tRNA processing and nuclear-cytoplasmic dynamics

tRNA 加工和核质动力学

• 批准号: 9284086
• 负责人: Anita K Hopper
• 金额: $ 32.52万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9284086
• 关键词: Actins; Address; Anabolism; Apoptosis; Area; Back; Biochemical; Biogenesis; Biological; Biological Process; Biology; Cell Nucleus; Cells; Complex; Cytoplasm; Cytoplasmic Protein; Cytoskeleton; Data; Defect; Disease; Event; Exportins; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Transcription; Genetic study; Genome; Genomics; Health; Human; Learning; Malignant Neoplasms; Mediating; Membrane Proteins; Messenger RNA; Metabolic Diseases; Methodology; Mitochondria; Modeling; Movement; Mutation; Myosin Type V; Neuromuscular Diseases; Nuclear; Nuclear Export; Nuclear Import; Nuclear Protein; Nutrient; Organelles; Organism; Outer Mitochondrial Membrane; Pathway interactions; Premature Birth; Process; Protein Biosynthesis; Proteins; Proteome; Quality Control; RNA; RNA Processing; RNA Splicing; Regulation; Research; Research Project Summaries; Reverse Transcription; Role; Saccharomycetales; Signal Transduction; Site; Stress; Surface; Testing; Transfer RNA; Yeast Model System; Yeasts; endonuclease; experimental study; gene product; genome-wide analysis; in vivo; mitochondrial membrane; novel; nuclease; piRNA; prevent; programs; protein degradation; repaired; response; tRNA Precursor; tool; trafficking

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.

项目摘要 该研究计划的重点是tRNA的生物合成及其亚细胞运输。除了它们 在蛋白质合成中起着重要作用，tRNA是营养信号传导、细胞凋亡调节、蛋白质合成和蛋白质合成所必需的。 降解和引发逆转录病毒逆转录。tRNA的生物合成需要一套复杂的保守的 用于转录后加工和亚细胞运输的基因产物。尽管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，我们共同发现，tRNA的双向运动， 在细胞核和细胞质之间的方向和动力学是保守的酵母和 脊椎动物细胞tRNA动力学包括3个步骤：tRNA从细胞核“初级输出”到细胞核， 细胞质中，细胞质tRNA的“逆行核输入”到细胞核中，以及输入的tRNA的“再输出”。 转运RNA回到细胞质。tRNA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核输出是必不可少的，已知的输出者是 不必要的是，我们在酵母中进行了公正的全基因组筛选，以寻找缺失的tRNA核 出口商。我们发现蛋白质（Crm 1）和mRNA（Mex 67-Mtr 2）两条途径的突变， 核输出也会导致有缺陷的tRNA核输出;数据支持Crm 1和Mex 67- Mtr 2在tRNA核输出中的功能。目的1采用体内生物化学分析来检验以下假设： tRNA直接与Crm 1和/或Mex 67-Mtr 2核输出机制相互作用， 替代的核输出途径识别tRNA底物。目标1还试图测试候选人是否 tRNA核输入子与tRNA发生物理相互作用。目的2阐明tRNA双链的生物学功能， 细胞核和细胞质之间的定向运输。我们发现一个功能是tRNA的质量 防止异常tRNA与蛋白质合成机器相互作用的控制。异常tRNA 到达细胞质，部分是由于容易出错的核输出。我们将分析平行tRNA的保真度， 核输出途径，并确定异常的tRNA是否被纠正和/或破坏后， 逆行输入细胞核。目标3解决线粒体上发生的RNA加工步骤 膜的酵母中的pre-tRNA剪接和后生动物中的皮尔纳加工发生在线粒体上 面我们鉴定了可能在指导tRNA剪接核酸内切酶和/或tRNA 线粒体，我们建议测试这些角色。所获得的信息应能说明如何以及为什么 线粒体表面充当RNA加工的“仓库”。因此，拟议的研究计划 对基因表达、质量控制和对人类健康重要的问题的多个方面的影响。