Reinitiation involving upstream ORFs regulates <i>ATF4</i> mRNA translation in mammalian cells

TLDR

During cellular stresses, phosphorylation of eIF2 triggers a gene expression program to mitigate the disturbance, with ATF4 as a key transcriptional regulator. The study investigates how two upstream ORFs in the ATF4 5′ leader regulate its expression. In nonstressed cells, ribosomes reinitiate at inhibitory uORF2, suppressing ATF4, whereas stress‑induced eIF2 phosphorylation delays reinitiation, allowing ribosomes to bypass uORF2 and translate ATF4. The conserved uORF‑mediated reinitiation mechanism enhances ATF4 expression, promoting stress‑response gene expression across species.

Abstract

During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance. Central to this stress response is the transcriptional regulator activating transcription factor, ATF4. Here we describe the mechanism regulating ATF4 expression involving the differential contribution of two upstream ORFs (uORFs) in the 5′ leader of the mouse ATF4 mRNA. The 5′ proximal uORF1 is a positive-acting element that facilitates ribosome scanning and reinitiation at downstream coding regions in the ATF4 mRNA. When eIF2-GTP is abundant in nonstressed cells, ribosomes scanning downstream of uORF1 reinitiate at the next coding region, uORF2, an inhibitory element that blocks ATF4 expression. During stress conditions, phosphorylation of eIF2 and the accompanying reduction in the levels of eIF2-GTP increase the time required for the scanning ribosomes to become competent to reinitiate translation. This delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead reinitiate at the ATF4 -coding region. Increased expression of ATF4 would contribute to the expression of genes involved in remediation of cellular stress damage. These results suggest that the mechanism of translation reinitiation involving uORFs is conserved from yeast to mammals.