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

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.