Function of TRM9L and tRNA wobble uridine modification in the nervous system

项目摘要/摘要 神经系统的功能高度依赖于翻译的动态调节。神经 电路组装需要轴突的平移，因为生长锥体导航到它们的目标和 突触可塑性是学习和记忆的细胞基础，需要依赖于活动的 突触定位的mRNA的翻译。TRNA曾经被认为是无处不在的适配器，但现在 在翻译的动态调控中成为关键的调节分子。TRNA稳定性， 效率和保真度由广泛的转录后修饰控制。在最近一次 筛选，我们发现果蝇tRNA甲基转移酶9样蛋白(TRM9L)是一种 突触生长和神经递质释放。TRM9L是酵母的两个动物类群之一 TRM9，它使tRNA反密码子环摆动位置的尿苷甲基化以调节 TRNA与同源密码子和摆动密码子的相互作用并调节 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旁侧ALKBH8甲基化可摆动尿苷。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在修正tRNA摆动中的作用 与Dragony Fu的实验室合作的尿苷。在这里，我们建议进行实验，以 以我们的发现为基础，全面了解TRM9L在神经细胞中的作用 系统。在目标1中，我们将遗传和成像研究与trna的生化分析相结合。 在信息性遗传背景中进行修饰以功能性地剖析TRM9L在 突触。在目标2中，我们将以我们的发现为基础，TRM9L在 对氧化应激的反应。有趣的是，一些tRNA修饰酶最近已经 已被证明在神经发育和氧化应激反应中都发挥作用，提高了 机械式连杆的可能性。要调查TRM9L的角色之间的关系，请访问 在突触和氧化应激抵抗中，我们提出了一种全面的功能基因 分析。在目标3中，我们提出了计算机和蛋白质组学方法来识别神经元 TRM9L目标转录本，随后对顶级候选进行体内功能验证。一个不断增长的 TRNA修饰与神经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。拟议中的实验将 从根本上洞察TRM9L在神经系统中的作用，并显著 扩大我们对蛋白质表达的动态调控及其如何 调节失调会改变神经系统功能。

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.