An Upstream Open Reading Frame Regulates Translation of GADD34 during Cellular Stresses That Induce eIF2α Phosphorylation

Cellular stress such as endoplasmic reticulum stress, hypoxia, and viral infection activates an integrated stress response, which includes the phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2 alpha) to inhibit overall protein synthesis. Paradoxically, this leads to translation of a subset of mRNAs, like transcription factor ATF4, which in turn induces transcription of downstream stress-induced genes such as growth arrest DNA-inducible gene 34 (GADD34). GADD34 interacts with protein phosphatase 1 to dephosphorylate eIF2 alpha, resulting in a negative feedback loop to recover protein synthesis and allow translation of stress-induced transcripts. Here, we show that GADD34 is not only transcriptionally induced but also translationally regulated to ensure maximal expression during eIF2 alpha phosphorylation. GADD34 mRNAs are preferentially associated with polysomes during eIF2 alpha phosphorylation, which is mediated by its 5'-untranslated region (5'UTR). The human GADD34 5'UTR contains two non-overlapping upstream open reading frames (uORFs), whereas the mouse version contains two overlapping and out of frame uORFs. Using 5'UTR GADD34 reporter constructs, we show that the downstream uORF mediates repression of basal translation and directs translation during eIF2 alpha phosphorylation. Furthermore, we show that the upstream uORF is poorly translated and that a proportion of scanning ribosomes bypasses the upstream uORF to recognize the downstream uORF. These findings suggest that GADD34 translation is regulated by a unique 5'UTR uORF mechanism to ensure proper GADD34 expression during eIF2 alpha phosphorylation. This mechanism may serve as a model for understanding how other 5'UTR uORF-containing mRNAs are regulated during cellular stress.