Dynamic interplay of eukaryotic translation and mRNA decay

真核翻译和 mRNA 衰减的动态相互作用

基本信息

批准号：
10884717
负责人：
Michael R Lawson
金额：
$ 24.9万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10884717
关键词：
ATP phosphohydrolase Affect Architecture Award Biological Assay Breast CRISPR screen Cryoelectron Microscopy Cystic Fibrosis Development Disease Duchenne muscular dystrophy Ensure Eukaryota Eukaryotic Cell Event Foundations Future Gene Expression Health Hereditary Neoplastic Syndromes Heritability Human In Vitro Individual Kinetics Language Learning Life Malignant Neoplasms Malignant neoplasm of ovary Mentors Messenger RNA Molecular Monitor Muscular Dystrophies Mutate Mutation Non-Stop Decay Nonsense Codon Open Reading Frames Outcome Pathway interactions Phase Postdoctoral Fellow Process Protein Biosynthesis Proteins Recycling Regulation Research Research Activity Ribosomes Signal Transduction Specificity Structural Biologist Structure System Techniques Terminator Codon Testing Therapeutic Time Training Training Activity Transcript Transfer RNA Translating Translations Work Yeasts genome-wide human disease inhibitor mRNA Decay novel therapeutics peptidyl-tRNA polypeptide premature programs reconstitution ribosome profiling single molecule skills time use translation factor

项目摘要

PROJECT SUMMARY / ABSTRACT Translation of eukaryotic mRNAs is highly regulated, and mutations that prematurely halt translation cause 11% of all heritable human diseases. Yet, it is unknown how ribosomes rapidly and accurately identify stop codons to halt protein synthesis and release the nascent polypeptide. It is also unclear how ribosomes trapped on an aberrant mRNA, such as those devoid of a stop codon, are liberated by decay machinery. An understanding of these fundamental processes could facilitate the discovery of novel therapeutics for diseases such as Cystic Fibrosis, Duchenne Muscular Dystrophy, and hereditary cancer syndromes. My central hypothesis is that the slow rate at which aberrant mRNAs are translated by ribosomes is exploited by slowly-acting decay factors to specifically degrade aberrant mRNAs and leave normal ones untouched. Examination of this hypothesis will require real-time tracking of ribosomes translating normal or aberrant mRNAs, capturing intricacies of pathway dynamics that are critical for regulation. As a postdoc in Joseph Puglisi’s lab at Stanford, I established single- molecule assays to directly track individual eukaryotic ribosomes throughout termination using an in vitro- reconstituted system. Co-mentored by Rachel Green, an expert in eukaryotic translation and mRNA decay, I will extend these assays to monitor other key events in termination, recycling, and mRNA decay. I will further assess the architecture of unique sub-states in translation and mRNA decay using cryo-EM. I propose the following specific aims: (I) Decipher the mechanisms that ensure fidelity in eukaryotic termination; (II) Define the dynamics that liberate ribosomes from normal and aberrant mRNAs; (III) Determine how termination, recycling, and mRNA decay are regulated in humans. Together, the proposed aims will reveal how eukaryotic cells distinguish between normal and aberrant mRNAs. Supported by my mentoring team, I will obtain expertise in cryo-EM, and the additional training necessary to expand beyond my initial studies of translational control into related mRNA decay mechanisms in yeast and humans. I will also become conversant in the language of genome-wide techniques such as CRISPR screening and ribosome profiling. The proposed research and training activities will provide me with the skills needed to establish an independent research program focused on the dynamic interplay of eukaryotic translation and mRNA decay, and reveal fundamental facets of gene expression with relevance to human health to be built upon in a future R01.

项目概要/摘要真核 mRNA 的翻译受到高度调控，过早停止翻译的突变会导致 11% 然而，目前尚不清楚核糖体如何快速准确地识别终止密码子。停止蛋白质合成并释放新生多肽目前还不清楚核糖体是如何被捕获的。异常的 mRNA，例如那些缺乏终止密码子的 mRNA，是通过衰变机制释放的。这些基本过程可以促进针对囊性囊肿等疾病的治疗小说的发现纤维化、杜氏肌营养不良症和遗传性癌症综合征我的中心假设是核糖体翻译异常 mRNA 的缓慢速度被缓慢作用的衰变因子利用特异性降解异常的 mRNA 并保持正常的 mRNA 不受影响。需要实时跟踪翻译正常或异常 mRNA 的核糖体，捕获通路的复杂性作为斯坦福大学 Joseph Puglisi 实验室的博士后，我建立了单一的动态机制。使用体外方法直接追踪整个终止过程中单个真核核糖体的分子测定在真核翻译和 mRNA 衰变方面的专家 Rachel Green 的共同指导下，我将重建系统。我将进一步评估这些检测的扩展，以监测终止、回收和 mRNA 衰变中的其他关键事件。使用冷冻电镜研究翻译和 mRNA 衰变中的独特亚状态的结构，我提出以下建议。具体目标： (I) 破译确保真核终止保真度的机制； (II) 定义动力学； (III) 确定如何终止、回收和 mRNA 总之，所提出的目标将揭示真核细胞如何区分。在我的指导团队的支持下，我将获得冷冻电镜和冷冻电镜方面的专业知识。需要额外的培训来扩展我对翻译控制的初步研究，扩展到相关的 mRNA 衰减我还将熟悉全基因组技术的语言。诸如 CRISPR 筛选和核糖体分析等拟议的研究和培训活动将为我提供帮助。具备建立独立研究计划所需的技能，重点关注以下领域的动态相互作用真核翻译和 mRNA 衰减，并揭示与相关的基因表达的基本方面未来 R01 的基础是人类健康。