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.

Department of Biology New York University New York NY USA

Department of Genetics and Genome Sciences Case Western Reserve University Cleveland OH USA

Institute of Genetics and Biotechnology Faculty of Biology University of Warsaw Warsaw Poland

Laboratory of Cellular and Molecular Immunology Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

Laboratory of Post transcriptional Control of Gene Expression Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

Laboratory of Regulation of Gene Expression Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

Laboratory of Structural Biology and Cell Signaling Institute of Microbiology of the Czech Academy of Sciences Prumyslova 595 252 50 Vestec Czech Republic

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bioRxiv. 2024 Feb 18:2023.07.12.548609. doi: 10.1101/2023.07.12.548609. PubMed

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Protocol for detecting and quantifying hATF4-HA in non-stress versus stress conditions using automated and quantitative Jess western blotting

Perturbations in eIF3 subunit stoichiometry alter expression of ribosomal proteins and key components of the MAPK signaling pathways