Cap‐independent translation of a subset of cellular mRNAs is driven by the direct recruitment of eIF4G or DAP5 to the 5′ UTR of these mRNAs

Apoptosis, hypoxia, and other stress conditions inhibit canonical, cap‐dependent translation by triggering overexpression of 4E‐binding protein (4E‐BP) and increasing 4E‐BP mediated sequestration of eIF4E. Under these conditions a subset of cellular mRNAs encoding proteins with important roles in human health and disease, including HIF‐1α, FGF‐9 and p53, are known to translate in a cap‐independent manner. Interestingly, eukaryotic initiation factor 4G (eIF4G) and/or DAP5 are also overexpressed under these stress conditions, suggesting eIF4G/DAP5 might play a role in the cap‐independent translation of cellular mRNAs during stress. Despite their physiological importance, however, the molecular mechanisms underlying cap‐independent initiation of this subset of cellular mRNAs remain unknown. To address this gap in our understanding, we have used fluorescence anisotropy‐based binding assays developed in our laboratories to demonstrate that two N‐terminal truncated forms of eIF4G (i) eIF4G 682‐1599 that cannot interact with eIF4E and (ii) eIF4G 557‐1599 that contains the eIF4E binding domain and or DAP5 can directly bind specifically and differentially to structural elements that are found in the 5′ UTRs of this subset of cellular mRNAs. We also report that the eIF4E binding domain on eIF4G 557‐1599 mutant increases the selectivity among the mRNAs even in the absence of eIF4E. Using a luciferase‐based gene expression reporter assay, we further demonstrate that these same structural elements can promote translation initiation in an eIF4GI or DAP5‐dependent manner in a nuclease‐treated rabbit reticulocyte lysate depleted of eIF4GI or DAP5. Collectively, our studies are providing quantitative binding data and in vitro translation data that are allowing us to elucidate how a subset of cellular mRNAs switch from cap‐dependent to cap‐independent modes of translation initiation; how the structural elements in these mRNAs recruit eIF4GI and/or DAP5 during cap‐independent translation initiation and how eIF4GI and/or DAP5 enables the subsequent assembly of the translation initiation complex. Support or Funding Information National Institutes of Health [NIH R01 GM 084288 to R.L.G]; National Center for Advancing Translational Sciences [1UL1TR002384‐01 Seed Project to D.J.G] and National Institutes of Health [NIH R01 GM 128239 to D.J.G]