Identifying mechanisms regulating neuronal homeostasis and cell-type specific tRNA expression in the mammalian brain

确定哺乳动物大脑中神经元稳态和细胞类型特异性 tRNA 表达的调节机制

• 批准号: 9759723
• 负责人: Michael Jacob Molumby
• 金额: $ 6.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9759723
• 关键词: Anticodon; Atlases; Biological; Brain; Candidate Disease Gene; Cells; ChIP-seq; Chromosomes, Human, Pair 3; Complex; Computer Simulation; DNA Polymerase III; DNA-Directed RNA Polymerase; Dependence; Environment; Epitopes; Ethylnitrosourea; Family; Genes; Genetic; Genetic Screening; Genetic Translation; Genome; Genomics; Homeostasis; Human; In Vitro; Inbred Strains Mice; Knock-in Mouse; Light; Malignant Neoplasms; Mammals; Maps; Mediating; Messenger RNA; Modification; Molecular; Mus; Mutagenesis; Mutate; Mutation; Nerve Degeneration; Neurons; Nuclear; Organism; Pathologic; Pathway interactions; Pattern; Phenotype; Population; Positioning Attribute; Prevalence; Process; Protein Biosynthesis; Proteins; RNA Splicing; RNA-Directed DNA Polymerase; Recombinants; Regulation; Reporting; Research; Ribosomes; Role; Specificity; Testing; Tissues; Training; Transfer RNA; Transgenic Organisms; Translational Regulation; Translations; Untranslated RNA; Vertebrates; Work; causal variant; cell type; experimental study; forward genetics; in vivo; insight; knockout gene; mouse model; neuron apoptosis; neuron loss; neuronal survival; novel; positional cloning; relating to nervous system; ribosome profiling; ribosome releasing factor

Growing evidence indicates that ribosome processivity during the translation of mRNA to proteins is exquisitely sensitive to changes in tRNA expression and processing. Perturbation of these processes contribute to aberrant cellular homeostasis that manifests as cancer or neurodegeneration. In a N-ethyl-N-nitrosourea (ENU) mutagenesis forward genetic screen in C57BL/6J (B6J) mice to identify novel genes regulating neuronal homeostasis, my lab identified a new ribosome rescue protein GTPBP2. Additionally, we identified a mutation in n-Tr20, a tRNAArgUCU that is specifically expressed in neurons, in B6J mice that epistatically interacts to cause neurodegeneration in Gtpbp2-/- mice. This mutation reduces the processing of n-Tr20 which in turn reduces the total pool of tRNAArgUCU in the brain causing ribosome stalling that is normally rescued by wild type GTPBP2. Interestingly, expression of n-Tr20 is restricted to the brain. These results established ribosome stalling as a novel mechanism for neurodegeneration and defined n-Tr20 as the first reported neuron-specific tRNA in vertebrates. Subsequent mapping crosses of B6J-Gtpbp-/- mice have identified a BALB/cByJ-derived locus that enhances neurodegeneration. Although we have narrowed the critical region containing this modifier gene, no causal gene or mechanism have been identified. In Aim 1 of this proposal, I have proposed a research strategy to identify the causative gene for this locus and elucidate the mechanisms by which this modifier gene augments neurodegeneration. In Aim 2, I will investigate the prevalence of cell-type tRNA expression in the brain by generating the first in vivo tRNA expression brain atlas. I will accomplish this aim by first generating a knock-in mouse line to conditionally epitope-tag RNA Polymerase III (Pol III), the RNA polymerase responsible for transcribing all tRNA, and then performing ChIP-sequencing to determine the occupancy of Pol III machinery on tRNA genes isolated from specific cell-types. With the completion of this proposal we expect to reveal the vast complexity of cell-type specific tRNA profiles in the mammalian brain, and new mechanisms of dysregulation that contribute to aberrant ribosome processivity and neuronal homeostasis in specific cell-types.

越来越多的证据表明，在 mRNA 翻译成蛋白质的过程中，核糖体的持续合成能力非常出色。 对 tRNA 表达和加工的变化敏感。这些过程的扰动有助于 异常的细胞稳态，表现为癌症或神经变性。在 N-乙基-N-亚硝基脲中 (ENU) C57BL/6J (B6J) 小鼠中的诱变正向遗传筛选，以确定调节神经元的新基因 为了平衡体内平衡，我的实验室发现了一种新的核糖体救援蛋白 GTPBP2。此外，我们还发现了一个突变 在 n-Tr20 中，一种在神经元中特异性表达的 tRNAArgUCU，在 B6J 小鼠中，上位性相互作用导致 Gtpbp2-/- 小鼠的神经退行性变。这种突变减少了 n-Tr20 的加工，从而减少了 大脑中导致核糖体停滞的 tRNAArgUCU 总量，通常由野生型 GTPBP2 来挽救。 有趣的是，n-Tr20 的表达仅限于大脑。这些结果确立了核糖体停滞作为 神经退行性变的新机制，并将 n-Tr20 定义为第一个报道的神经元特异性 tRNA 脊椎动物。 B6J-Gtpbp-/- 小鼠的后续作图杂交已鉴定出 BALB/cByJ 衍生的基因座，该基因座增强了 神经变性。尽管我们已经缩小了包含该修饰基因的关键区域，但没有因果关系 基因或机制已被确定。在本提案的目标 1 中，我提出了一项研究策略 识别该位点的致病基因并阐明该修饰基因增强的机制 神经变性。在目标 2 中，我将通过以下方式研究大脑中细胞型 tRNA 表达的普遍性： 生成第一个体内 tRNA 表达脑图谱。我将通过首先生成一个敲入来实现这个目标 条件性表位标记 RNA 聚合酶 III (Pol III) 的小鼠品系，该 RNA 聚合酶负责 转录所有 tRNA，然后进行 ChIP 测序以确定 Pol III 机器的占用情况 tRNA 基因从特定细胞类型中分离出来。随着本提案的完成，我们预计将公布 哺乳动物大脑中细胞类型特异性 tRNA 谱的巨大复杂性，以及新的机制 导致特定细胞类型中核糖体持续合成能力和神经元稳态异常的失调。