Stem-loop induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. Since its discovery, the delayed translation reinitiation mechanism, with reinitiation-permissive uORF1 playing a key role, was thought to underlie the ATF4 stress-induced upregulation. However, mounting evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional means of its regulation. Contrasting the original model, we show that inhibitory uORF2 is inducible and translated even under stress; that it encompasses two post-transcriptionally modified adenines performing antagonistic roles; and that the canonical ATF4 translation start site is robustly leaky-scanned. Most importantly, we demonstrate that the presence of a stable stem-loop in the uORF2/ ATF4 overlap, immediately preceded by near-cognate CUG, introduces another layer of regulation in the form of ribosome queuing. Thus, ATF4 translational control is much more complex than originally described underpinning its key role in diverse biological processes.