tRNA processing and nuclear-cytoplasmic dynamics

项目摘要 该研究计划的重点是tRNA生物学及其亚细胞运输。tRNA是一种小的非编码RNA 根据密码子将氨基酸传递到翻译核糖体， mRNA的方向。tRNA生物学的缺陷导致许多人类疾病，从代谢疾病， 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， 转录后加工、亚细胞运输和内含子周转。我们使用芽殖酵母， 发现tRNA生物学未知的重要方面的技术。在本提案的目标1中，我们将研究 tRNA核输出。目前还不完全清楚在细胞中转录和部分加工的tRNA是如何在细胞中表达的。 细胞核被输出到细胞质，用于它们在翻译中的迭代功能。一种途径利用 保守的b-importin Los 1/Exportin，其致力于tRNA核输出，但在所有测试的细胞中都是不必要的。 有机体采用无偏见的全基因组筛选与tRNA生物学有关的基因产物， 发现了另外三种将tRNA输出到细胞质的基因产物：异二聚体Mex 67-Mtr 2和 Crm1。然而，Mex 67-Mtr 2和Crm 1并不专用于tRNA，它们在mRNA和蛋白质中起主要作用。 蛋白质核输出。因此，不知道它们是如何识别tRNA的。此外，Mex 67-Mtr 2似乎 易于出错，在去除前导/尾随序列之前将tRNA递送到细胞质。我们将确定 适配器需要复杂的Mex 67-Mtr 2和Crm 1与tRNA，并了解如何错误的易错 出口商在处理。在本提案的目标2中，我们将研究tRNA在细胞核和细胞核之间的运输。 细胞质尽管几十年来人们一直认为tRNA是单向的，从细胞核到细胞质，但我们发现， 共同发现，tRNA在细胞核和细胞质之间双向移动， 在酵母和脊椎动物细胞中是保守的。我们开发了一种新的方法，盐酸/苯胺测定， 报告tRNA逆行核输入和再输出到细胞质。我们将采用这种方法， 表征在tRNA逆行途径中起作用的基因产物，并评估tRNA是否 逆行交通是迭代的。目标3涉及tRNA内含子。tRNA内含子在tRNA亚类中的占有 基因是保守的，从古细菌到人类。尽管从前体tRNA中去除内含子的机制是 尽管如此，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 内含子周转;然而，至少有四种额外的未知机制来破坏tRNA内含子， 我们建议进行表征。令人惊讶的是，我们还了解到，在特定的压力下， 积累到高水平。此外，tRNA内含子含有与mRNA互补的长片段。我们 初步的数据支持这一假设，即它们通过互补碱基配对调节基因表达 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、质量控制和对人类健康重要的问题的各个方面。

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.