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

• 批准号: 10662193
• 负责人: Thomas J Begley
• 金额: $ 35.86万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 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/10662193
• 关键词: Alkynes; Antibiotics; Anticodon; Bacteria; Base Pairing; Binding; Biochemistry; Biological Assay; Biology; Biotin; Carbon; Cells; Chemicals; Chloramphenicol; Code; Codon Nucleotides; Complement; Complex; Computer Simulation; 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; Visualization; Yeasts; biological adaptation to stress; biomarker identification; cancer cell; cofactor; design; environmental stressor; epitranscriptomics; in vivo; insight; mutant; novel; response; selenate; simulation; stem; tool; transcriptome sequencing

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.

摘要 细胞通过调节基因表达来应对环境压力。最近的研究 表明压力促进了反密码子中酶修饰核苷水平的变化 许多tRNA，调节翻译的应激反应转录与特定的密码子使用模式。在 细菌中tRNA的摆动U34残基可以被酶促修饰以产生巯基化， 在位置2的香叶基化或硒化产物，以及对位置5的不同修饰，以产生12 不同的修饰尿苷，其中许多预测在翻译和应激反应中发挥关键作用。Mnm 簇合酶可以催化这些修饰的尿苷的形成 以及相应的摆动尿苷在应激反应中的重要性，我们提出它们可以被 用来开发标记修饰RNA的技术。我们利用化学生物学和结构研究， 证明2-硫代尿苷（s2 U）、香叶基-2-硫代尿苷（ges 2U）和硒基-2-硫代尿苷（se 2U）具有不同的 碱基配对特异性此外，利用密码子分析法对所有E.大肠杆菌基因，我们已经确定密码子偏向 s2 U、ges 2U和se 2U修饰可调控的转录本。我们还表明 缺乏摆动U修饰酶MnmE和MnmH的细胞对通过以下途径的杀伤敏感： 氯霉素（CAM）和干扰翻译。我们假设s2 U、ges 2U和se 2U修饰 水平的变化，以响应环境压力，调节翻译的反应蛋白质。在这 应用中，我们将合成和表征9个与MnmE和MnmH相关的摆动修饰。我们还 建议表征Mnm连接的tRNA修饰中应激诱导的变化，并确定翻译是否 密码子特异性转录物的延长与12种尿苷tRNA修饰之一有关。此外，我们会 开发基于MnmH的分子工具，以标记和可视化酵母和人类细胞中的巯基化tRNA。的 拟议的研究是重要的，因为它们将定义细菌中一种新的翻译调控形式， 新的试剂和技术标记表位组学标记，并将提供统一的理解， 细菌tRNA中12种不同的摆动尿苷。