Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response

TLDR

Stress‑induced eIF2α phosphorylation paradoxically enhances ATF4 translation, activating the ISR, and this regulation involves the 5′ mRNA uORFs, a mechanism shared with yeast GCN4 and resembling the yeast general control response. Low eIF2α phosphorylation in unstressed cells promotes ribosome reinitiation at the inhibitory uORF2, suppressing ATF4, whereas high phosphorylation in stressed cells delays capacitation, favoring reinitiation at ATF4 and activating the ISR. Mutation analysis showed ribosomes initiate efficiently at both uORFs and reinitiate at downstream AUGs after translating uORF1.

Abstract

Stress-induced eukaryotic translation initiation factor 2 (eIF2) α phosphorylation paradoxically increases translation of the metazoan activating transcription factor 4 (ATF4), activating the integrated stress response (ISR), a pro-survival gene expression program. Previous studies implicated the 5′ end of the ATF4 mRNA, with its two conserved upstream ORFs (uORFs), in this translational regulation. Here, we report on mutation analysis of the ATF4 mRNA which revealed that scanning ribosomes initiate translation efficiently at both uORFs and ribosomes that had translated uORF1 efficiently reinitiate translation at downstream AUGs. In unstressed cells, low levels of eIF2α phosphorylation favor early capacitation of such reinitiating ribosomes directing them to the inhibitory uORF2, which precludes subsequent translation of ATF4 and represses the ISR. In stressed cells high levels of eIF2α phosphorylation delays ribosome capacitation and favors reinitiation at ATF4 over the inhibitory uORF2. These features are common to regulated translation of GCN4 in yeast. The metazoan ISR thus resembles the yeast general control response both in its target genes and its mechanistic details.