Regulation of tRNA fragment biogenesis and function by nucleotide modifications

项目概要/摘要 tRNA 是众所周知的遗传密码、桥接密码子的物理适配器。 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来调节，包括调节 细胞氨基酸水平的 tRNA 氨酰化。两个之前被低估的 最近出现的 tRNA 生物学关键调节机制——共价 tRNA 核苷酸的修饰和 tRNA 的切割，从而产生稳定且 潜在功能性 tRF。 在这里，我们建议研究 tRNA 切割的调控，以及 所得 tRNA 片段在芽殖酵母和哺乳动物胚胎中的功能 干细胞。首先，tRNA 核苷酸修饰已被证明会干扰 tRNA 裂解在几种模式生物中，我们将首先调查所有已知的影响 酵母中 tRNA 切割的 tRNA 修饰。小RNA的深度测序将 对大量酵母缺失突变体进行了研究——涵盖了所有非必需的 目前已知的 tRNA 修饰酶 – 在各种 tRNA 过度表达后 核酸酶。与此同时，我们将开发一个全面的条件突变体面板 鼠 ES 细胞中的 tRNA 修饰机制将这些研究扩展到哺乳动物 模型。 补充工作将重点关注 tRNA 片段在翻译中的作用 控制。再次比较酵母和哺乳动物，我们将翻译特征描述为 野生型和缺乏 tRNA 切割或各种 tRNA 的突变体中的核糖体足迹 修改。为了允许生化获取特定 tRNA 片段的活性，我们将 使用体外翻译系统直接研究各种纯化或 蛋白质翻译明确阶段的合成 tRNA 片段。 总之，这些实验系统地阐述了 tRNA 的作用 通过产生的 tRNA 片段修改 tRNA 切割和翻译控制， 提供与神经退行性疾病等疾病相关的重要见解 到癌症。

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.