THE ROLE OF THE mRNA-ENTRY-CHANNEL ARM OF eIF3 IN mRNA RECRUITMENT

项目摘要/摘要 翻译启动为蛋白质合成建立了阅读框架，并致力于翻译 根据细胞需要来生产特定的RNA的机械。毫不奇怪，翻译 启动阶段是翻译的限速阶段，也是最严格的控制阶段。翻译启动过程中的误区 是人类癌症的致病因素；翻译启动因子水平的改变与癌症有关 改变启动途径的发展和进展以及具体步骤以使快速 癌细胞的增殖。真核细胞翻译起始因子3(EIF3)是最大、最复杂的翻译起始因子 这些启动因子，并在启动途径的每一步发挥作用。五个基本亚单位 包括酿酒酵母中的eIF3复合体，构成在其他真核生物中保守的核心复合体。 这些亚基中的每一个亚基的表达改变都会引发癌症的发展或进展，以及几个 亚基已成为原癌基因或治疗靶点。然而，一个机械的框架 了解这些与癌症之间的因果联系还不存在。事实上，我们在理解上的根本差距 EIF3及其对翻译启动的机械性贡献仍然存在。特别是，eIF3如何为 核糖体对信使核糖核酸的募集仍然是个谜。最近的高分辨率结构揭示了eIF3 通过与核糖体小亚基结合并在信使核糖核酸进出通道附近伸出手臂 哪个mRNA进入和离开核糖体前起始复合体(PIC)。这些结构还表明， EIF3进入通道臂的动态重排发生在PIC结合的mRNA的反应中。 然而，这种重排的机械性作用和eIF3各个亚基的具体作用 仍然不为人所知。我们正在结合强大的基因组规模和体外生化方法来解决 这些基本问题。使用核糖体图谱，我们已经识别出特定的mRNA，其翻译是 对整个eIF3复合体或其进入通道臂的破坏高度敏感，并将解剖机械 使用重组的体外系统重述关键启动事件的这种敏感性的来源(目标1)。 我们还将利用先前描述的eIF3和小核糖体功能变异体的库 子单元，以使用检测来说明进入通道ARM和PIC之间的机械性协作 它探索了PIC进出通道中的mRNA结合的稳定性(目标2)。最后，我们会 利用一种现有的重组表达和纯化eIF3的方法，使第一个体外 研究eIF3复合体的致命性突变，从而消除该领域的一个关键挑战(目标3)。 综上所述，这些努力将阐明mRNA招募的机制以及eIF3和eIF3的作用 亚单位。这一新的理解还将有助于建立一个框架，以解释eIF3在 癌症的发展和进展。

PROJECT SUMMARY/ABSTRACT Translation initiation establishes the reading frame for protein synthesis and dedicates the translational machinery to the production of specific mRNAs depending on cellular need. Not surprisingly, translation initiation is the rate-limiting and most highly regulated phase of translation. Misregulation of translation initiation is a causative factor in human cancers; altered levels of translation initiation factors are implicated in cancer development and progression and specific steps of the initiation pathway are altered to enable the rapid proliferation of cancerous cells. Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these initiation factors and plays a role in every step of the initiation pathway. Five essential subunits comprise the eIF3 complex in S. cerevisiae, constituting a core complex conserved in other eukaryotes. Altered expression of each of these subunits provokes cancer development or progression, and several subunits have emerged as proto-oncogenes or therapeutic targets. However, a mechanistic framework for understanding these causal links to cancer does not yet exist. In fact, fundamental gaps in our understanding of eIF3 and its mechanistic contributions to translation initiation remain. In particular, how eIF3 contributes to mRNA recruitment by the ribosome remains a mystery. Recent high-resolution structures have revealed eIF3 binding to the small ribosomal subunit and projecting arms near the mRNA-entry- and exit channels through which mRNA enters and exits the ribosomal pre-initiation complex (PIC). These structures also suggest that a dynamic rearrangement of the eIF3 entry-channel arm occurs in response to mRNA binding by the PIC. However, the mechanistic role of this rearrangement and the specific roles of the individual subunits of eIF3 remain unknown. We are combining powerful genome-scale and in vitro biochemical approaches to address these fundamental questions. Using ribosome profiling, we have identified specific mRNAs whose translation is hypersensitive to disruption of the entire eIF3 complex or its entry-channel arm and will dissect the mechanistic origins of this sensitivity using a reconstituted in vitro system that recapitulates key initiation events (Aim 1). We will also leverage a library of previously-characterized functional variants of eIF3 and the small ribosomal subunit to illuminate the mechanistic collaboration between the entry-channel arm and the PIC using an assay that probes the stability of mRNA binding in the entry- and exit-channels of the PIC (Aim 2). Finally, we will make use of an existing approach for recombinant expression and purification of eIF3 to enable the first in vitro investigation of lethal mutations to the eIF3 complex, thereby removing a key challenge in the field (Aim 3). Together, these efforts will shed light on the mechanism of mRNA recruitment and the role of eIF3 and its subunits. This new understanding will also contribute to a framework for interpreting the critical role of eIF3 in cancer development and progression.