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).

这一建议侧重于哺乳动物蛋白质合成和核糖体相关mRNA的机制。 监测(不进行和不停止腐烂(分别为NGD和NSD))和蛋白质质量控制途径。 翻译是一个循环的过程，包括起始、延伸、终止和核糖体循环。而当 伸长是高度保守的，其他阶段在王国之间存在显著差异。印心是最重要的 哺乳动物翻译的复杂和受调控的阶段，涉及多个 启动因子(EIF)，而终止和再循环与细胞的mRNA和 由延伸复合体的异常停滞引起的蛋白质质量控制系统，例如稳定 二级结构、稀有密码子、RNA碱基受损(NGD)或缺少终止密码子(NSD)。我们的 整个哺乳动物翻译过程的体外重建和OUR整合的最新进展 在J.Frank实验室制造的冷冻电子显微镜技术进步方面的专业知识(HHMI， 哥伦比亚大学)现在给了我们一个独特的机会来弥合在理解 哺乳动物翻译的机制，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 规范翻译过程中的关键阶段(例如核糖体)尚未解决的机械性问题 Met-tRNAiMet的募集及ABC50在其中的作用和eIF4F介导的43S附着机制 核糖体与封端mRNAs的预引发复合体，DHX29在核糖体过程中的作用机制 这一过程的扫描和动力学，以及哺乳动物终止的动力学)，(Ii)体外和 确定具有高度生理重要性的非规范启动的特定病例的机制或 临床相关性，如Leu-tRNALeu介导的启动，重复相关的非Aug(RAN)翻译， 不同细胞IRESS的启动和5‘-端寡嘧啶(TOP)mRNAs的调控，以及(Iii)发育 我们在核糖体相关mRNA的机制和蛋白质质量控制方面的最新进展， 包括肽tRNA水解酶PTRH1在核糖体相关新生链肽释放中的作用 中断翻译产生的tRNA及其成员GTPBP1和GTPBP2的作用机制 一组相对不同的翻译GTP酶。我们将继续使用体外重建 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.Hasem(法国斯特拉斯堡IBMC)和M.Rodnina合作 (MPI Göttingen，德国)