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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要转移 RNA (tRNA) 是关键的接头分子，可将氨基酸与密码子物理连接，解码 mRNA 翻译过程中的文字记录。哺乳动物基因组包含数百个 tRNA 基因，这些基因被分类为根据反密码分为家庭。每个家族包含多个 tRNA 基因，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-Tr20（一种 tRNAArgUCU 基因）的加工或其完全丧失，改变基因表达并尽管存在另外四种，小鼠基因组中的功能性 tRNAArgUCU 基因。更具体地说，这种高度表达的神经元的丢失 tRNAArgUCU 家族的特定成员可降低小鼠癫痫发作的易感性并改变海马体的兴奋-抑制平衡。 n-Tr20 缺失导致核糖体在同源 AGA 上停滞密码子，以及转录和翻译景观的变化，其特征是减少 mTORC1 信号传导和整合应激反应的激活。其他的转基因过度表达 tRNAArgUCU 家族基因的成员恢复了癫痫易感性，其方式与转基因的 tRNA 表达水平，表明 n-Tr20-/- 小鼠的表型是由于 tRNAArgUCU 神经元库减少，其中 n-Tr20 是主要贡献者。我们的结果首次证明多拷贝、核编码的个体成员的突变 tRNA家族可以改变神经元的分子结构和生理学，并为未来提供动力研究维持细胞稳态和疾病中的 tRNA 突变。这个提议以多种方式扩展了我们的发现。在目标 1 中，我们将确定潜在的细胞机制通过有条件地删除抑制或抑制中的 n-Tr20，改变 n-Tr20 丢失时的兴奋性-抑制平衡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-Tr20-/- 神经元中 mTOR 信号传导突触传递。为了进一步了解这些生理变化，我们将分析 n-Tr20-/- 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-Tr20 删除是否会破坏本地翻译。在目标 2 中，我们将检验我们的假设，即表型源自 tRNA 丢失的原因是具有相同反密码子的 tRNA 库水平下降，我们将研究耗尽的 tRNA 家族的身份是否影响这些表型。我们将进行 ChIP- 利用一种新型小鼠模型，对大脑中的几种主要细胞类型进行测序，该模型可以有条件地表达 RNA 聚合酶 III 的表位标记等位基因。根据这些数据，我们将识别并删除其他高度表达 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，我们将通过研究 tRNA 丢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。