The UPR (Unfolded Protein Response) is a well-orchestrated response to ER protein folding and processing overload, integrating both transcriptional and translational outputs. Its three arms in mammalian cells, the PERK translational response arm, together with the ATF6 and IRE1-XBP1-mediated transcriptional arms, have been thoroughly investigated. Using ribosome footprint profiling, we performed a deep characterization of gene expression programs involved in the early and late ER stress responses, within WT or PERK −/− Mouse Embryonic Fibroblasts (MEFs). We found that both repression and activation gene expression programs, affecting hundreds of genes, are significantly hampered in the absence of PERK. Specifically, PERK −/− cells do not show global translational inhibition, nor do they specifically activate early gene expression programs upon short exposure to ER stress. Furthermore, while PERK −/− cells do activate/repress late ER-stress response genes, the response is substantially weaker. Importantly, we highlight a widespread PERK-dependent repression program, consisting of ER targeted proteins, including transmembrane proteins, glycoproteins, and proteins with disulfide bonds. This phenomenon occurs in various different cell types, and has a major translational regulatory component. Moreover, we revealed a novel interplay between PERK and the XBP1-ATF6 arms of the UPR, whereby PERK attenuates the expression of a specific subset of XBP1-ATF6 targets, further illuminating the complexity of the integrated ER stress response.

Summary The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a complex cellular response to ER protein misfolding that involves transcriptional regulatory branches and a PERK-mediated translational regulatory branch. Here we revealed that amino acid biosynthesis regulation is coupled to protein synthesis demands during ER stress. Specifically, we demonstrated that the UPR leads to PERK-dependent induction in the biosynthesis of specific amino acids, and to upregulation of their corresponding tRNA synthetases. Furthermore, we found that sequences of UPR-upregulated proteins are significantly enriched with these UPR-induced amino acids. Interestingly, whereas the UPR leads to repression of ER target proteins, we showed that secreted proteins tended to escape this repression and were highly enriched for the UPR-induced amino acids. Our results unravel coordination between amino acid supply, namely, biosynthesis and tRNA loading, and demand from UPR-induced proteins under ER stress, thus revealing an additional regulatory layer of protein synthesis.

The UPR (Unfolded Protein Response) is a well-orchestrated response to ER protein folding and processing overload, integrating both transcriptional and translational outputs. Its three arms in mammalian cells, the PERK translational response arm, together with the ATF6 and IRE1-XBP1-mediated transcriptional arms, have been thoroughly investigated.Using ribosome footprint profiling, we performed a deep characterization of gene expression programs involved in the early and late ER stress responses, within WT or PERK -/-Mouse Embryonic Fibroblasts (MEFs). We found that both repression and activation gene expression programs, affecting hundreds of genes, are significantly hampered in the absence of PERK. Specifically, PERK -/-cells do not show global translational inhibition, nor do they specifically activate early gene expression programs upon short exposure to ER stress. Furthermore, while PERK -/-cells do activate/repress late ER-stress response genes, the response is substantially weaker. Importantly, we highlight a widespread PERK-dependent repression gene expression program, consisting of ER targeted proteins, including transmembrane proteins, glycoproteins, and proteins with disulfide bonds. This phenomenon occurs in various different cell types, and has a major translational regulatory component. Moreover, we revealed a novel interplay between PERK and the XBP1-ATF6 arms of the UPR, whereby PERK attenuates the expression of a specific subset of XBP1-ATF6 targets, further illuminating the complexity of the integrated ER stress response.