The Function of the Cytoplasmic tRNA Repertoire in the Cellular and Molecular Homeostasis of the Mammalian Brain

项目摘要/摘要 转移RNAs(TRNAs)是将氨基酸物理连接到密码子、解码mrna的关键适配分子。 翻译过程中的文字记录。哺乳动物的基因组包含数百个tRNA基因，它们被归类为 根据他们的反密码子进化成家族。每个家族包含多个tRNA基因，这表明这些基因 可以缓冲有害突变的影响。最近，我们已经证明了一种突变 损害tRNAArgUCU基因n-TR20的加工或完全缺失，改变基因表达和 在细胞和组织水平上的生理反应，尽管存在另外四个， 小鼠基因组中的功能性tRNAArgUCU基因。更具体地说，失去这种高度表达的神经元- TRNAArgUCU家族的特定成员降低了小鼠对癫痫的敏感性，并改变了 海马区的兴奋性-抑制性平衡。N-TR20缺失导致同源AGA核糖体停滞 密码子，随着转录和翻译格局的变化，特征是减少 MTORC1信号转导和整合应激反应的激活。另一种的转基因过表达 TRNAArgUCU家族基因的成员恢复了癫痫的易感性，其方式与 转基因小鼠的tRNA表达水平，表明n-TR20-/-小鼠的表型是由于 TRNAArgUCU神经元池减少，其中n-TR20是主要贡献者。 我们的结果首次证明了核编码的多拷贝个体成员的突变 TRNA家族可以改变神经元的分子格局和生理，为未来提供动力 研究tRNA突变在维持细胞内稳态和疾病中的作用。这项建议 从几个方面对我们的发现进行了扩展。在目标1，我们将确定潜在的细胞机制 在n-TR20丢失时改变兴奋-抑制平衡，有条件地删除抑制性或 兴奋性神经元在发育过程中或发育后。我们还将调查基因增加的影响 N-TR20-/-神经元突触传递中的mTOR信号。为了进一步了解这些生理上的 变化，我们将分析n-TR20-/-和野生型小鼠兴奋性和抑制性神经元的翻译组 确定n-TR20删除是否会中断本地翻译。在目标2中，我们将检验我们的假设：表型 来自tRNA丢失的是由于具有相同反密码子的tRNA池的水平降低，并且我们 将调查耗尽的tRNA家族的身份是否会影响这些表型。我们将表演芯片- 利用一种新的小鼠模型，该模型可以有条件地表达一种 RNA聚合酶III表位标记的等位基因。根据这一数据，我们将鉴定和删除其他高度 表达tRNA，并研究它们的丢失对小鼠大脑中主要细胞类型的影响。最后，我们会 通过研究tRNA丢失对人的翻译组和生理学的影响，我们的工作扩展到人类 IPSC来源的神经元。

PROJECT SUMMARY/ABSTRACT Transfer RNAs (tRNAs) are critical adaptor molecules that physically link amino acids to codons, decoding mRNA transcripts during translation. The mammalian genome contains hundreds of tRNA genes which are classified into families based on their anticodon. Each family contains multiple tRNA genes, suggesting that these genes may be buffered against the impact of deleterious mutations. Recently, we have demonstrated that a mutation that impairs processing of n-Tr20, a tRNAArgUCU gene, or its complete loss, alters gene expression and physiological responses at both the cellular and organismal level, despite the existence of four additional, functional tRNAArgUCU genes in the mouse genome. More specifically, loss of this highly expressed, neuron- specific member of the tRNAArgUCU family decreases the susceptibility of mice to seizures and alters the excitatory-inhibitory balance in the hippocampus. Loss of n-Tr20 leads to ribosome stalling on cognate AGA codons, along with changes in the transcriptional and translational landscape, characterized by decreased mTORC1 signaling and activation of the integrated stress response. Transgenic overexpression of the other members of the tRNAArgUCU family genes restored seizure susceptibility, in a manner which correlated with the level of tRNA expression from the transgene, suggesting that the phenotypes in n-Tr20-/- mice are due to a decrease in the tRNAArgUCU neuronal pool, to which n-Tr20 is the major contributor. Our results provide the first demonstration that mutation of an individual member of a multicopy, nuclear-encoded tRNA family can alter the molecular landscape and physiology of neurons and provide an impetus for future investigations of tRNA mutations in the maintenance of cellular homeostasis and in disease. This proposal expands upon our findings in several ways. In Aim 1, we will determine the cellular mechanisms underlying the altered excitatory-inhibitory balance upon n-Tr20 loss by conditionally deleting n-Tr20 in either inhibitory or excitatory neurons during or post-development. We will also investigate the effect of genetically increasing mTOR signaling in n-Tr20-/- neurons on synaptic transmission. To further understand these physiological changes, we will analyze the translatome in excitatory and inhibitory neurons of n-Tr20-/- and wild-type mice and determine whether n-Tr20 deletion disrupts local translation. In Aim 2, we will test our hypothesis that phenotypes derived from tRNA loss are due to the decreased level of the pool of tRNAs with the same anticodon, and we will investigate whether the identity of the depleted tRNA family impacts these phenotypes. We will perform ChIP- Seq from several major cell types in the brain, utilizing a novel mouse model that can conditionally express an epitope-tagged allele of RNA Polymerase III. Based on this data, we will identify and delete other highly expressed tRNAs and investigate the effect of their loss on major cell types in the mouse brain. Finally, we will extend our work into humans by investigating the impact of tRNA loss on the translatome and physiology of iPSC-derived neurons.