The mechanism through which mRNA translation enhancer elements drive cap-independent translation

项目摘要/摘要 选择mRNAs以翻译成蛋白质或翻译启动的过程， 在基因表达中起主要作用，对这一过程的调控在癌症等疾病中至关重要， 糖尿病和神经系统疾病。在此推动下，我们研究的长期目标是了解和 调节在这些疾病的生物学中发挥重要作用的非规范启动机制。为 大多数细胞mRNA，起始涉及识别5‘端的N7-甲基鸟苷-三磷酸’帽‘。 通过帽结合蛋白eIF4E结束mRNA，这一事件导致额外的EIF的招募， 包括eIF4G，最终是一个核糖体起始前复合体(PIC)，它可以启动 MRNA.值得注意的是，在细胞应激条件下，如肿瘤缺氧、病毒感染、营养 限制等，其中eIF4E招募eIF4G的能力以及因此依赖帽的启动的能力是 受到损害，编码有助于在这种压力条件下生存的基因的mRNAs子集仍然可以 高效翻译。这些mRNAs以某种方式从规范的、依赖帽子的启动途径切换到 一种非正则的、帽不依赖的启动途径。调节CAP和CAP之间切换的机制- 依赖和帽不依赖的这些mRNAs的启动仍然未知，阻碍了我们对 调节人类健康和疾病的这一方面的能力。 先前对一组候选α的研究，包括缺氧诱导因子-1、成纤维细胞生长因子-9、血管内皮生长因子-A和p53，它们可以从 作为eIF4E抑制的结果，帽依赖到帽非依赖性的启动已经表明，开关 与eIF4G或其同系物DAP5(eIF4G/DAP5)水平升高相关。有趣的是，这些mRNA 也被证明含有作为翻译增强剂的高度结构化的5‘非翻译区 (TES)，这可能在调节帽依赖和帽非依赖性启动之间的切换中发挥作用。 基于我们强大的初步数据，我们假设含有TE的mRNAs(TE-mRNAs)中的TES 通过直接招募eIF4G/DAP5来调节这种切换，从而使TE-mRNAs甚至能够招募PIC 当eIF4E被抑制时。以我们的初步数据为指导，使用系综和单分子 荧光方法结合互补的细胞和分子生物学方法，我们 建议：(1)表征eIF4G/DAP5与TE-mRNAs结合的强度和选择性；(2)确定 EIF4G/DAP5识别TE的结构基础，以及(3)阐明 EIF4G/DAP5与TE-mRNAs的结合及其PIC募集和翻译效率 这些TE-mRNA。总体而言，这里提出的研究将通过提供一个模型来广泛影响该领域 通过它，mRNAs的一个基本子集TE-mRNAs在规范和非规范之间切换 引爆机制。拟议的研究结果可能为深入了解CAP-C的机制提供参考。 依赖的启动以及其他帽不依赖的启动途径的机制。

PROJECT SUMMARY/ABSTRACT The process through which mRNAs are selected for translation into proteins, or translation initiation, plays a major role in gene expression, and regulation of this process is critical in diseases such as cancer, diabetes, and neurological diseases. Motivated by this, the long-term goal of our studies is to understand and modulate non-canonical initiation mechanisms that play a significant role in the biology of these diseases. For most cellular mRNAs, initiation involves recognition of the N7-methylguanosine-triphosphate `cap' at the 5' end of the mRNA by eIF4E, the cap-binding protein, an event that results in the recruitment of additional eIFs, including eIF4G, and, ultimately, a ribosomal pre-initiation complex (PIC) that can initiate translation of the mRNA. Remarkably, under cellular stress conditions, such as tumor hypoxia, viral infection, nutrient limitation, etc., where the ability of eIF4E to recruit eIF4G and, consequently, cap-dependent initiation are compromised, a subset of mRNAs encoding genes that aid survival under such stress conditions can still be translated efficiently. These mRNAs somehow switch from the canonical, cap-dependent initiation pathway to a non-canonical, cap-independent initiation pathway. The mechanisms that regulate switching between cap- dependent and cap-independent initiation of these mRNAs remain unknown, impeding our understanding and ability to modulate this aspect of human health and disease. Previous studies of a candidate set of mRNAs, HIF-1α, FGF-9, VEGF-A and p53, that can switch from cap-dependent to cap-independent initiation as a result of eIF4E inhibition, have shown that switching correlates with increased levels of eIF4G or its homolog, DAP5 (eIF4G/DAP5). Interestingly, these mRNAs have also been shown to contain highly structured 5' untranslated regions that act as translational enhancers (TEs) that might play a role in regulating the switch between cap-dependent and cap-independent initiation. Based on our strong preliminary data, we hypothesize that the TEs in TE-containing mRNAs (TE-mRNAs) regulate this switch by directly recruiting eIF4G/DAP5, thereby enabling TE-mRNAs to recruit PICs even when eIF4E is inhibited. Guided by our preliminary data and using ensemble and single-molecule fluorescence methods in combination with complementary cellular and molecular biology approaches, we propose to: (1) characterize the strength and selectivity of eIF4G/DAP5 binding to TE-mRNAs, (2) determine the structural basis for TE recognition by eIF4G/DAP5, and (3) elucidate the causal relationship between the binding of eIF4G/DAP5 to the TEs of TE-mRNAs and the efficiency of PIC recruitment and translation of these TE-mRNAs. Collectively, the studies proposed here will broadly impact the field by providing a model through which an essential subset of mRNAs, TE-mRNAs, switch between a canonical and a non-canonical mechanism of initiation. The results of the proposed studies may provide insights into the mechanism of cap- dependent initiation as well as the mechanisms of other cap-independent initiation pathways.