Translational quality control by trans-editing domains

总结 氨酰-tRNA 合成酶 (ARS) 建立了遗传密码的规则，其中每个氨基酸都是 连接到同源 tRNA。这个过程中的错误会导致翻译错误，这可能对细胞有毒。最近 研究表明，细胞特异性要求和环境条件所施加的选择力 潜在地形成质量控制机制。大约一半的 ARS 拥有校对（或编辑）功能 水解错误带电的 aa-tRNA 的功能以及非蛋白质氨基酸构成最大的证据 对忠诚度的威胁开始出现。 Musier-Forsyth 实验室在翻译质量领域的早期工作 控制重点是我们发现的 II 类脯氨酰-tRNA 合成酶 (ProRS) 编辑。这导致了详细的 对新型细菌 ProRS 转移后编辑域 (INS) 的机制理解和演示 INS 结构域在 ARS 环境之外单独纯化时，在 tRNA 中具有完全功能 脱酰基化。我们随后发现单域 INS 同源物广泛存在于细菌和 近年来，我们在这一领域的重点几乎完全转向了解这些类 INS 的功能 tRNA 编辑中的结构域。然而，关于这些假定的生理功能的许多悬而未决的问题 反式编辑蛋白仍然存在。 MIRA 应用程序中描述的研究的总体目标是 揭示不断增长的反式编辑蛋白家族（称为 INS 超家族）的特定功能。这个 多样化但普遍保守的家族现在拥有扎实且不断积累的体外结构功能知识 基础，它强烈支持维持翻译保真度的作用。我们的知识更广泛 这些蛋白质的生理作用，特别是在真核生物中，仍处于起步阶段，刚刚开始揭示 角色比之前预期的更广泛。这项工作将解决这一重大差距。虽然古典敲门- 仅定义必需基因与非必需基因的筛选不能立即识别编辑域 至关重要的是，这些领域的强有力的保护意味着它们发挥着重要作用，并且在大多数情况下仍然 尚未发现，在细胞生存和竞争力中的作用。拟议的研究旨在解决一些问题 关于生理转编辑功能和潜在兼职的许多悬而未决的问题 INS 超家族的功能。这些领域在真核生物中很大程度上尚未被探索，包括一个新的亚 许多单细胞真核病原体中编码的家族簇。反式编辑的治疗潜力 域尚未被利用，这代表了该领域的另一个重大差距，我们希望通过我们的解决方案来解决这一问题 计划的研究。从长远来看，将针对新型翻译保真机制的药物与 已知的核糖体靶向蛋白质合成抑制剂，例如氨基糖苷类药物，可能会更有效 治疗策略。

Summary Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. Recent studies suggest that the selective forces exerted by cell-specific requirements and environmental conditions potentially shape quality control mechanisms. Approximately half of the ARSs possess a proofreading (or editing) function to hydrolyze mischarged aa-tRNAs and evidence that non-proteinaceous amino acids pose the greatest threat to fidelity is beginning to emerge. Early work in the Musier-Forsyth lab in the field of translational quality control focused on our discovery of Class II prolyl-tRNA synthetase (ProRS) editing. This led to a detailed mechanistic understanding of the novel bacterial ProRS posttransfer editing domain (INS) and the demonstration that the INS domain, when purified on its own outside the context of the ARS, was fully functional in tRNA deacylation. We subsequently discovered that single-domain INS homologs are widespread in Bacteria and in recent years, our focus in this area has turned almost entirely to understanding the function of these INS-like domains in tRNA editing. However, many open questions regarding the physiological function of these putative trans-editing proteins remain. The overarching goal of the research described in this MIRA application is to uncover the specific functions of a growing family of trans-editing proteins known as the INS superfamily. This diverse yet universally conserved family now has a solid and accumulating in vitro structure-function knowledge base, which strongly supports a role in maintaining translational fidelity. Our knowledge of the broader physiological roles of these proteins, especially in eukaryotes, is still in its infancy and is just beginning to reveal wider roles than previously anticipated. This major gap will be addressed in this work. While classical knock- down screens that only define essential versus non-essential genes do not immediately identify editing domains as essential, the strong conservation of these domains implies they play important, and in most cases still undiscovered, roles in cell survival and competitiveness. Proposed studies are designed to address some of the many open questions with regard to both physiological trans-editing functions and potential moonlighting functions of the INS superfamily. These domains are largely unexplored in eukaryotes, including a novel sub- family cluster that is encoded in many unicellular eukaryotic pathogens. The therapeutic potential of trans-editing domains has not been exploited and represents another major gap in the field that we hope to address by our planned studies. In the long term, combining drugs that target novel translational fidelity mechanisms along with known ribosome-targeting protein synthesis inhibitors such as aminoglycosides, may results in more effective therapeutic strategies.