tRNA processing and nuclear-cytoplasmic dynamics

tRNA 加工和核质动力学

• 批准号: 9920190
• 负责人: Anita K Hopper
• 金额: $ 32.76万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920190
• 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; Messenger RNA; Metabolic Diseases; Methodology; Mitochondria; Mitochondrial Membrane Protein; 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 screen; 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 该研究计划着重于tRNA生物合成及其亚细胞运输。除了他们 在蛋白质合成中的重要作用，营养信号传导，凋亡调节，蛋白质需要TRNA 降解和启动逆转录病毒逆转录。 tRNA生物发生需要一组复杂的保守 转录后处理和亚细胞流量的基因产品。虽然几十年来一直认为 tRNA运动是单向的，核向细胞质的核，我们共同发现，trnas移动bi- 在细胞核和细胞质之间方向，并且动力学在酵母和 脊椎动物细胞。 tRNA动力学由3个步骤组成：tRNA的“主要出口”从细胞核到 细胞质，细胞质tRNA的“逆行核进口”，进口的“重新出口” TRNA回到细胞质。 TRNA在细胞核和细胞质之间移动的机制 不完全理解。因为tRNA核导出是必不可少的，并且已知的出口商是 不必要的，我们在酵母中进行了无偏基因组筛查，以寻找缺失的tRNA核 出口商。我们发现用于蛋白质（CRM1）和mRNA的两种途径的突变（MEX67-MTR2） 核出口还会导致tRNA核出口缺陷；数据支持CRM1和MEX67-的模型 MTR2在tRNA核输出中的功能。 AIM 1员工在体内生化分析，以检验以下假设 TRNA直接与CRM1和/或MEX67-MTR2核出口机械相互作用，并了解这些 替代核输出途径识别tRNA底物。 AIM 1还试图测试候选人是否 tRNA核进口商与tRNA物理相互作用。 AIM 2解决了tRNA bi-的生物学功能 细胞核和细胞质之间的方向流量。我们发现一个功能是用于trna质量的 防止异常tRNA与蛋白质合成机制相互作用的控制。异常的trnas 由于容易出错的核出口，部分到达细胞质。我们将分析平行tRNA的保真度 核出口途径并确定是否纠正和/或破坏异常的TRNA 逆行进口到核中。 AIM 3解决了线粒体上发生的RNA处理步骤 膜。线粒体上发生酵母中的tRNA剪接和后生动物的piRNA加工 表面。我们确定了可能在指导tRNA剪接内核酸酶和/或trNA的蛋白质 线粒体，我们建议测试这些角色。获得的信息应告知如何以及为什么 线粒体表面充当RNA处理的“仓库”。那是拟议的研究计划 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。