Dynamic interplay of eukaryotic translation and mRNA decay

项目总结/摘要 真核生物mRNA的翻译受到高度调控，过早停止翻译的突变导致11%的 所有人类遗传疾病的源头然而，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困在 通过衰变机制释放出异常mRNA，如缺乏终止密码子的mRNA。了解 这些基本过程可以促进发现新的治疗疾病的方法， 纤维化、杜氏肌营养不良症和遗传性癌症综合征。我的核心假设是 缓慢作用的衰变因子利用核糖体翻译异常mRNA的缓慢速率， 特异性地降解异常的mRNA而不影响正常的mRNA。对这一假设的检验将 需要实时跟踪核糖体翻译正常或异常的mRNA，捕获复杂的途径 对监管至关重要的动态。作为斯坦福大学约瑟夫·普格利西实验室的博士后，我建立了单- 分子测定法，使用体外- 重建系统。由雷切尔绿色，真核生物翻译和mRNA衰变专家共同指导，我将 扩展这些检测以监测终止、再循环和mRNA衰变中的其他关键事件。我会进一步评估 使用cryo-EM的翻译和mRNA衰变中的独特子状态的架构。我提出以下 具体目标：（I）破译确保真核生物终止保真度的机制;（II）定义动力学 从正常和异常mRNA中释放核糖体;（III）确定终止，回收和mRNA 在人体内是受调节的。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的专业知识， 额外的培训是必要的，以扩大我的翻译控制到相关的mRNA衰变的初步研究 在酵母和人类中的机制。我也将精通全基因组技术的语言 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 与建立一个独立的研究计划，重点是动态的相互作用， 真核生物翻译和mRNA衰变，并揭示了基因表达的基本方面与相关的 人类健康将在未来R 01中建立。

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.