During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4 gamma I (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5’ UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5’ UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity, but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5’ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

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Protein translation is an energy demanding process in the cells and plays a vital role in regulation of gene expression. During canonical translation initiation, eukaryotic initiation factor 4G (eIF4GI) plays a central role in recruitment of various initiation factors to mRNA. During cellular stress conditions, canonical cap‐dependent translation is suppressed by triggering overexpression of 4E binding protein (4EBP) and increasing 4EBP mediated sequestration of eIF4E. Under this condition, a subset of cellular mRNAs (e.g., FGF‐9, p53A) are known to translate in a cap‐independent manner. Recently, work in our lab has shown that eIF4GI directly interacts with the structured 5’UTRs of a subset of mRNAs and stimulates their translation in a cap independent manner. Yet, the detailed thermodynamics for the binding of RNA oligos corresponding to the 5’UTRs of FGF‐9 and p53A mRNAs, to two different truncated forms of recombinant eIF4GI protein, one lacking the eIF4E binding domain (eIF4GI682‐1599), and the other containing the eIF4E binding domain (eIF4GI557‐1599) remains unexplored. Here, our study revealed that the eIF4E binding domain of eIF4GI557‐1599 enhances the binding affinity among the mRNAs. The van't Hoff analysis of temperature dependent binding revealed that binding of eIF4GI557‐1599 to mRNAs is enthalpy‐driven, whereas for the shorter mutant of eIF4GI682‐1599 the binding is entropy driven. These thermodynamic data suggest that, eIF4GI557‐1599•mRNAs complex formation is potentially accompanied by an elevated hydrogen bonding and weakened hydrophobic interactions, favored in terms of its Gibbs free energy. It has been thought that the eIF4E domain of eIF4GI557‐1599 contributes a greater number of lysine and arginine moieties, which are responsible for formation of hydrogen bonds between eIF4GI to mRNAs and ultimately stabilize this interaction via conformational changes. Together this study provided quantitative data that highlight the contribution of the eIF4E‐binding domain of eIF4GI to the stability of the eIF4GI‐mRNA complex and gives us better insights into the molecular mechanisms for recruitment of eIFs to mRNA and subsequent translation of those mRNAs.