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

项目概要/摘要 神经系统功能高度依赖于翻译的动态调节。神经 当生长锥导航到它们的目标时，电路组装需要在轴突中平移， 突触可塑性，学习和记忆的细胞基础，需要活动依赖性 突触定位mRNA的翻译。一旦被认为是无处不在的衔接子，tRNA 在翻译的动态调节中作为关键调节分子出现。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 筛选，我们确定果蝇tRNA甲基转移酶9样（TRM 9 L）作为调节剂， 突触生长和神经递质释放。TRM 9 L是酵母的两种动物旁系同源物之一 TRM 9，它甲基化tRNA反密码子环摆动位置的尿苷，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。生化和遗传研究 证明TRM 9与ALKBH 8甲基化摆动尿苷。相比之下，TRM 9 L 在任何系统中都没有生物化学特征。随着第一代 TRM 9 L功能丧失模型，我们已经确定了TRM 9 L在修饰tRNA摆动中的作用 与Dragony Fu的实验室合作。在这里，我们提出实验， 基于我们的研究结果，对TRM 9 L在神经系统中的作用有了全面的了解。 系统在目标1中，我们将联合收割机遗传学和影像学研究与tRNA的生化分析相结合 修改信息遗传背景，功能解剖TRM 9 L的作用， 突触在目标2中，我们将基于我们的发现，即TRM 9 L在细胞内也起着保守的作用。 氧化应激反应。有趣的是，一些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， 机械连接的可能性。为了研究TRM 9 L的作用之间的关系 突触和抗氧化应激，我们提出了一个全面的功能遗传 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， TRM 9 L靶转录物，然后是最佳候选物的体内功能验证。越来越 tRNA修饰和神经系统疾病之间的联系列表强调了以下方面的重要性： 了解tRNA在神经元功能调节中的作用。拟议的实验将 产生对TRM 9 L在神经系统中的作用的基本见解， 扩展我们对蛋白质表达的动态调节的理解， 失调改变神经系统功能。

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.