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

eIF3 的 mRNA 进入通道臂在 mRNA 招募中的作用

基本信息

批准号：
10390011
负责人：
Colin Aitken
金额：
$ 5万
依托单位：
VASSAR COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-11 至 2024-08-31
项目状态：
已结题

项目摘要

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.

项目总结/摘要翻译起始建立了蛋白质合成的阅读框架，并将翻译起始转化为蛋白质合成。根据细胞的需要，细胞可以通过机械来产生特定的mRNA。毫不奇怪，翻译起始是翻译的限速和最高度调节的阶段。翻译起始失调是人类癌症的致病因素;翻译起始因子水平的改变与癌症有关改变起始途径的发展和进展以及特定步骤，癌细胞的增殖。真核翻译起始因子3（eIF 3）是目前已知的最大、最复杂的翻译起始因子。这些启动因子的作用，并在启动途径的每一步发挥作用。五个基本亚基包含S中的eIF 3复合物。酿酒酵母，构成在其他真核生物中保守的核心复合物。这些亚基中的每一个的改变的表达引起癌症的发展或进展，并且几个亚基的改变引起癌症的发展或进展。亚基已成为原癌基因或治疗靶点。然而，一个机械的框架，对这些与癌症的因果关系的理解尚不存在。事实上，我们对人类行为的理解的eIF 3和它的翻译起始机制的贡献仍然存在。特别是，eIF 3如何有助于核糖体募集mRNA仍然是一个谜。最近的高分辨率结构揭示了eIF 3 结合小核糖体亚基和突出臂附近的mRNA进入和退出通道，通过其中mRNA进入和离开核糖体前起始复合物（PIC）。这些结构还表明， eIF 3进入通道臂的动态重排响应PIC的mRNA结合而发生。然而，这种重排的机制作用和eIF 3单个亚基的特定作用，仍然未知。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。使用核糖体分析，我们已经确定了特定的mRNA，其翻译是对整个eIF 3复合物或其进入通道臂的破坏高度敏感，并将剖析其机制。使用重现关键起始事件的体外重建系统来研究这种敏感性的起源（目的1）。我们还将利用以前表征的eIF 3和小核糖体的功能变体库，亚基来阐明进入通道臂和PIC之间的机械协作，探测PIC入口和出口通道中mRNA结合的稳定性（目的2）。最后我们将利用现有的重组表达和纯化eIF 3的方法，研究eIF 3复合物的致死突变，从而消除该领域的关键挑战（目标3）。总之，这些努力将阐明mRNA募集的机制和eIF 3及其受体的作用。亚单位。这一新的理解也将有助于建立一个框架，解释eIF 3在以下方面的关键作用：癌症的发展和进展。