tRNA processing and nuclear-cytoplasmic dynamics

项目摘要 这项研究的重点是tRNA生物学及其亚细胞运输。TRNAs是一种小的非编码RNA 它们是通过根据密码子将氨基酸输送到翻译核糖体来解码基因组所必需的 MRNAs中的方向。TRNA生物学缺陷导致许多人类疾病，从代谢性疾病到 神经肌肉疾病，以及癌症。TRNA生物学需要一套复杂的保守基因产物来 转录后加工、亚细胞交通和内含子周转。我们使用发芽酵母和活体酵母 发现tRNA生物学未知重要方面的技术。在本提案的目标1中，我们将研究 核糖核糖核酸核出口。目前还不完全清楚转录和部分加工的tRNA是如何在 细胞核被输出到细胞质中，因为它们在翻译中具有迭代功能。一种途径是利用 保守的b-importin LOS1/Exportin，致力于tRNA核输出，但在所有测试中都不是必需的 有机体。通过对tRNA生物学中涉及的基因产物进行全基因组无偏见筛选，我们 发现了另外三种将tRNA输出到细胞质的基因产物：异源二聚体Mex67-mtr2和 CRM1。然而，Mex67-mtr2和CRM1并不专用于tRNA，它们在mRNA和CRM1中具有重要作用。 蛋白质核出口。因此，人们不知道它们是如何识别tRNA的。此外，Mex67-MTR2似乎 容易出错，在去除前导/拖尾序列之前将tRNA递送到细胞质。我们将确定 将Mex67-MTR2和CRM1与tRNA复杂化所需的适配器，并了解容易出错的人如何出错 与出口商打交道。在本提案的第二部分中，我们将研究tRNA在细胞核和 细胞质。虽然几十年来一直认为trna的运动是单向的，从细胞核到细胞质，但我们 共同发现tRNAs在细胞核和细胞质之间双向移动，并且动力学 在酵母和脊椎动物细胞之间是保守的。我们开发了一种新的方法，盐酸/苯胺法， 它报告了tRNA逆行核输入和再输出到细胞质。我们将采用这种方法来 鉴定在tRNA逆行途径中起作用的基因产物，并评估tRNA 逆行的交通是反复的。AIM 3针对tRNA内含子。在tRNA亚群中拥有tRNA内含子 从古生物到人类，基因都是保守的。尽管从前tRNA中去除内含子的机制是 要知道，tRNA内含子的命运和功能在很大程度上是个谜。我们发现了tRNA的一种机制 内含子周转；然而，至少还有四种额外的未知机制来破坏tRNA内含子，这些机制 我们建议将其描述为。令人惊讶的是，我们还了解到在特定的压力下，tRNA内含子 积累到很高的水平。此外，tRNA内含子包含与mRNAs互补的长片段。我们的 初步数据支持它们通过互补碱基配对来调控基因表达的假设 我们将在目标3中检验这一假设。因此，拟议的研究计划对多个 基因表达的方方面面，质量控制，以及对人类健康重要的问题。

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.