Chemical Modifications to Wobble Uridines in tRNA Regulate Responses to Stress

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 证明压力促进反密码子中发现的酶修饰核苷水平的变化 在许多tRNA中，调节具有特定密码子使用模式的应激反应转录本的翻译。在……里面 细菌tRNA的摆动U34残基可以被编写者酶修饰以产生硫代化， 2位的香叶化或硒化产物，以及对5位的不同修饰，以产生12 不同的修饰尿苷，其中许多被预测在翻译和应激反应中发挥关键作用。MNM 簇状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，我们认为它们可以 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来 证明了2-硫代尿苷(S2U)、香叶基-2-硫脲(Ges2U)和硒-2-硫脲(Se2U)具有不同的结构 碱基配对的专一性。此外，利用对所有大肠杆菌基因的密码子分析，我们已经确定了偏向密码子的 可以通过s2U、ges2U和se2U修饰进行翻译调控的转录本。我们还展示了 缺乏摆动U修饰酶MnmE和MnmH的细胞对 氯霉素(CAM)，并有扰动翻译。我们假设s2U、ges2U和se2U修饰 水平会随着环境压力的变化而变化，以调节反应蛋白的翻译。在这 应用方面，我们将合成并表征与MnmE和MnmH相关的9种摆动修饰。此外，我们 建议表征压力引起的Mnm连接tRNA修饰的变化，并确定是否翻译 密码子特异转录本的延长与12个尿苷tRNA修饰之一有关。此外，我们还将 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代化tRNA。这个 拟议的研究意义重大，因为它们将定义细菌中一种新的翻译调控形式，产生 用于标记表位转录标记的新试剂和技术，并将提供对 细菌tRNA中12种不同的摆动尿苷。

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.