Yeast genes in RNA processing and nucleus/cytoplasm exchange

描述（由申请人提供）：该研究计划的重点是转移RNA生物合成和亚细胞运输。除了它们在蛋白质合成中的重要作用之外，tRNA还需要营养信号传导、细胞凋亡调节、蛋白质降解和引发逆转录病毒逆转录。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双向运动，并且这种动力学在酵母和脊椎动物细胞之间是保守的。tRNA动力学由3个步骤组成：tRNA从细胞核到细胞质的“初始输出”，细胞质tRNA的“逆行”核输入，以及输入的tRNA“再输出”回到细胞质。tRNA在细胞核和细胞质之间转运的机制还不完全清楚，但它们需要2-importin家族的至少3个成员。然而，仍然需要确定一个主要的参与者，因为如果缺少含有内含子的tRNA的唯一已知的核输出者，则细胞是健康的。另一方面，有一些蛋白质通过未知的机制影响核输入和再输出步骤。因此，目标1旨在发现缺失的基因产物，并了解已知蛋白质在tRNA亚细胞动力学中的功能。tRNA双向运输的生物学作用仍然未知;然而，我们发现，当细胞受损或缺乏营养时，tRNA会在细胞核中积累。我们认为，在细胞核中发生的众多tRNA加工步骤与tRNA核输出之间存在竞争，有时会导致异常tRNA错误地传递到细胞质中，并且tRNA逆行过程可能已经进化，部分地，以纠正这种错误。由于细胞质的tRNA积累在细胞核中时，细胞被急性剥夺的营养，我们提出，逆行过程提供了一个额外的功能，以调节蛋白质合成，以响应营养的可用性。因此，目的2探讨了tRNA亚细胞动力学是否在tRNA质量控制和/或翻译调控中发挥作用。除了将tRNA递送到其正确的亚细胞目的地的蛋白质之外，tRNA生物发生需要一组复杂的~80个独立的基因产物用于转录后加工。大多数加工活动在整个真核生物中是保守的。我们的数据表明，保守的酵母前tRNA剪接核酸内切酶提供了一个额外的必要的新的细胞质功能（S）和Aim 3探针推定的作用（S）。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髓肌肉疾病，而不会导致前tRNA剪接缺陷。因此，拟议的研究计划影响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.