Yeast genes in RNA processing and nucleus/cytoplasm exchange

描述（申请人提供）：本研究项目主要研究转移RNA生物合成和亚细胞运输。除了在蛋白质合成中发挥重要作用外，trna还参与营养信号传导、细胞凋亡调节、蛋白质降解和启动逆转录病毒反转录。虽然几十年来人们一直认为tRNA的运动是单向的，从细胞核到细胞质，但我们和其他人发现tRNA在细胞核和细胞质之间是双向运动的，并且这种动态在酵母和脊椎动物细胞之间是保守的。tRNA动力学包括三个步骤：tRNA从细胞核“初始输出”到细胞质，tRNA从细胞核“逆行”输入到细胞质，tRNA输入“再输出”到细胞质。trna在细胞核和细胞质之间转运的机制尚不完全清楚，但它们至少需要2输入蛋白家族的3个成员。然而，一个主要的参与者仍然需要确定，因为如果唯一已知的含内含子trna的核输出缺失，细胞是健康的。另一方面，有一些蛋白质以未知的机制影响核的进口和再出口步骤。因此，Aim 1旨在发现缺失的基因产物，并了解已知蛋白质如何在tRNA亚细胞动力学中起作用。tRNA双向转运的生物学作用仍然未知；然而，我们发现trna在细胞核受损或细胞缺乏营养时积累。我们提出，在细胞核中发生的许多tRNA加工步骤和tRNA核输出之间存在竞争，有时导致异常tRNA错误地传递到细胞质，tRNA逆行过程可能已经进化，部分是为了纠正这些错误。由于当细胞严重缺乏营养时，胞质trna在细胞核中积累，我们提出逆行过程在响应营养可用性时调节蛋白质合成方面具有额外的功能。因此，Aim 2探索tRNA亚细胞动力学是否在tRNA质量控制和/或翻译调控中起作用。除了将tRNA传递到正确的亚细胞目的地的蛋白质外，tRNA的生物发生还需要一组复杂的约80个独立的基因产物来进行转录后处理。大多数加工活动在真核生物中是保守的。我们的数据表明，保守的酵母前trna剪接内切酶具有额外的重要的新细胞质功能，Aim 3探索了可能的作用。酵母酶的新作用的发现可能有助于解释人类tRNA剪接酶的改变如何导致致命的脊柱肌肉疾病而不引起tRNA前剪接缺陷。因此，拟议的研究计划影响基因表达，对环境信号的反应以及对人类健康重要的问题的多个方面。

DESCRIPTION (provided by applicant): This research program focuses on transfer RNA biosynthesis and subcellular trafficking. In addition to their essential role in protein synthesis, tRNAs are required for nutrient signaling, regulation of apoptosis, protein degradation, and priming retroviral reverse transcription. Although for decades it was thought that tRNA movement is unidirectional, nucleus to cytoplasm, we and others discovered that tRNAs move bi-directionally between the nucleus and the cytoplasm and that the dynamics are conserved between yeast and vertebrate cells. tRNA dynamics is comprised of 3 steps: "initial export" of tRNA from the nucleus to the cytoplasm, "retrograde" nuclear import of cytoplasmic tRNAs, and "re-export" of the imported tRNAs back to the cytoplasm. The mechanisms by which tRNAs transit between the nucleus and the cytoplasm are not completely understood but they require at least 3 members of the 2-importin family. However, a major player(s) still needs to be identified since cells are healthy if the only known nuclear exporter for intron-containing tRNAs is absent. On the other hand, there are proteins that affect the nuclear import and re-export steps by unknown mechanisms. Thus, Aim 1 seeks to discover missing gene products and to learn how the known proteins function in tRNA subcellular dynamics. The biological role(s) of tRNA bi-directional trafficking remains unknown; however, we showed that tRNAs accumulate in nuclei when damaged or when cells are deprived of nutrients. We propose that there is competition between the numerous tRNA processing steps that occur in the nucleus and tRNA nuclear export, sometimes resulting in erroneous delivery of aberrant tRNAs to the cytoplasm and that the tRNA retrograde process may have evolved, in part, to correct such errors. Since cytoplasmic tRNAs accumulate in the nucleus when cells are acutely deprived of nutrients, we propose that the retrograde process serves an additional function to regulate protein synthesis in response to nutrient availability. Thus, Aim 2 explores whether tRNA subcellular dynamics serve roles in tRNA quality control and/or regulation of translation. In addition to the proteins that deliver tRNAs to their correct subcellular destinations, tRNA biogenesis requires a complex set of ~80 independent gene products for post- transcriptional processing. Most of the processing activities are conserved throughout eukaryotes. Our data indicate that the conserved yeast pre-tRNA splicing endonuclease serves an additional essential novel cytoplasmic function(s) and Aim 3 probes the putative role(s). Discovery of a novel role(s) of the yeast enzyme may help explain how alterations of human tRNA splicing enzyme cause a fatal spinal muscular disease without causing defects in pre-tRNA splicing. Thus, the proposed research program impacts upon multiple facets of gene expression, response to environmental signals, and issues important to human health.