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

细胞质 tRNA 库在哺乳动物大脑细胞和分子稳态中的功能

• 批准号: 10550207
• 负责人: SUSAN L ACKERMAN
• 金额: $ 43.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-15 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10550207
• 关键词: Absence Epilepsy; Acute; Alleles; Amino Acids; Anticodon; Astrocytes; Brain; Brain region; Buffers; Cell Death; Cell physiology; Cells; ChIP-seq; Chemicals; Classification; Codon Nucleotides; Cytoplasm; Data; Development; Disease; Epilepsy; Epitopes; Equilibrium; Eukaryota; FRAP1 gene; Family; Family member; Future; Gene Expression; Gene Family; Genes; Genetic; Genetic Transcription; Goals; Hippocampus; Homeostasis; Human; Impairment; Individual; Induced pluripotent stem cell derived neurons; Investigation; Laboratories; Link; Maintenance; Messenger RNA; Microglia; Modeling; Molecular; Mus; Mutation; Nature; Nerve Degeneration; Neurons; Nuclear; Phenotype; Physiological; Physiology; Predisposition; Process; Property; Proteins; RNA; RNA Polymerase III; Regulation; Ribosomes; Role; Seizures; Severities; Signal Pathway; Signal Transduction; Sirolimus; Small RNA; Synapses; Synaptic Transmission; Synaptosomes; Testing; Tissues; Transcript; Transfer RNA; Transgenic Organisms; Translations; United States; Variant; Vertebrates; Wild Type Mouse; Work; biological adaptation to stress; cell type; differential expression; excitatory neuron; frontier; genome-wide; granule cell; in vivo; inhibitory neuron; mRNA Translation; mTOR Inhibitor; mammalian genome; member; mouse genome; mouse model; nervous system disorder; neuronal cell body; novel; null mutation; overexpression; patch clamp; response; restoration; transcriptome; transgene expression; translatome

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.

项目总结/摘要 转运RNA（transferRNA，tRNA）是一种重要的衔接分子，它将氨基酸与密码子连接起来，解码mRNA 翻译过程中的记录。哺乳动物基因组包含数百个tRNA基因， 根据反密码子分为不同的家族每个家族都包含多个tRNA基因，这表明这些基因 可以缓冲有害突变的影响。最近，我们证明了一种突变 损害n-Tr 20（一种tRNAArgUCU基因）的加工或其完全丢失，改变基因表达， 在细胞和生物体水平的生理反应，尽管存在四个额外的， 功能性tRNAArgUCU基因。更具体地说，这种高度表达的神经元的缺失- tRNAArgUCU家族的一个特定成员降低了小鼠对癫痫发作的易感性， 海马体中的兴奋-抑制平衡。n-Tr 20的缺失导致核糖体在同源阿加上停滞 密码子，沿着转录和翻译景观的变化，其特征是减少 mTORC 1信号传导和整合应激反应的激活。另一种转基因过表达 tRNAArgUCU家族基因的成员恢复了癫痫发作的易感性，其方式与 转基因的tRNA表达水平，表明n-Tr 20-/-小鼠的表型是由于 tRNAArgUCU神经元池的减少，其中n-Tr 20是主要贡献者。 我们的研究结果首次证明了多拷贝核编码基因的单个成员的突变， tRNA家族可以改变神经元的分子景观和生理学，并为未来的神经元生长提供动力。 研究tRNA突变在维持细胞稳态和疾病中的作用。这项建议 从几个方面扩展了我们的发现。在目标1中，我们将确定潜在的细胞机制， 通过在抑制性或非抑制性细胞中有条件地缺失n-Tr 20， 在发育过程中或发育后的兴奋性神经元。我们还将研究基因增加的影响， n-Tr 20-/-神经元中的mTOR信号传导对突触传递的影响。为了进一步了解这些生理 变化，我们将分析n-Tr 20-/-和野生型小鼠的兴奋性和抑制性神经元中的翻译组， 确定n-Tr 20缺失是否破坏局部翻译。在目标2中，我们将检验我们的假设， 来自tRNA丢失的突变是由于具有相同反密码子的tRNA库的水平降低，我们 将研究耗尽的tRNA家族的身份是否影响这些表型。我们将执行ChIP- 从大脑中的几种主要细胞类型的Seq，利用一种新的小鼠模型， RNA聚合酶III的表位标记的等位基因。根据这些数据，我们将识别并删除其他高度 表达的tRNA，并研究其损失对小鼠脑中主要细胞类型的影响。最后我们将 通过研究tRNA缺失对翻译组和生理学的影响，将我们的工作扩展到人类 iPSC衍生的神经元。