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).

该建议的重点是哺乳动物蛋白质合成和核糖体相关mRNA的机制 监测（No-Go和不停衰变（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、终止和核糖体再循环。而 延伸是高度保守的，其它阶段在界之间显著不同。入会仪式是最 哺乳动物翻译的一个复杂和受调控的阶段，涉及多种 起始因子（eIF），而终止和再循环与细胞mRNA密切相关， 蛋白质质量控制系统，其由延伸复合物的异常停滞诱导，例如通过稳定的 二级结构、稀有密码子、受损的RNA碱基（NGD）或终止密码子的缺失（NSD）。我们 整个哺乳动物翻译过程的体外重建的发展和最近整合我们的 J. Frank实验室在低温电子显微镜方面的技术进步的专业知识（HHMI， 哥伦比亚大学）现在给了我们一个独特的机会，以缩小在理解的几个关键差距， 哺乳动物翻译机制，研究翻译调控的生理学重要案例， 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 经典翻译过程中关键阶段未解决的机制方面（例如核糖体翻译） Met-tRNAiMet的募集和ABC 50在其中的作用，eIF 4F介导的43 S附着的机制 核糖体前起始复合物加帽的mRNA，DHX 29的作用机制，在核糖体前起始复合物加帽的mRNA，DHX 29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）体外重现， 确定具有高生理重要性的非经典启动的特定情况的机制，或 临床相关性，如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和调节5 '-末端寡嘧啶（TOP）mRNA，和（iii）发展 我们最近在核糖体相关mRNA和蛋白质质量控制机制方面的进展， 包括肽基-tRNA水解酶PTRH 1在核糖体相关新生链肽基-tRNA的释放中的作用。 翻译中断引起的tRNA，以及GTPBP 1和GTPBP 2成员的功能机制 一组相对不同的翻译GTPases。我们将继续使用体外重组 方法，结合最先进的生物化学技术，以及动力学和结构方法， Frank（HHMI，哥伦比亚大学），Y. Hashem（IBMC斯特拉斯堡，法国）和M.罗德妮娜 (MPI Göttingen，德国）。