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，许多细胞重新编程其蛋白质组以增殖并适应新的条件。蛋白质合成的调节被改变，不仅蛋白质的数量会改变，而且产生的蛋白质也会改变。了解mRNA如何通过非经典蛋白质合成机制翻译对于确定基因表达如何控制至关重要，特别是在压力，发育或疾病条件下。我们对非经典翻译机制的理解主要来自于全局分析，着眼于整个细胞的输出。这些全球性研究没有在分子水平上提供详细的机制见解，这对于确定可能的治疗干预的中间体、限速步骤和调节机制是必要的。本计画将利用单分子萤光、稳态萤光、动力学及分子生物学来鉴定分子间的相互作用，以建立详细的转译调控模式。此外，该项目将为不同的博士后、研究生和本科生科学家提供极好的培训机会。对于大多数细胞mRNA，起始涉及eIF 4 E（帽结合蛋白）识别mRNA 5'末端的N7-甲基鸟苷三磷酸“帽”，导致招募额外的蛋白质，包括eIF 4G，最终，启动翻译的核糖体前起始复合物（PIC）。在细胞应激条件下，当eIF 4 E募集eIF 4G的能力以及因此的帽依赖性起始受到损害时，含有高度结构化的5'UTR（非翻译区）和帮助细胞存活的编码基因的mRNA的子集仍然可以被有效地翻译。先前对从帽依赖性起始转换为帽非依赖性起始的mRNA（HIF-1α、FGF-9、VEGF-A和p53）的研究表明，转换与eIF 4G或其同系物DAP 5（eIF 4G/DAP 5）和eIF 4 E结合蛋白4 E-BP水平的增加相关。在初步数据的指导下，使用集成和单分子荧光方法以及互补的细胞和分子生物学方法，将研究调节这些mRNAs表达的三个关键方面：1）其他eIFs在mRNAs特异性和选择中的作用; 2）eIF 3帽结合和募集DAP 5的机制; 3）4 E-BP在选择性增强或抑制mRNA翻译中的作用。该奖项反映了NSF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.