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

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4’s induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4’s translational control is more complex than originally described, underpinning its key role in diverse biological processes. [Display omitted] •A highly conserved stem-loop (SL3) occurs in the uORF2/ATF4 overlap•SL3 enables ribosome queuing to provide another layer of ATF4 translational control•SL3 is preceded by a near-cognate CUG in the ATF4 frame with which it genetically interacts•The canonical ATF4 translation start site is a subject of substantial leaky scanning Traditionally, ATF4 translational control was thought to comprise only delayed reinitiation. Smirnova et al. describe additional layers of ATF4 translational regulation, including substantial leaky scanning of ATF4’s AUG1 and ribosome queuing implemented by a stem-loop (SL3). SL3 co-operates with an upstream near-cognate start codon and localizes to the uORF2/ATF4 overlap.