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

项目总结/摘要 mRNA被选择翻译成蛋白质或翻译起始的过程， 在基因表达中起主要作用，并且该过程的调节在诸如癌症的疾病中是关键的， 糖尿病和神经系统疾病。受此启发，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起重要作用的非典型起始机制。为 大多数细胞mRNA，起始涉及在5'端识别N7-甲基鸟苷-三磷酸'帽'， 通过eIF 4 E（帽结合蛋白）终止mRNA，这是导致额外eIFs募集的事件， 包括eIF 4G，以及最终的核糖体前起始复合物（PIC），其可以启动eIF 4G的翻译。 mRNA。值得注意的是，在细胞应激条件下，如肿瘤缺氧、病毒感染、营养不良等， 限制等，其中eIF 4 E募集eIF 4G的能力以及因此的帽依赖性起始是 即使受到损害，在这种压力条件下帮助生存的mRNA编码基因的子集仍然可以被破坏。 高效翻译这些mRNA以某种方式从典型的帽依赖性起始途径转换为 一种非典型的帽独立起始途径。调节帽之间转换的机制- 这些mRNA的依赖性和帽非依赖性启动仍然未知，阻碍了我们的理解和 调节人类健康和疾病的这方面的能力。 先前的研究发现，HIF-1α、FGF-9、VEGF-A和p53等一组候选mRNA可以从 作为eIF 4 E抑制的结果的帽依赖性到帽非依赖性起始，已经表明， 与eIF 4G或其同源物DAP 5（eIF 4G/DAP 5）水平升高相关。有趣的是，这些mRNA 还显示含有高度结构化的5 ′非翻译区， (TEs)这可能在调节帽依赖性和帽非依赖性起始之间的转换中起作用。 基于我们强有力的初步数据，我们假设含TE mRNA（TE-mRNA）中的TE 通过直接募集eIF 4G/DAP 5来调节这种开关，从而使TE-mRNAs能够募集PIC， 当eIF 4 E被抑制时。在我们初步数据的指导下， 结合互补的细胞和分子生物学方法，我们 建议：（1）表征eIF 4G/DAP 5与TE-mRNA结合的强度和选择性，（2）确定 eIF 4G/DAP 5识别TE的结构基础，以及（3）阐明 eIF 4G/DAP 5与TE-mRNA的TE的结合以及PIC募集和翻译的效率 这些TE-mRNA。总的来说，这里提出的研究将通过提供一个模型来广泛影响该领域。 通过这个机制，一个重要的mRNA亚群TE-mRNA在规范和非规范之间转换， 启动机制。这些研究结果可能有助于深入了解CAP的机制。 依赖性起始以及其他帽非依赖性起始途径的机制。

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.