Dynamic interplay of eukaryotic translation and mRNA decay

真核翻译和 mRNA 衰减的动态相互作用

• 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
• 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

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.

项目摘要 /摘要 真核mRNA的翻译受到高度调节，过早停止翻译的突变导致11％ 在所有可遗传的人类疾病中。然而，核糖体如何快速，准确地识别停止密码子 停止蛋白质合成并释放新生的多肽。还不清楚核糖体如何被困在一个 异常的mRNA，例如没有终止密码子的mRNA，被腐烂的机械释放。对 这些基本过程可以促进发现诸如囊性疾病的新型疗法 纤维化，Duchenne肌肉营养不良和遗传性癌症综合征。我的中心假设是 核糖体将异常mRNA翻译的异常mRNA被缓慢作用的衰减因子所利用 对该假设的检查将特别降解异常的mRNA，并使正常的mRNA未经触摸。 需要对核糖体进行实时跟踪，这些核糖体翻译正常或异常mRNA，以捕获途径的复杂性 对于调节至关重要的动力学。作为在斯坦福大学约瑟夫·普利利斯（Joseph Puglisi）实验室的博士后，我建立了单一 分子测定，直接使用体外 - 重构系统。由真核翻译和mRNA Decay专家雷切尔·格林（Rachel Green）共同出庭，我将 扩展这些评估以监视终止，回收和mRNA衰变中的其他关键事件。我将进一步评估 使用Cryo-Em在翻译和mRNA衰减中独特子群的结构。我建议以下 具体目的：（i）破译了确保真核终止的忠诚的机制； （ii）定义动力学 从正常和异常的mRNA中释放出核糖体； （iii）确定终止，回收和mRNA如何 衰减受到人类的调节。共同的目标将揭示真核细胞如何区分 正常和异常的mRNA。在我的心理团队的支持下，我将获得Cryo-EM的专业知识， 除了我对翻译控制到相关mRNA衰变的最初研究所需的额外培训 酵母和人类的机制。我还将以全基因组技术的语言变得熟悉 例如CRISPR筛选和核糖体分析。拟议的研究和培训活动将为我提供 凭借建立独立研究计划所需的技能，重点是动态相互作用 真核翻译和mRNA衰变，并揭示了基因表达的基本相，与 人类健康将在未来的R01中建立。