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

酿酒酵母中参与核 mRNP 组装的蛋白质中 RNA 结合位点的鉴定和功能分析

• 批准号: 313454814
• 负责人: Professorin Dr. Katja Sträßer
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/313454814?language=en
• 关键词: Identification; functional; analysis; RNA; binding

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.

基因表达的“赫尔墨斯”是信使RNA (mRNA)，它将蛋白质编码基因的信息传递给核糖体。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（rbp）不仅在其结合时起作用，而且经常影响基因表达的后期步骤。因此，mRNP在细胞核中的组装过程是必不可少的，同时也非常复杂。在这里提出的项目中，我们结合质谱和生化方法来揭示核mRNP组装过程和所涉及的rbp的功能。在过去的三年中，我们鉴定了rna结合位点，开发了鉴定蛋白质- rna交联的新技术，并对第一个核RBP Npl3的rna结合突变体进行了功能表征。我们将继续通过“经典”光交联和化学交联结合高分辨率质谱法系统地鉴定葡萄球菌这些rbp中的rna结合位点。利用由此确定的rna结合位点的突变体，我们将评估每个RBP在mRNP组装、核mRNA输出和mRNA稳定性方面的功能。我们期望这些结果将阐明核mRNP组装过程以及mRNP组成对细胞中mRNA生命周期的影响。