Dynamic interplay of eukaryotic translation and mRNA decay

项目摘要/摘要 真核生物mRNAs的翻译受到高度调控，过早停止翻译的突变会导致11% 所有可遗传的人类疾病中。然而，核糖体如何快速而准确地识别终止密码子以 停止蛋白质合成，释放新生多肽。目前也不清楚核糖体是如何被困在 异常的信使核糖核酸，如那些缺少终止密码子的信使核糖核酸，会被腐烂的机器释放出来。一种对 这些基本过程可能有助于发现囊性等疾病的新疗法 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 核糖体翻译异常的mRNAs的缓慢速度被缓慢作用的衰变因子利用，以 特别是降解变异的mRNAs，让正常的mRNAs保持不变。对这一假设的检验将 需要实时跟踪翻译正常或异常mRNAs的核糖体，捕捉复杂的途径 对监管至关重要的动态。作为约瑟夫·普格利西在斯坦福大学实验室的博士后，我建立了单身- 分子分析直接跟踪单个真核细胞核糖体在整个终止过程中使用体外- 重组后的系统。由真核细胞翻译和信使核糖核酸衰变专家雷切尔·格林共同指导，我将 将这些分析扩展到监测终止、再循环和信使核糖核酸衰变的其他关键事件。我会进一步评估 翻译和信使核糖核酸中独特亚态的结构利用低温EM衰变。我提出以下建议 具体目标：(1)破译确保真核终止的保真度的机制；(2)确定动态 将核糖体从正常和异常的mRNAs中解放出来；(Iii)决定终止、再循环和mRNAs 腐烂在人类中是受调节的。总之，提出的目标将揭示真核细胞如何区分 正常和异常的mRNA。在我的指导团队的支持下，我将获得冷冻-EM方面的专业知识，以及 除了我最初研究的翻译控制到相关的信使核糖核酸衰变之外，还需要额外的培训 酵母菌和人类的机制。我还将精通全基因组技术的语言 例如CRISPR筛查和核糖体图谱。拟议的研究和培训活动将为我提供 具备建立独立研究计划所需的技能，专注于 真核翻译和信使核糖核酸的衰退，并揭示了基因表达的基本方面，与 人类健康将在未来的R01中建立起来。

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.