Regulation of tRNA fragment biogenesis and function by nucleotide modifications

项目摘要/摘要 TRNA是众所周知的遗传密码的物理适配器，桥接密码子- 基于蛋白质翻译所需的适当氨基酸的信息。TRNA 功能可以通过多种机制进行调节，包括调节 TRNA氨酰化受细胞内氨基酸水平的影响。之前被低估的两个 最近，机制已成为tRNA生物学的关键调节因素--共价 TRNA核苷酸的修饰和tRNAs的切割，产生稳定和 潜在功能的TRFs。 在这里，我们建议研究tRNA裂解的调节，以及 TRNA片段在发芽酵母和哺乳动物胚胎中的功能 干细胞。首先，由于tRNA的核苷酸修饰已被证明会干扰 TRNA在几种模式生物中的裂解，我们将首先调查所有已知的影响 TRNA修饰对酵母中tRNA切割的影响。小RNA的深度测序将是 对大量酵母缺失突变体进行了测试-涵盖了所有非必需的 目前已知的tRNA修饰酶--在各种tRNA过表达后 核酸酶。同时，我们将开发一个全面的条件突变小组 小鼠胚胎干细胞中的tRNA修饰机制将这些研究扩展到哺乳动物 模特。 一项补充工作将集中在tRNA片段在翻译中的作用 控制力。再次比较酵母和哺乳动物，我们将通过以下方式来描述翻译 野生型和缺乏tRNA裂解或各种tRNA的突变体中的核糖体足迹 修改。为了允许生化访问特定的tRNA片段活动，我们将 使用体外翻译系统直接研究各种纯化或 在蛋白质翻译的明确阶段合成tRNA片段。 总之，这些实验系统地解决了tRNA的作用 TRNA裂解的修饰和由此产生的tRNA片段对翻译的控制， 提供与从神经退行性变等疾病相关的重要见解 致癌。

Project Summary/Abstract tRNAs are well-known as the physical adaptors of the genetic code, bridging codon- based information with the appropriate amino acids required for protein translation. tRNA function can be regulated by a wide variety of mechanisms, including the modulation of tRNA aminoacylation by cellular amino acid levels. Two previously underappreciated mechanisms have recently emerged as key regulators of tRNA biology – covalent modifications of tRNA nucleotides, and cleavage of tRNAs, which generates stable and potentially functional tRFs. Here, we propose to investigate the regulation of tRNA cleavage, and the functions of resulting tRNA fragments, in budding yeast and in mammalian embryonic stem cells. First, as tRNA nucleotide modifications have been shown to interfere with tRNA cleavage in several model organisms, we will first survey the effects of all known tRNA modifications on tRNA cleavage in yeast. Deep sequencing of small RNAs will be carried out for a large collection of yeast deletion mutants – covering all nonessential tRNA-modifying enzymes known at present – following overexpression of various tRNA nucleases. In parallel, we will develop a comprehensive panel of conditional mutants in tRNA modification machinery in murine ES cells to extend these studies to a mammalian model. A complementary effort will focus on the roles for tRNA fragments in translational control. Again comparing yeast and mammals, we will characterize translation by ribosome footprinting in wild-type and in mutants lacking tRNA cleavage or various tRNA modifications. To allow biochemical access to specific tRNA fragment activities, we will use in vitro translation systems to directly investigate the roles for various purified or synthetic tRNA fragments at well-defined stages of protein translation. Together, these experiments systematically address the roles for tRNA modifications in tRNA cleavage and control of translation by resulting tRNA fragments, providing important insights with relevance to diseases ranging from neurodegeneration to cancer.