Mining the tRNA genome by live-cell imaging

通过活细胞成像挖掘 tRNA 基因组

• 批准号: 10005950
• 负责人: Christopher A Ahern
• 金额: $ 23.49万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005950
• 关键词: Address; Amino Acids; Amino Acyl Transfer RNA; Amino Acyl-tRNA Synthetases; Anticodon; Bacteria; Binding; Biological Assay; Biology; Cell Cycle; Cell division; Cell fusion; Cells; Charge; Chemicals; Chimeric Proteins; Codon Nucleotides; Color; Complex; Crowding; Development; Disadvantaged; Dyes; Electroporation; Elongation Factor; Energy Transfer; Escherichia coli; Eukaryota; Evolution; Fluorescence; Fluorescence Resonance Energy Transfer; Fostering; Free Ribosome; Genes; Genetic; Genetic Code; Genetic Translation; Genome; Human; Human Genome; Image; In Vitro; Individual; Label; Libraries; Life; Light; Mango - dietary; Mining; Monitor; Nucleic Acids; Nucleotides; Peptides; Pharmacotherapy; Population; Process; Protein Biosynthesis; Proteins; RNA; Reporter Genes; Research; Resources; Ribosomes; Sense Codon; Side; Site; Spinach - dietary; Structure; System; Techniques; Technology; Terminator Codon; Transfection; Transfer RNA; Translating; Translations; Work; aptamer; base; design; environmental change; fluorophore; genome wide screen; genome-wide; human disease; imaging approach; innovative technologies; live cell imaging; member; new technology; novel; novel strategies; peptidyl-tRNA; premature; reconstitution; response; success; tool; translation factor

Project Summary Transfer RNAs (tRNAs) are central to translation of the genetic code to amino acid building blocks during protein synthesis on the ribosome. The human genome encodes 417 tRNA genes (gtrnadb.ucsc.edu), more than what is needed to translate the 61 sense codons. The diversity of tRNA genes in the human genome is previously unanticipated. We do not yet know which tRNA genes support protein synthesis and how we can image their activity and dynamics. While there is a strong need for robust labeling and imaging of tRNAs in live cells, progress has been slow. Without the convenience of making genetic fusions, such as protein fusions with fluorescent tags (GFP, YFP, etc), the current technology of tRNA labeling is limited to ex vivo conjugation with a fluorophore, followed by transfection or electroporation of the labeled tRNA into a cell. The disadvantage of the ex vivo approach is that the labeled tRNA is not synchronized with cell division. We were the first to develop a genetic fusion technology of tRNA with an RNA aptamer in an approach that is entirely based on nucleic acid replication to express and monitor tRNA for live-cell imaging. We have shown that an E. coli tRNA fused with a “Spinach” aptamer emits spinach-like fluorescence when expressed in E. coli. We have further shown that this Spinach- tRNA is accommodated by the E. coli endogenous protein synthesis machinery, including amino-acid charging by an aminoacyl-tRNA synthetase, access to the ribosome by translation factors, and interaction with the ribosome to make a peptide bond at both the A (aminoacyl-tRNA)- and P (peptidyl-tRNA)-site. The success of the Spinach-tRNA technology was unexpected, given that both the tRNA and the aptamer are of a similar size and that each has a well-defined tertiary structure. We propose to bring this technology to human cells and explore additional aptamers, such as “Mango” that emits a mango-like color. In Aim 1, we will use our genome- wide screening platform to identify tRNA genes that support protein synthesis by the ability to suppress a pre- mature termination codon in a reporter gene. All of the 417 tRNA genes will be screened for suppression at all three stop codons (UAG, UGA, and UAA) to identify the subset that are active in protein synthesis as tools for genome research. In Aim 2, we will perform another genome-wide screen to identify tRNAs that can be fused with an aptamer for live-cell imaging. We will generate a Spinach- and a Mango-library and screen for fusions in each that are active for protein synthesis. This will allow us to pair a Spinach- with a Mango-tRNA in a novel design that monitors FRET (Foster resonance energy transfer) when they occupy adjacent sites on the same ribosome during the making of a nascent peptide bond. By using FRET to focus on tRNAs in association with ribosomes, rather than those non-associated, we will quantify levels of protein synthesis in response to drug treatment and determine how protein synthesis may oscillate in the progression of a cell cycle. This project is at the forefront of powerful developments of new technologies for live-cell imaging of tRNA in the human genome.

项目摘要 转移RNA（TRNA）是将遗传密码转换为氨基酸构建块的核心 核糖体上的蛋白质合成。人类基因组编码417个tRNA基因（gtrnadb.ucsc.edu），而不是 翻译61个感官密码子需要什么。人类基因组中tRNA基因的多样性以前是 意外。我们尚不知道哪些tRNA基因支持蛋白质的合成以及如何成像它们 活动和动态。虽然强烈需要在活细胞中对TRNA进行强大的标记和成像，但仍在 一直很慢。没有使遗传融合的便利性，例如带有荧光标签的蛋白质融合 （GFP，YFP等），当前的tRNA标记技术仅限于与荧光团的离体共轭， 然后将标记的tRNA转染或电穿孔到细胞中。前体内的灾难 方法是标记的tRNA与细胞分裂不同步。我们是第一个发展遗传的人 tRNA的融合技术与RNA APATMER完全基于核酸复制的方法 表达和监视tRNA进行活细胞成像。我们已经表明，大肠杆菌trna与“自旋”融合 当在大肠杆菌中表达时，Apatmer会排放菠菜样荧光。我们进一步表明了这个菠菜 tRNA由大肠杆菌内源性蛋白质合成机制容纳，包括氨基酸充电 通过氨基酰基-TRNA合成酶，通过翻译因子访问核糖体，并与 核糖体在A（氨基酰基-TRNA）和P（肽基-TRNA）位置形成肽键。成功的成功 鉴于tRNA和座席的大小都相似 每个都有一个定义明确的三级结构。我们建议将这项技术带到人类细胞，并 探索其他的适体，例如发出类似漫画的颜色的“芒果”。在AIM 1中，我们将使用我们的基因组 - 广泛的筛选平台，以识别通过抑制预先抑制蛋白质合成的tRNA基因 记者基因中的成熟终止密码子。所有417个tRNA基因都将筛选以抑制 三个终止密码子（UAG，UGA和UAA）识别活性在蛋白质合成中的子集作为工具 基因组研究。在AIM 2中，我们将执行另一个全基因组屏幕以识别可以融合的TRNA 使用用于活细胞成像的APATMER。我们将生成一个菠菜和芒果图书馆和屏幕，以进行融合 每个活性用于蛋白质合成。这将使我们能够将菠菜与小说中的芒果trna搭配 当他们在同一地点占据相邻站点时，可以监视fret（促进共振能量传递）的设计 核糖体在新生的胡椒键建立过程中。通过使用FRET专注于与TRNA有关 核糖体，而不是非相关的核糖体，我们将根据药物量化蛋白质合成水平 处理并确定蛋白质合成如何在细胞周期的进展中振荡。这个项目在 人类基因组中tRNA的新技术有力发展的最前沿。