Yeast genes in RNA processing and nucleus/cytoplasm exchange

描述(申请者提供)：该研究项目的重点是转移RNA的生物合成和亚细胞运输。除了在蛋白质合成中的重要作用外，tRNAs还需要营养信号、调节细胞凋亡、蛋白质降解和启动逆转录病毒的逆转录。尽管几十年来一直认为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双向运动，而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3个步骤：tRNA从细胞核到细胞质的“初始输出”，胞质tRNA的“逆行”核输入，以及输入的tRNA回到细胞质的“再输出”。TRNAs在细胞核和细胞质之间传递的机制尚不完全清楚，但它们至少需要2-Importin家族的3个成员。然而，一个主要的参与者(S)仍然需要确定，因为如果唯一已知的含有内含子的tRNA的核输出子缺失，细胞是健康的。另一方面，还有一些蛋白质通过未知的机制影响核的进口和再出口步骤。因此，目标1试图发现缺失的基因产物，并了解已知的蛋白质如何在tRNA亚细胞动力学中发挥作用。TRNA双向运输的生物学作用尚不清楚(S)；然而，我们证明了当tRNA受损或细胞缺乏营养时，tRNA在细胞核中积累。我们认为，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，有时会导致异常tRNA错误地输送到细胞质，并且tRNA逆行过程可能已经进化，部分地是为了纠正这些错误。由于当细胞严重缺乏营养物质时，细胞质tRNA在细胞核中积累，我们认为逆行过程具有调节蛋白质合成的额外功能，以响应营养的可获得性。因此，目标2探索tRNA亚细胞动力学是否在tRNA质量控制和/或翻译调节中发挥作用。除了将tRNA运送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套复杂的~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Pre-tRNA剪接内切酶具有额外的重要的新的细胞质功能(S)，而Aim 3探针可能具有这种作用(S)。酵母酶的一个新作用的发现(S)可能有助于解释人类tRNA剪接酶的变化如何在不导致tRNA前剪接缺陷的情况下导致致命的脊椎肌肉疾病。因此，拟议的研究计划影响到基因表达的多个方面，对环境信号的反应，以及对人类健康重要的问题。

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.