tRNA processing and nuclear-cytoplasmic dynamics

tRNA 加工和核质动力学

• 批准号: 10296430
• 负责人: Anita K Hopper
• 金额: $ 34.07万
• 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 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10296430
• 关键词: Adaptor Signaling Protein; Address; Affect; Amino Acids; Aniline; Apoptosis Regulation Gene; Archaea; Back; Biochemical; Biological; Biological Assay; Biological Process; Biology; Cell Nucleus; Cells; Cleaved cell; Code; Codon Nucleotides; Complex; Cytoplasm; Data; Defect; Disease; Ensure; Equilibrium; Eukaryotic Cell; Excision; Explosion; Exportins; Family; Formaldehyde; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Transcription; Genetic study; Genome; Genomics; Growth; Health; Human; Importins; Individual; Introns; Knowledge; Learning; Licensing; Malignant Neoplasms; Messenger RNA; Metabolic Diseases; Methodology; Microscopy; Modification; Molecular; Movement; Neuromuscular Diseases; Nuclear; Nuclear Export; Nuclear Import; Nuclear Protein; Nutrient; Organism; Pathway interactions; Phenylalanine-Specific tRNA; Play; Procedures; Process; Proteins; Proteome; Quality Control; RNA; RNA Processing; RNA Splicing; Regulation; Regulator Genes; Reporting; Research; Research Project Summaries; Ribosomes; Role; Saccharomycetales; Signal Transduction; Specific qualifier value; Specificity; Stress; Stretching; Technology; Testing; Time; Transfer RNA; Translating; Translations; Travel; Untranslated RNA; Vertebrates; Yeast Model System; Yeasts; base; cell preparation; crosslink; gene product; genome wide screen; human disease; in vivo; mRNA Stability; mutant; novel; nuclease; preference; programs; response; tRNA Precursor; trafficking

Project Summary This research program focuses on tRNA biology and its subcellular trafficking. tRNAs are small noncoding RNAs that are essential for decoding the genome by delivering amino acids to translating ribosomes according to codon directions in mRNAs. Defects in tRNA biology cause numerous human disorders from metabolic diseases, to neuromuscular diseases, and to cancer. tRNA biology requires a complex set of conserved gene products for post-transcriptional processing, subcellular traffic, and intron turnover. We employ budding yeast and in vivo technologies to discover unknown important aspects of tRNA biology. In Aim 1 of this proposal we will study tRNA nuclear export. It is not completely understood how tRNAs that are transcribed and partially processed in the nucleus are exported to the cytoplasm for their iterative function in translation. One pathway utilizes the conserved b-importin Los1/Exportin that is dedicated to tRNA nuclear export, but it is unessential in all tested organisms. Employing an unbiased genome-wide screen for gene products involved in tRNA biology, we discovered three additional gene products that export tRNA to the cytoplasm: the heterodimer, Mex67-Mtr2 and Crm1. However, Mex67-Mtr2 and Crm1 are not dedicated to tRNA and they have major roles in mRNA and protein nuclear export. Thus, it is not understood how they recognize tRNAs. Moreover, Mex67-Mtr2 appears to be error-prone, delivering tRNA to the cytoplasm prior to removal of leader/trailer sequences. We will identify adapters needed to complex Mex67-Mtr2 and Crm1 with tRNAs and learn how mistakes by the error-prone exporters are dealt with. In Aim2 of this proposal we will study trafficking of tRNAs between the nucleus and the cytoplasm. 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. We developed a new methodology, the HCl/aniline assay, that reports tRNA retrograde nuclear import and re-export to the cytoplasm. We will employ this methodology to characterize the gene products that function in the tRNA retrograde pathway and assess whether tRNA retrograde traffic is iterative. Aim 3 addresses tRNA introns. Possession of tRNA introns in subsets of tRNA genes is conserved from Archaea to humans. Although the mechanism to remove introns from pre-tRNAs is understood, the fate and function of tRNA introns is largely mysterious. We discovered one mechanism for tRNA intron turnover; however, there are at least four additional unknown mechanisms to destroy tRNA introns which we propose to characterize. Surprisingly, we also learned that under particular stresses, tRNA introns accumulate to high levels. Furthermore, tRNA introns contain long stretches of complementarity to mRNAs. Our preliminary data support the hypothesis that they regulate gene express through complementary base paring with mRNAs; we will test this hypothesis in Aim 3. 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生物学及其亚细胞贩运。 TRNA是小的非编码RNA 通过将氨基酸传递到翻译核糖体的基因组至关重要的是必不可少的 mRNA的指示。 tRNA生物学的缺陷导致许多人类疾病因代谢疾病而导致 神经肌肉疾病和癌症。 tRNA生物学需要一组复杂的保守基因产物 转录后处理，亚细胞流量和内含子营业额。我们使用萌芽的酵母和体内 发现tRNA生物学的未知重要方面的技术。在本提案的目标1中，我们将学习 tRNA核出口。尚未完全了解如何转录和部分处理的trnas 将细胞核导出到细胞质中的迭代功能。一个途径利用 专用于tRNA核导出的保守的B元素LOS1/Exportin，但在所有经过测试中都是无用的 有机体。我们采用无偏见的全基因组筛选，用于参与tRNA生物学的基因产品，我们 发现了将tRNA导出到细胞质的三个其他基因产物：异二聚体，mex67-mtr2和 CRM1。但是，MEX67-MTR2和CRM1并不专用于tRNA，它们在mRNA和 蛋白质核出口。因此，尚不理解他们如何识别TRNA。而且，MEX67-MTR2似乎 容易出错，在去除领导者/拖车序列之前，将tRNA输送到细胞质。我们将确定 需要将MEX67-MTR2和CRM1与TRNA复杂化的适配器，并了解错误的错误 出口商被处理。在该提案的AIM2中，我们将研究细胞核与 细胞质。尽管数十年来一直认为tRNA运动是单向的，核向细胞质的核，但我们 共同发现，tRNA在核和细胞质之间双向移动，动力学 在酵母和脊椎动物细胞之间保守。我们开发了一种新方法，即HCL/苯胺测定法， 这报告了tRNA逆行核进口并重新出口细胞质。我们将采用这种方法来 表征在tRNA逆行途径中起作用的基因产物并评估tRNA是否 逆行流量是迭代的。 AIM 3解决了tRNA内含子。在tRNA子集中拥有tRNA内含子 从古细菌到人类，基因是保守的。虽然从pre-trnas中去除内含子的机制是 理解，tRNA内含子的命运和功能在很大程度上是神秘的。我们发现了一种tRNA的机制 内含子营业额；但是，至少还有四种杀死tRNA内含子的其他未知机制 我们建议表征。令人惊讶的是，我们还了解到在特定的压力下，tRNA内含子 积累高水平。此外，tRNA内含子包含对mRNA的互补性。我们的 初步数据支持以下假设，即它们通过互补碱基削减调节基因表达基因表达 与mrnas;我们将在目标3中检验这一假设。因此，拟议的研究计划影响了多个 基因表达，质量控制和对人类健康重要的问题的方面。