During unfavorable cellular conditions (e.g., tumor hypoxia, viral infection, nutrient deprivation, etc.), the canonical, cap-dependent translation initiation pathway in human cells is suppressed by sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, by 4E-binding proteins. Additionally, the expression levels of eIF4G and its cellular homolog, death associated protein 5 (DAP5), are elevated. Under these conditions, a subset of cellular mRNAs, including many encoding proteins with important roles in human health and disease, (e.g. HIF-1α, FGF-9, and p53) is translated in a cap-independent manner. Despite their physiological importance, however, the molecular mechanisms underlying ca... More
During unfavorable cellular conditions (e.g., tumor hypoxia, viral infection, nutrient deprivation, etc.), the canonical, cap-dependent translation initiation pathway in human cells is suppressed by sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, by 4E-binding proteins. Additionally, the expression levels of eIF4G and its cellular homolog, death associated protein 5 (DAP5), are elevated. Under these conditions, a subset of cellular mRNAs, including many encoding proteins with important roles in human health and disease, (e.g. HIF-1α, FGF-9, and p53) is translated in a cap-independent manner. Despite their physiological importance, however, the molecular mechanisms underlying cap-independent initiation of this subset of cellular mRNAs remain unknown. Here, we have used fluorescence anisotropy-based equilibrium binding assays developed in our laboratories to demonstrate that an N-terminal truncated form of human eIF4G1 that cannot interact with eIF4E (ΔN-4G1) or DAP5 directly bind to the 5’ untranslated regions (UTRs) of these mRNAs. Specifically, we have measured the differential affinities with which ΔN-4G1 and DAP5 interact with the 5’ UTRs of four distinct members of this subset of cellular mRNAs. Using a luciferase-based gene expression reporter assay, we further demonstrate that these same 5’ UTRs can promote cap-independent initiation in an ΔN-4G1 or DAP5-dependent manner in a rabbit reticulocyte lysate-based in vitrotranslation system. Integrating the results of our quantitative binding- and in vitrotranslation studies, we propose a model specifying how a subset of cellular mRNAs switch from cap-dependent to cap-independent modes of translation initiation
