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: 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
• 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

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应激。最近的研究 证明应力促进了在反密码子中发现的酶修饰核苷的水平的变化 许多TRNA，以使用特定的密码子使用模式来调节应力响应转录本的翻译。在 细菌摇摆的tRNA的摇摆U34残基可以通过作者酶变化以产生硫醇化， 位置2处的香烷基化或硒化产物以及对位置5的不同修改，以产生12 不同的修改尿苷，许多预计许多人在翻译和压力反应中起关键作用。 MNM 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 以及相应摇摆尿苷在压力反应中的重要性，我们建议它们可以是 利用开发技术来标记修改的RNA。我们已经使用化学生物学和结构研究来 证明2-硫脲（S2U），Geranyl-2-硫岛（GES2U）和Seleno-2- thiouridine（SE2U）具有不同的 基础配对特异性。此外，使用所有大肠杆菌基因的密码子分析，我们已经确定了偏见的密码子 可以通过S2U，GES2U和SE2U修改来翻译调节的转录本。我们也显示了 摇摆不定的细胞修饰酶MNME和MNMH对被杀死敏感 氯霉素（CAM）并具有干扰的翻译。我们假设S2U，GES2U和SE2U修改 响应环境压力的水平变化，以调节反应蛋白的翻译。在这个 应用程序，我们将合成并表征与MNME和MNMH链接的9个Wobble修改。此外，我们 提出表征应力诱导的MNM链接tRNA修饰的变化，并确定是否翻译 密码子特异性转录物的伸长与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 拟议的研究很重要，因为它们将定义细菌中一种新的翻译调节形式 新试剂和技术，用于标记上映标记，并将提供统一的理解 细菌tRNA中的12种不同的尿尿苷。