Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• MeSH
• iniciace translace peptidového řetězce MeSH
• malé podjednotky ribozomu eukaryotické * metabolismus   genetika   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• otevřené čtecí rámce * MeSH
• proteosyntéza MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• transkripční faktory bZIP * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• messenger RNA * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• transkripční faktory bZIP * MeSH

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.

• Klíčová slova
• ATF4, CP: Molecular biology, integrated stress response, ribosome, ribosome queuing, translation reinitiation, translational control, unfolded protein response,
• MeSH
• fyziologický stres MeSH
• HEK293 buňky MeSH
• lidé MeSH
• otevřené čtecí rámce * genetika   MeSH
• proteosyntéza * MeSH
• ribozomy * metabolismus   MeSH
• sekvence nukleotidů MeSH
• transkripční faktor ATF4 * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• ATF4 protein, human MeSH Prohlížeč
• transkripční faktor ATF4 * MeSH

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S subunit from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation at short upstream open reading frames (uORFs) harboring penultimate codons that confer dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited reinitiation at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on reinitiation at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) reinitiation. We found that the Tma proteins generally impede reinitiation at native uORF4 and uORF4 variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on reinitiation at native uORF1, and equipping uORF1 with Tma-dependent penultimate codons generally did not confer Tma-dependent reinitiation; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the reinitiation potential of the uORF and the penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• Klíčová slova
• DENR, MCTS1, Tma, eIF2D, reinitiation, ribosome recycling,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH