变体。我们将继续系统地鉴定S.通过“经典的”光交联和另外通过化学交联结合高分辨率质谱法来鉴定酿酒酵母。使用突变体的RNA结合位点，从而确定，我们将评估每个RBP的功能mRNP组装，核mRNA的出口和mRNA的稳定性。我们希望这些结果将阐明核mRNP组装的过程和mRNP组成对细胞中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.