tRNA processing and nuclear-cytoplasmic dynamics

项目摘要 这项研究的重点是tRNA的生物合成及其亚细胞转运。除了他们的 在蛋白质合成中起重要作用，tRNAs是营养信号、细胞凋亡调节、蛋白质所必需的 降解，并启动逆转录病毒的逆转录。TRNA的生物发生需要一套复杂的保守的 用于转录后加工和亚细胞交通的基因产品。尽管几十年来人们一直认为 TRNA运动是单向的，从细胞核到细胞质，我们共同发现tRNA运动是双向的。 在细胞核和细胞质之间的方向上，并且在酵母和 脊椎动物细胞。TRNA动力学包括3个步骤：tRNA从细胞核到核内的“初级输出” 细胞质，“逆行核进口”的细胞质tRNA进入细胞核，并“再出口”的进口 TRNA回到细胞质。TRNAs在细胞核和细胞质之间移动的机制 还没有完全被理解。因为tRNA核出口是必不可少的，而已知的出口商是 不必要的是，我们在酵母中进行了无偏见的全基因组筛查，以寻找缺失的tRNA核 出口商(S)。我们发现蛋白质(CRM1)和信使核糖核酸(Mex67-MTR2)两条途径的突变 核出口也会导致有缺陷的tRNA核出口；数据支持CRM1和Mex67- Mtr2在tRNA核输出中发挥作用。目的1使用体内生化分析来检验这一假设 TRNA直接与CRM1和/或Mex67-MTR2核出口机制相互作用，并了解这些 另一种核出口途径识别tRNA底物。AIM 1还试图测试候选人是否 TRNA核进口体与tRNA发生物理相互作用。目的2阐明tRNA bi-tRNA的生物学功能。 胞核和胞质之间的定向运输。我们发现其中一个功能是tRNA质量 防止异常tRNA与蛋白质合成机制相互作用的控制。异常tRNAs 到达细胞质，部分原因是容易出错的核出口。我们将分析平行tRNA的保真度 并确定异常的tRNA是否被纠正和/或在 逆行传入核内。AIM 3介绍了发生在线粒体上的RNA处理步骤 薄膜。酵母中的前tRNA剪接和后生动物中的piRNA加工发生在线粒体上 浮出水面。我们确定了可能在引导tRNA剪接内切酶和/或tRNAs到 线粒体，我们建议测试这些作用。所获得的信息应告知如何以及为什么 线粒体表面是RNA加工的“仓库”。因此，拟议的研究方案 对基因表达、质量控制和对人类健康至关重要的问题的多个方面的影响。

Project Summary This research program focuses on tRNA biosynthesis and its 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. tRNA biogenesis requires a complex set of conserved gene products for post-transcriptional processing and subcellular traffic. Although for decades it was thought that tRNA movement is unidirectional, nucleus to cytoplasm, we co-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 consist of 3 steps: “primary export” of tRNA from the nucleus to the cytoplasm, “retrograde nuclear import” of cytoplasmic tRNA into the nucleus, and “re-export” of the imported tRNAs back to the cytoplasm. The mechanisms by which tRNAs move between the nucleus and the cytoplasm are not completely understood. Because tRNA nuclear export is essential and the known exporters are unessential, we conducted an unbiased genome-wide screen in yeast to search for the missing tRNA nuclear exporter(s). We discovered that mutations of two pathways utilized for protein (Crm1) and mRNA (Mex67-Mtr2) nuclear export also cause defective tRNA nuclear export; the data support the model that Crm1 and Mex67- Mtr2 function in tRNA nuclear export. Aim 1 employs in vivo biochemical analyses to test the hypothesis that tRNAs directly interact with the Crm1 and/or Mex67-Mtr2 nuclear export machinery and to learn how these alternative nuclear export pathways recognize tRNA substrates. Aim 1 also seeks to test whether a candidate tRNA nuclear importer physically interacts with tRNA. Aim 2 addresses the biological function for tRNA bi- directional traffic between the nucleus and cytoplasm. We discovered that one function is for tRNA quality control that prevents aberrant tRNA from interacting with the protein synthesis machinery. Aberrant tRNAs reach the cytoplasm, in part, due to error-prone nuclear export. We will analyze the fidelity of the parallel tRNA nuclear export pathways and determine whether aberrant tRNAs are corrected and/or destroyed upon retrograde import into the nucleus. Aim 3 addresses RNA processing steps that occur on the mitochondrial membrane. Pre-tRNA splicing in yeast and piRNA processing in metazoans occur on the mitochondrial surface. We identified proteins that likely function in directing the tRNA splicing endonuclease and/or tRNAs to mitochondria and we propose to test these roles. The information gained should inform how and why the mitochondrial surface functions as a “warehouse” for RNA processing. Thus, the proposed research program impacts upon multiple facets of gene expression, quality control, and issues important to human health.