mRNA Template-free Protein Elongation: a New Paradigm for Quality Control at the Ribosome

 描述（由申请人提供）： 将 mRNA 翻译成蛋白质是所有生命都必需的过程。最近，导致翻译或相关反应途径（核糖体质量控制复合体（RQC））停滞的突变被证明会导致神经变性，尽管驱动病理学的机制尚不清楚。该研究计划的重点是了解这种质量控制途径的机制及其在疾病中的作用。在酵母中，RQC 途径包括四种高度保守的蛋白质：Ltn1p、Rqc1p、Tae2p 和 Cdc48p，它们与 60S 核糖体新生链复合物结合，这些复合物使 mRNA 的翻译中期停滞并从 40S 核糖体亚基和 mRNA 中分离出来。我们之前发现，破坏 RQC 组件 Ltn1p 或 Rqc1p 会导致蛋白毒性应激，可能与 Ltn1p 小鼠直系同源物的亚型引起的神经退行性表型有关。然而，我们还发现 RQC 介导的蛋白毒性应激依赖于 Tae2p。我们的新初步数据揭示了蛋白质 Tae2p 在翻译失败反应中的惊人作用：Tae2p 与解离的 60S 新生链复合物结合，接合停滞的新生链，并招募带有丙氨酸和苏氨酸的 tRNA，以便 60S 催化新生链的 C 端添加丙氨酸和苏氨酸尾（“CAT 尾”）。这种蛋白质延伸反应在没有 mRNA 模板或 40S 核糖体亚基的情况下发生，因此是一种以前从未在体内观察到的新型延伸形式。 RQC 介导的蛋白毒性应激依赖于 CAT 尾的合成，因此 CAT 尾可能有毒。与这一假设一致，CAT 尾蛋白形成内含物，这可能是由丙氨酸引起的，丙氨酸形成淀粉样蛋白，是至少九种疾病的原因，其中许多疾病包括神经认知表型。 CAT 尾巴的发现引发了许多关于它们参与的质量控制机制及其在疾病中的作用的直接问题。 CAT 加尾的底物有哪些？ CAT 尾巴如何引起蛋白毒性应激？ RQC 靶向的核糖体会发生什么情况？为了探索这些领域，将实现以下具体目标（1）确定 CAT 加尾底物的特征和身份。 (2) 鉴定CAT尾蛋白的命运。 (3) 鉴定核糖体的命运 Tae2p 和 RQC 的目标。为了实现这些目标，将进行各种实验，包括纯化的 RQC 新生链复合物的生化分析、对细胞内蛋白质质量控​​制系统的遗传扰动、体内成像、合成 RQC 底物的生化研究、测量翻译的测定（核糖体分析）以及 RQC 靶向的停滞核糖体的共价标记。相关性：拟议的研究将为未来的基础生物学和治疗应用研究奠定基础 与疾病相关的蛋白质延伸的新范例：无 mRNA 模板的蛋白质延伸。这项工作可能会增加我们对基础生物学、蛋白质质量控​​制和神经退行性疾病病因的了解。

 DESCRIPTION (provided by applicant): Translating mRNA into protein is a process that is essential to all life. Recently, mutations that cause stalls in translation or to an associated response pathway, the Ribosome Quality control Complex (RQC), were shown to cause neurodegeneration, although the mechanisms driving pathology are unknown. This research proposal is focused on understanding the mechanism of this quality control pathway and its role in disease. In yeast, the RQC pathway includes four highly conserved proteins, Ltn1p, Rqc1p, Tae2p, and Cdc48p that bind to 60S ribosome-nascent chain complexes that have stalled mid-translation of an mRNA and split off from the 40S ribosomal subunit and mRNA. We previously found that disrupting the RQC components Ltn1p or Rqc1p causes proteotoxic stress, perhaps relating to the neurodegenerative phenotype caused by a hypomorph of the mouse ortholog of Ltn1p. However, we also found RQC-mediated proteotoxic stress is dependent upon the Tae2p. Our new preliminary data reveals a surprising role for the protein Tae2p in the response to translation failure: Tae2p binds to the dissociated 60S-nascent chain complex, engages the stalled nascent chain, and recruits tRNAs charged with alanine and threonine so that the 60S catalyzes the C terminal addition of the nascent chain with Alanine and Threonine tails ("CAT tails"). This protein elongation reaction happens without an mRNA-template or the 40S ribosomal subunit, and is therefore a novel form of elongation never before observed in vivo. RQC-mediated proteotoxic stress is dependent on synthesis of CAT tail synthesis, and therefore CAT tails may be toxic. Consistent with this hypothesis, CAT tailed proteins form inclusions and this may be caused by alanine, which form amyloid and are the cause of a at least nine diseases, many of which include neurocognitive phenotypes. The discovery of CAT tails raises a number of immediate questions about the quality control mechanisms they take part in and their role in disease. What substrates are CAT tailed? How do CAT tails cause proteotoxic stress? What happens to ribosomes targeted by the RQC? In order to explore these areas the following specific aims will be carried out (1) identify the characteristics and identity of CAT tailed substrates. (2) Identify the fate of CAT tailed proteins. (3) Identify the fate of ribosomes targeted by Tae2p and the RQC. To address achieve these goals, a variety of experiments will be performed, including biochemical analysis of purified RQC-nascent chain complexes, genetic perturbations to intracellular protein quality control systems, in vivo imaging, biochemical studie of synthetic RQC substrates, assays measuring translation (ribosome profiling), and covalent labeling of stalled ribosomes targeted by the RQC. Relevance: The proposed investigations will lay the foundation for future investigations into the basic biology and therapeutic applications of a new paradigm of protein elongation that is linked to disease: mRNA template- free protein elongation. This work will likely increase our knowledge of fundamental biology, protein quality control, and the cause of neurodegenerative diseases.