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

气候变化、营养不足和病毒感染都会对植物和动物细胞造成压力。在压力下，许多细胞重新编程他们的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，而且产生哪些蛋白质也会改变。了解mRNAs是如何通过非规范的蛋白质合成机制翻译的，对于确定基因表达是如何控制的至关重要，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在很大程度上源于全球分析，着眼于整个单元的输出。这些全球性研究没有在分子水平上提供详细的机械性见解，这对于确定中间体、限速步骤和可能的治疗干预的调节机制是必要的。该项目将使用单分子荧光、稳态荧光、动力学和分子生物学来识别分子相互作用，以建立翻译调控的详细模型。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mRNAs，起始涉及到帽结合蛋白eIF4E识别mRNA 5‘端的N7-甲基鸟苷-三磷酸’帽‘，导致招募包括eIF4G在内的额外蛋白质，并最终招募核糖体起始前复合体(PIC)来启动翻译。在细胞应激条件下，当eIF4E招募eIF4G的能力受到损害，从而影响帽依赖的启动时，含有高结构的5‘UTRs(非翻译区)和编码有助于细胞生存的基因的mRNA子集仍然可以有效地翻译。以往对从帽依赖到帽非依赖性启动的α(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)的研究表明，这种转换与eIF4G或其同系物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，将研究这些mRNAs表达的三个关键方面：1)其他EIF在mRNAs特异性和选择中的作用；2)eIF3帽结合和DAP5的募集机制；3)4E-BP在选择性增强或抑制mRNAs翻译中的作用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.