Abstract
A variety of cellular stresses lead to global translation attenuation due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2), which decreases the availability of the eIF2-GTP-Met-tRNAi ternary complex. However, a subset of mRNAs continues to be translated by non-canonical mechanisms under these conditions. In fact, although translation initiation of activating transcription factor 4 (ATF4) is normally repressed by an upstream open reading frame (uORF), a decreased availability of ternary complex leads to increased translation of the main ATF4-coding ORF. We show here that siRNA-mediated depletion of eIF5B—which can substitute for eIF2 in delivering Met-tRNAi—leads to increased levels of ATF4 protein in mammalian cells. This de-repression is not due to phosphorylation of eIF2α under conditions of eIF5B depletion. Although eIF5B depletion leads to a modest increase in the steady-state levels of ATF4 mRNA, we show by polysome profiling that the depletion of eIF5B enhances ATF4 expression primarily at the level of translation. Moreover, eIF5B silencing increases the expression of an ATF4-luciferase translational reporter by a mechanism requiring the repressive uORF2. Further experiments suggest that eIF5B cooperates with eIF1A and eIF5, but not eIF2A, to facilitate the uORF2-mediated repression of ATF4 translation.
Highlights
Translation of mRNA is critical yet highly energy-intensive, necessitating its stringent regulation [1]
We identify a role for eukaryotic initiation factor 5B (eIF5B) in upstream open reading frame (uORF)-mediated repression of activating transcription factor 4 (ATF4) translation initiation
Depletion of eIF5B leads to increased translation of the ATF4 transcript, and eIF5B-imposed repression of an ATF4-luciferase translational reporter fusion requires the repressive uORF2 to be intact (Figures 1–3)
Summary
Translation of mRNA is critical yet highly energy-intensive, necessitating its stringent regulation [1]. The translation of some mRNAs is increased under conditions of eIF2α phosphorylation, such as activating transcription factor 4 (ATF4). EIF5B was shown to act as an essential translation factor during hypoxia by facilitating Met-tRNAi delivery to ribosomes for efficient cap-dependent translation of hypoxia-response proteins in glioblastoma cells [20]. EIF5B has been shown to regulate cell cycle progression via regulating uORF-containing mRNAs such as p27 and p21 [6] These findings suggest a role for eIF5B in non-canonical mechanisms of translation initiation under cellular stress conditions. As eIF5B can apparently substitute for eIF2α in delivering Met-tRNAi during translation initiation [17,18,19,20], we hypothesized that eIF5B might play a role in the uORF-mediated regulation of ATF4 translation. Our data suggest that eIF5B facilitates the uORF2-mediated repression of ATF4 translation
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