The role of eIF3 and 4E-BP in non-canonical translation of a subset of human mRNAs

气候变化、营养缺乏和病毒感染都会对植物和动物细胞产生压力。在压力下，许多细胞重新编程它们的蛋白质组来增殖和适应新的条件。蛋白质合成的调控发生了改变，不仅蛋白质的数量发生了变化，而且产生的蛋白质种类也发生了变化。了解mrna如何通过非规范蛋白合成机制翻译，对于确定基因表达如何受到控制至关重要，特别是在应激、发育或疾病条件下。我们对非规范翻译机制的理解主要来自全局分析，着眼于整个细胞的输出。这些全球性的研究并没有在分子水平上提供详细的机制见解，而分子水平是识别中间体、限速步骤和可能的治疗干预调节机制所必需的。该项目将利用单分子荧光、稳态荧光、动力学和分子生物学来识别分子相互作用，建立一个详细的翻译调节模型。此外，该项目将为各类博士后、研究生和本科生提供良好的培训机会。对于大多数细胞mRNA，起始涉及到帽结合蛋白eIF4E对mRNA 5 '端n7 -甲基鸟苷-三磷酸“帽”的识别，导致额外蛋白质的募集，包括eIF4G，最终，核糖体起始前复合物（PIC）启动翻译。在细胞应激条件下，当eIF4E招募eIF4G的能力受到损害，从而导致帽依赖性起始时，含有高度结构化的5 ' utr（非翻译区）和编码有助于细胞存活的基因的mrna子集仍然可以有效地翻译。先前对mrna （HIF-1α、FGF-9、VEGF-A和p53）从帽依赖性起始转换为帽非依赖性起始的研究表明，这种转换与eIF4G或其同源物DAP5 （eIF4G/DAP5）和eIF4E结合蛋白4E-BP水平的升高有关。在初步数据的指导下，利用细胞生物学和分子生物学相辅相成的集合和单分子荧光方法，将研究这些mrna表达调控的三个关键方面：1)其他eif在mrna特异性和选择中的作用；2) eIF3帽结合和DAP5募集的机制；3)4E-BP在选择性增强或抑制mRNA翻译中的作用。结果有望产生新的机制见解帽独立翻译启动。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Climate change, nutritional deficiencies, and viral infections all produce stress on plant and animal cells. Under stress, many cells reprogram their proteome to proliferate and adapt to the new conditions. Regulation of protein synthesis is altered, and not only can the quantity of proteins change, but also which proteins are produced. Understanding how mRNAs are translated through non-canonical protein synthesis mechanisms is critical for determining how gene expression is controlled, especially under stress, development or disease conditions. Our understanding of non-canonical translation mechanisms has derived largely from global analysis, looking at whole cell outputs. These global studies do not provide detailed mechanistic insights on a molecular level, which are necessary to identify intermediates, rate limiting steps and mechanisms of regulation for possible therapeutic intervention. This project will identify molecular interactions using single molecule fluorescence, steady-state fluorescence, kinetics and molecular biology to build a detailed model of translational regulation. In addition, this project will provide excellent training opportunities for a diverse group of post-doctoral, graduate and undergraduate scientists.For most cellular mRNAs, initiation involves recognition of the N7-methylguanosine-triphosphate ‘cap’ at the 5’ end of mRNA by eIF4E, the cap-binding protein, resulting in recruitment of additional proteins, including eIF4G, and, ultimately, a ribosomal pre-initiation complex (PIC) to initiate translation. Under cellular stress conditions, when the ability of eIF4E to recruit eIF4G and, consequently, cap-dependent initiation is compromised, a subset of mRNAs containing highly structured 5’ UTRs (untranslated regions) and encoding genes that aid cell survival can still be translated efficiently. Previous studies of mRNAs (HIF-1α, FGF-9, VEGF-A and p53) that switch from cap-dependent to cap-independent initiation have shown that switching correlates with increased levels of eIF4G or its homolog, DAP5 (eIF4G/DAP5) and eIF4E binding protein, 4E-BP. Guided by preliminary data and using ensemble and single-molecule fluorescence methods with complementary cellular and molecular biology approaches, three critical aspects of regulating expression of these mRNAs will be investigated: 1) the role of other eIFs in specificity and selection of mRNAs; 2) the mechanism of eIF3 cap-binding and recruitment of DAP5, and 3) the role of 4E-BP in selectively enhancing or repressing the translation of mRNA. The outcomes are expected to yield new mechanistic insights into cap-independent translation initiation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.