Mechanisms of eukaryotic translation and ribosome-associated mRNA surveillance and protein quality control

真核翻译机制和核糖体相关 mRNA 监测和蛋白质质量控​​制

• 批准号: 9912787
• 负责人: TATYANA V PESTOVA
• 金额: $ 67.83万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9912787
• 关键词: Address; Aminoacyl-tRNA hydrolase; Biochemical; Codon Nucleotides; Collaborations; Complex; Cryoelectron Microscopy; Defect; Development; Disease; Ensure; France; GTPBP1 gene; Germany; Guanosine Triphosphate Phosphohydrolases; In Vitro; Interruption; Intervention; Kinetics; Laboratories; Leucine-Specific tRNA; Malignant Neoplasms; Mediating; Messenger RNA; Metabolic; Methods; Molecular; Nerve Degeneration; Non-Stop Decay; Pathway interactions; Peptide Initiation Factors; Periodicity; Physiological; Process; Protein Biosynthesis; Proteins; Quality Control; RNA; Recycling; Regulation; Ribosomes; Role; Scanning; Structure; System; Techniques; Terminator Codon; Translation Initiation; Translation Process; Translational Regulation; Translations; Universities; base; clinically relevant; mRNA Surveillance; mRNA capping; member; peptidyl-tRNA; reconstitution; recruit; therapeutic development

This proposal focuses on the mechanisms of mammalian protein synthesis and ribosome-associated mRNA surveillance (No-Go and non-stop decay (NGD and NSD, respectively)) and protein quality control pathways. Translation is a cyclical process, consisting of initiation, elongation, termination and ribosome recycling. While elongation is highly conserved, other stages differ significantly between kingdoms. Initiation is the most complicated and regulated stage of mammalian translation involving a complex interplay between multiple initiation factors (eIFs), whereas termination and recycling are intimately connected with cellular mRNA and protein quality control systems that are induced by aberrant stalling of elongation complexes by e.g. stable secondary structures, rare codons, damaged RNA bases (NGD), or the absence of a stop codon (NSD). Our development of in vitro reconstitution of the entire mammalian translation process and recent integration of our expertise with technical advances in cryo-electron microscopy made in the laboratory of J. Frank (HHMI, Columbia University) now gives us a unique opportunity to close several critical gaps in understanding of the mechanism of mammalian translation, to investigate physiologically important cases of translational regulation, and to extend our studies to associated mRNA and protein quality control pathways. We will (i) address the unresolved mechanistic aspects of the key stages in the canonical translation process (e.g. ribosomal recruitment of Met-tRNAiMet and the role in it of ABC50, the mechanism of eIF4F-mediated attachment of 43S ribosomal preinitiation complexes to capped mRNAs, the mechanism of action of DHX29 during ribosomal scanning and kinetics of this process, and kinetics of mammalian termination), (ii) recapitulate in vitro and determine mechanisms of specific cases of non-canonical initiation that have high physiological importance or clinical relevance, such as initiation mediated by Leu-tRNALeu, repeat-associated non-AUG (RAN) translation, initiation on distinct cellular IRESs and regulation of 5’-terminal oligopyrimidine (TOP) mRNAs, and (iii) develop our recent advances concerning mechanisms of ribosome-associated mRNA and protein quality control, including the role of peptidyl-tRNA hydrolase PTRH1 in release of ribosome-associated nascent chain peptidyl- tRNAs arising from interrupted translation, and the mechanism of function of GTPBP1 and GTPBP2, members of a relatively divergent group of translational GTPases. We will continue to use the in vitro reconstitution approach, integrated with state-of-art biochemical techniques, as well as kinetic and structural methods in collaboration with J. Frank (HHMI, Columbia University), Y. Hashem (IBMC Strasbourg, France), and M. Rodnina (MPI Göttingen, Germany).

该提案重点关注哺乳动物蛋白质合成和核糖体相关 mRNA 的机制 监测（No-Go 和不间断腐烂（分别为 NGD 和 NSD））和蛋白质质量控​​制途径。 翻译是一个循环过程，包括起始、延伸、终止和核糖体回收。尽管 伸长高度保守，不同王国之间的其他阶段存在显着差异。起始是最 哺乳动物翻译的复杂且受调控的阶段，涉及多种因素之间的复杂相互作用 起始因子（eIF），而终止和循环与细胞 mRNA 密切相关， 由延伸复合物的异常失速诱导的蛋白质质量控​​制系统，例如稳定的 二级结构、稀有密码子、RNA 碱基受损 (NGD) 或终止密码子缺失 (NSD)。我们的 整个哺乳动物翻译过程的体外重建的发展以及我们最近的整合 J. Frank 实验室（HHMI， 哥伦比亚大学）现在为我们提供了一个独特的机会来弥合理解上的几个关键差距 哺乳动物翻译机制，研究翻译调节的生理学重要案例， 并将我们的研究扩展到相关的 mRNA 和蛋白质质量控​​制途径。我们将 (i) 解决 规范翻译过程中关键阶段的未解决的机制方面（例如核糖体 Met-tRNAiMet 的募集以及 ABC50 在其中的作用，eIF4F 介导的 43S 附着机制 核糖体预起始复合物与加帽 mRNA，DHX29 在核糖体过程中的作用机制 该过程的扫描和动力学，以及哺乳动物终止的动力学），（ii）在体外重述和 确定具有高度生理重要性的非规范启动的特定案例的机制，或 临床相关性，例如由 Leu-tRNALeu 介导的起始、重复相关的非 AUG (RAN) 翻译、 启动不同的细胞 IRES 并调节 5'-末端寡嘧啶 (TOP) mRNA，以及 (iii) 开发 我们在核糖体相关 mRNA 和蛋白质质量控​​制机制方面的最新进展， 包括肽基-tRNA 水解酶 PTRH1 在核糖体相关新生链肽基-释放中的作用 翻译中断产生的 tRNA 以及 GTPBP1 和 GTPBP2 的功能机制 一组相对不同的翻译 GTP 酶。我们将继续使用体外重构 方法，与最先进的生化技术以及动力学和结构方法相结合 与 J. Frank（HHMI、哥伦比亚大学）、Y. Hashem（IBMC 斯特拉斯堡，法国）和 M. Rodnina 合作 （MPI 哥廷根，德国）。