Chemical Modifications to Wobble Uridines in tRNA Regulate Responses to Stress

tRNA 中摆动尿苷的化学修饰可调节应激反应

基本信息

批准号：
10387039
负责人：
Thomas J Begley
金额：
$ 35.77万
依托单位：
STATE UNIVERSITY OF NEW YORK AT ALBANY
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：
2022-07-08 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10387039
关键词：
Alkynes Antibiotics Anticodon Bacteria Base Pairing Binding Biochemistry Biological Assay Biological Markers Biology Biotin Carbon Cells Chemicals Chloramphenicol Code Codon Nucleotides Complement Complex Computer Simulation Crystallization Data Detection Diphosphates Disease Enzymes Escherichia coli Exposure to Gene Expression Gene Expression Regulation Genes Human In Vitro Kinetics Label Lead Ligand Binding Ligands Link Lipids Modification Molecular Molecular Biology Nucleosides Pathway interactions Pattern Play Polyribosomes Positioning Attribute Proteins RNA Reaction Reagent Regulation Reporter Research Ribosomes Role Site Specificity Stress Structure System Technology Thermodynamics Thiouridine Time Transcript Transfer RNA Translational Regulation Translations Uridine Validation Variant Yeasts base biological adaptation to stress cancer cell cofactor design environmental stressor epitranscriptomics in vivo insight mutant novel response simulation stem tool transcriptome sequencing

项目摘要

Abstract Cells respond to environmental stresses by regulating gene expression. Recent studies have demonstrated that stress promotes changes in the levels of enzyme-modified nucleosides found in the anticodon of many tRNAs, to regulate the translation of stress-response transcripts with specific codon usage patterns. In bacteria the wobble U34 residue of tRNA can be enzymatically modified by writers to generate thiolation, geranylation or selenation products at position 2, as well as distinct modifications to position 5, to produce 12 different modified uridines, with many predicted to play key roles in translation and stress responses. The Mnm cluster enzymes can catalyze the formation of these modified uridines and due to their uniqueness to bacteria and the importance of corresponding wobble uridines in the stress response, we propose that they can be exploited to develop technology to tag modified RNA. We have used chemical biology and structural studies to demonstrate that 2-thiouridine (s2U), geranyl-2-thiouridine (ges2U) and seleno-2-thiouridine (se2U) have different base pairing specificity. In addition, using codon analytics of all E. coli genes, we have identified codon-biased transcripts that could be translationally regulated by s2U, ges2U and se2U modifications. We have also shown that cells deficient in the wobble U modifying enzymes MnmE and MnmH are sensitive to killing by chloramphenicol (CAM) and have perturbed translation. We hypothesize that s2U, ges2U and se2U modification levels change in response to environmental stress, to regulate the translation of response proteins. In this application, we will synthesize and characterize 9 wobble modifications linked to MnmE and MnmH. Further, we propose to characterize stress-induced changes in Mnm linked tRNA modifications and determine if translation elongation of codon specific transcripts is linked to one of the 12 uridine tRNA modifications. In addition, we will develop MnmH-based molecular tools to tag and visualize thiolated tRNAs in yeast and human cells. The proposed studies are significant, as they will define a new form of translational regulation in bacteria, generate new reagents and technologies for tagging epitranscriptomic marks and will provide a unified understanding of the 12 different wobble uridines in bacterial tRNA.

抽象的细胞通过调节基因表达来应对环境压力。最近的研究有证明压力会促进反密码子中酶修饰核苷水平的变化许多 tRNA 的序列，以调节具有特定密码子使用模式的应激反应转录物的翻译。在细菌 tRNA 的摆动 U34 残基可以被作者酶促修饰以产生硫醇化，在位置 2 处香叶基化或硒化产物，以及对位置 5 的明显修饰，产生 12 不同的修饰尿苷，其中许多预计在翻译和应激反应中发挥关键作用。曼尼姆簇酶可以催化这些修饰的尿苷的形成，并且由于它们对细菌的独特性以及相应的摆动尿苷在应激反应中的重要性，我们建议它们可以是用于开发标记修饰 RNA 的技术。我们利用化学生物学和结构研究证明 2-硫尿苷 (s2U)、香叶基-2-硫尿苷 (ges2U) 和硒代-2-硫尿苷 (se2U) 具有不同的碱基配对特异性。此外，通过对所有大肠杆菌基因进行密码子分析，我们发现了密码子偏倚可以通过 s2U、ges2U 和 se2U 修饰进行翻译调节的转录本。我们还展示了缺乏摆动 U 修饰酶 MnmE 和 MnmH 的细胞对杀伤敏感氯霉素 (CAM) 并干扰翻译。我们假设 s2U、ges2U 和 se2U 修饰水平因环境压力而变化，以调节反应蛋白的翻译。在这个应用程序中，我们将合成并表征与 MnmE 和 MnmH 相关的 9 种摆动修改。此外，我们建议描述应激诱导的 Mnm 连接 tRNA 修饰变化，并确定翻译是否有效密码子特异性转录本的延伸与 12 个尿苷 tRNA 修饰之一有关。此外，我们将开发基于 MnmH 的分子工具来标记和可视化酵母和人类细胞中的硫醇化 tRNA。这拟议的研究意义重大，因为它们将定义细菌翻译调控的新形式，产生用于标记表观转录组标记的新试剂和技术，并将提供统一的理解细菌 tRNA 中 12 种不同的摆动尿苷。