Temporal Dynamics of mRNP Granules in Relation to Cellular Metabolic State

描述(申请人提供)：在压力下，真核细胞抑制翻译并聚集成细胞质mRNP颗粒，称为压力颗粒和p-小体。关于这些颗粒的功能意义，仍有许多未知之处。我建议利用酵母代谢周期(YMC)系统来研究这些mRNP颗粒，在YMC系统中，细胞的代谢状态与mRNA转录、翻译和代谢的同步变化精确协调。我的初步结果表明，mRNP颗粒在整个YMC中动态形成和分解。在此，我建议剖析mRNP颗粒在调节代谢循环所带来的mRNA水平变化中的作用。使用强大的生化、LC/MS/MS和基于RNA-SEQ的技术相结合，我将测定这些mRNP颗粒的RNA和蛋白质含量，并调查颗粒蛋白质的翻译后修饰是否与颗粒的形成或拆解有关。通过从基因上扰乱mRNP颗粒的形成，我将进一步确定它们如何在周期的每个阶段协调mRNA和蛋白质的表达。最后，我将研究mRNAs与mRNP颗粒的关联是否以及如何决定它们作为细胞代谢状态的函数进入和逃离活跃的翻译池。

DESCRIPTION (provided by applicant): Under stress, eukaryotic cells repress translation and aggregate mRNAs into cytoplasmic mRNP-granules termed stress-granules and p-bodies. Much remains unknown about the functional significance of these granules. I propose to investigate these mRNP-granules by utilizing the yeast metabolic cycle (YMC) system where the metabolic state of the cell is precisely coordinated with synchronous changes in mRNA transcription, translation, and metabolism. My preliminary results suggest that mRNP granules dynamically form and disassemble throughout the YMC. Herein, I propose to dissect the role of mRNP granules in mediating the changes to mRNA levels brought about by metabolic cycling. Using a combination of robust biochemical, LC/MS/MS, and RNA-seq based techniques, I will determine the RNA and the protein contents of these mRNP granules and investigate whether any 'signature' posttranslational modification of the granule proteins is associated with granule formation or disassembly. By genetically disrupting formation of mRNP granules, I will further determine how they coordinate mRNA and protein expression with each stage of the cycle. Lastly, I will investigate whether, and how, the association of mRNAs with mRNP granules dictates their entry into, and escape from, the active translation pool as a function of the metabolic state of the cell.