Function of TRM9L and tRNA Wobble Uridine Modification in the Nervous System

TRM9L 和 tRNA 摆动尿苷修饰在神经系统中的功能

• 批准号: 10597004
• 负责人: Kathaleen M O'Connor-Giles
• 金额: $ 37.05万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597004
• 关键词: Animals; Anticodon; Attenuated; Axon; Biochemical; Codon Nucleotides; Collaborations; Complex; Drosophila genus; Engineering; Enzymes; Epilepsy; Family; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Screening; Genetic study; Growth; Growth Cones; Intellectual functioning disability; Laboratories; Learning; Link; Links List; Memory; Messenger RNA; Methylation; Modeling; Modification; Nerve Degeneration; Nervous System; Nervous System Physiology; Neuronal Plasticity; Neurons; Oxidative Stress; Play; Positioning Attribute; Proteomics; Regulation; Resistance; Role; Stress; Synapses; Synaptic plasticity; System; Testing; Transcript; Transfer RNA; Translational Regulation; Translations; Uridine; Validation; Yeasts; biological adaptation to stress; experimental study; genetic analysis; imaging study; in vivo; insight; loss of function; nervous system development; nervous system disorder; neural circuit; neurodevelopment; neurotransmitter release; paralogous gene; posttranscriptional; protein expression; response; spatiotemporal; synaptic function; tRNA Methyltransferases; tool

Project Summary/Abstract Nervous system function is highly dependent on the dynamic regulation of translation. Neural circuit assembly requires translation in axons as growth cones navigate to their targets and synaptic plasticity, the cellular basis of learning and memory, requires the activity-dependent translation of synaptically localized mRNAs. Once thought of as ubiquitous adaptors, tRNAs are emerging as key regulatory molecules in the dynamic regulation of translation. tRNA stability, efficiency and fidelity are controlled by extensive posttranscriptional modification. In a recent screen, we identified Drosophila tRNA methyltransferase 9-like (TRM9L) as a regulator of synaptic growth and neurotransmitter release. TRM9L is one of two animal paralogs of yeast TRM9, which methylates uridines in the wobble position of tRNA anticodon loops to modulate tRNA interactions with cognate vs. wobble codons and regulate the dynamic translation of stress response genes enriched for specific codons. Biochemical and genetic studies demonstrate that the TRM9 paralog ALKBH8 methylate wobble uridines. In contrast, TRM9L has remained biochemically uncharacterized in any system. With the generation of the first TRM9L loss-of-function model, we have identified a role for TRM9L in modifying tRNA wobble uridines in collaboration with the laboratory of Dragony Fu. Here, we propose experiments to build on our findings to gain an integrated understanding of TRM9L's role in the nervous system. In Aim 1, we combine genetic and imaging studies with biochemical analysis of tRNA modification in informative genetic backgrounds to functionally dissect TRM9L's role at synapses. In Aim 2, we will build on our finding that TRM9L also plays a conserved role in the response to oxidative stress. Interestingly, a number of tRNA modifying enzymes have recently been shown to play roles in both neurodevelopment and oxidative stress response, raising the possibility of mechanistic links. To investigate the relationship between TRM9L's roles at synapses and in oxidative stress resistance, we propose a comprehensive functional genetic analysis. In Aim 3, we propose computational and proteomic approaches to identify neuronal TRM9L target transcripts, followed by in vivo functional validation of top candidates. A growing list of links between tRNA modifications and neurological disorders underlines the importance of understanding the role of tRNA regulation in neuronal function. The proposed experiments will generate fundamental insight into the role of TRM9L in the nervous system and significantly expand our understanding of the dynamic regulation of protein expression and how its dysregulation alters nervous system function.

项目概要/摘要 神经系统功能高度依赖翻译的动态调节。神经 电路组装需要轴突的平移，因为生长锥导航到它们的目标并 突触可塑性是学习和记忆的细胞基础，需要依赖于活动 突触定位 mRNA 的翻译。 tRNA 曾经被认为是普遍存在的接头 成为翻译动态调节中的关键调节分子。 tRNA 稳定性， 效率和保真度由广泛的转录后修饰控制。在最近的一次 通过筛选，我们鉴定出果蝇 tRNA 甲基转移酶 9 样 (TRM9L) 作为调节因子 突触生长和神经递质释放。 TRM9L 是酵母的两种动物旁系同源物之一 TRM9，甲基化 tRNA 反密码子环摆动位置的尿苷以进行调节 tRNA 与同源密码子和摆动密码子相互作用并调节 富含特定密码子的应激反应基因。生化和遗传学研究 证明 TRM9 旁系同源物 ALKBH8 甲基化会摆动尿苷。相比之下，TRM9L 在任何系统中都没有生化特征。随着第一代的诞生 TRM9L 功能丧失模型，我们已经确定了 TRM9L 在修改 tRNA 摆动中的作用 与 Dragony Fu 实验室合作研究尿苷。在这里，我们建议进行实验 以我们的发现为基础，全面了解 TRM9L 在神经系统中的作用 系统。在目标 1 中，我们将遗传和成像研究与 tRNA 的生化分析结合起来 修改信息遗传背景，以功能性剖析 TRM9L 的作用 突触。在目标 2 中，我们将基于我们的发现，即 TRM9L 在 对氧化应激的反应。有趣的是，许多 tRNA 修饰酶最近 已被证明在神经发育和氧化应激反应中发挥作用，提高 机械联系的可能性。研究 TRM9L 的角色之间的关系 突触和氧化应激抵抗，我们提出了全面的功能遗传 分析。在目标 3 中，我们提出了计算和蛋白质组学方法来识别神经元 TRM9L 目标转录本，然后对顶级候选者进行体内功能验证。一个不断成长的 tRNA 修饰与神经系统疾病之间的联系列表强调了 了解 tRNA 调节在神经元功能中的作用。拟议的实验将 深入了解 TRM9L 在神经系统中的作用，并显着 扩大我们对蛋白质表达动态调节及其如何进行的理解 失调会改变神经系统功能。