Dynamics of Ribosome Biogenesis in Yeast

描述(由申请人提供)： 本研究计划旨在研究酵母中核糖体组装的动力学，以及核糖体生物发生与生长和细胞周期控制之间的关系。真核生物中核糖体的合成需要&gt；200组装因子来指导核糖体RNA的加工和&gt；100核糖体蛋白的有序组装。虽然rRNA加工的框架已经建立得很好，但对于核糖体蛋白的生物发生因子结合和释放或结合的动力学没有连贯的机制。此外，对核糖体生物发生程序的控制在两个关键方面知之甚少。首先，细胞质中核糖体蛋白的合成与核糖体在核中组装的动力学相协调的机制还知之甚少。其次，尽管有充分的证据表明核糖体生物发生缺陷与细胞生长和分裂缺陷之间存在关联，但通过监测核糖体生物发生来调节细胞周期的生长和进展的机制尚不清楚。在这项提案中，我们将应用质谱仪和酵母遗传分析对核糖体生物发生进行定量分析，以解决这两个关键问题。这将通过三个具体目标来实现：1)实施同位素脉冲定量质谱仪(IP-QMS)来监测酵母中核糖体的生物发生。2)通过定义核糖体蛋白的组装途径(S)，阐述了核糖体生物发生的机制。3)应用IP-QMS分析核糖体生物发生因子的作用，并将其放置在目标2中开发的组装路径图上，阐明调控核糖体生物发生与生长和细胞周期相协调的途径。这个建议的主要目的是结合酵母遗传学和QMS来询问核糖体生物发生中涉及核糖体蛋白和生物发生因子的中间体，并确定这些中间体与细胞生长和细胞周期调节之间的联系。

DESCRIPTION (provided by applicant): Project Summary/Abstract This proposal outlines a plan to study the dynamics of ribosome assembly in yeast, and the relationship between ribosome biogenesis and growth and cell cycle control. The synthesis of ribosomes in eukaryotes requires > 200 assembly factors to direct the ribosomal RNA processing and ordered assembly of >100 ribosomal proteins. While the framework for rRNA processing is well established, there is no coherent mechanism for the dynamics of biogenesis factor binding and release or association of the ribosomal proteins. Furthermore, the control of the ribosome biogenesis program is poorly understood in two key respects. First, the mechanism by which synthesis of the ribosomal proteins in the cytoplasm is coordinated with the dynamics for ribosome assembly in the nucleus is poorly understood. Second, although there is ample evidence for the association between defects in ribosome biogenesis and defects in cell growth and division, the mechanism by which ribosome biogenesis is monitored to regulate growth and progression through the cell cycle is unclear. In this proposal, we will apply quantitative analysis of ribosome biogenesis using mass spectrometry with yeast genetic analysis to address both of these key questions. This will be accomplished with three Specific Aims: 1) Implement Isotope Pulse-Quantitative Mass Spectrometry (IP-QMS) for monitoring ribosome biogenesis in yeast. 2) Elaborate the mechanism of ribosome biogenesis by defining the pathway(s) for assembly of ribosomal proteins. 3) To apply IP-QMS to analyze the roles of ribosome biogenesis factors and place them on the assembly pathway map developed in Aim 2 and to elucidate the pathways coordinating regulated ribosome biogenesis with growth and the cell cycle. The main goal of this proposal is to combine yeast genetics and QMS to interrogate the intermediates in ribosome biogenesis involving ribosomal proteins and biogenesis factors and to identify connections between those intermediates and the regulation of cell growth and the cell cycle.