eIF4F complex dynamics are important for the activation of the integrated stress response.

In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.

eIF4E integrates into stress response.

In this issue, Diamond et al.1 and Kim et al.2 report that depletion of eIF4E leads to translational upregulation of GCN4, a key player in the integrated stress response, in an eIF2α phosphorylation-independent manner, suggesting a new mode of translational adaptation.

eIF2ß zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding.

The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2ß subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2ß-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2ßS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2ßS264Y mutation has eIF2ß-γ interaction defect. Consistently, the eIF2ßT238A intragenic suppressor mutation restored the eIF2ß-γ and Met-tRNAiMet binding. The eIF2ß-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2ßT238A mutation, suggesting the eIF2ß-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2ßF217A/Q221A double mutation in the HTH domain. The eIF2ßT238A suppressor mutation could not rescue the eIF2ß binding defect of the eIF2γV281K mutation; however, combining the eIF2ßS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2ß with the eIF2γ subunit via its α1-helix, the eIF2ß-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2ß-γ interacts via two distinct binding sites.

A new G3BP1-GFP reporter system for assessing skin toxicity by real-time monitoring of stress granules in vitro.

The skin, the organ with the largest surface area in the body, is the most susceptible to chemical exposure from the external environment. In this study, we aimed to establish an in vitro skin toxicity monitoring system that utilizes the mechanism of stress granule (SG) formation induced by various cellular stresses. In HaCaT cells, a keratinocyte cell line that comprises the human skin, a green fluorescent protein (GFP) was knocked in at the C-terminal genomic locus of Ras GTPase-activating protein-binding protein 1 (G3BP1), a representative component of SGs. The G3BP1-GFP knock-in HaCaT cells and wild-type (WT) HaCaT cells formed SGs containing G3BP1-GFP upon exposure to arsenite and household chemicals, such as bisphenol A (BPA) and benzalkonium chloride (BAC), in real-time. In addition, the exposure of G3BP1-GFP knock-in HaCaT cells to BPA and BAC promoted the phosphorylation of eukaryotic initiation factor 2 alpha and protein kinase R-like endoplasmic reticulum kinase, which are cell signaling factors involved in SG formation, similar to WT HaCaT cells. In conclusion, this novel G3BP1-GFP knock-in human skin cell system can monitor SG formation in real-time and be utilized to assess skin toxicity to various substances.

LC3A-mediated autophagy elicits PERK-eIF2α-ATF4 axis activation and mitochondrial dysfunction: Exposing vulnerability in aggresome-positive cancer cells.

The unfolded protein response pathways (UPR), autophagy, and compartmentalization of misfolded proteins into inclusion bodies are critical components of the protein quality control network. Among inclusion bodies, aggresomes are particularly intriguing due to their association with cellular survival, drug resistance, and aggresive cancer behavior. Aggresomes are molecular condensates formed when collapsed vimentin cages encircle misfolded proteins before final removal by autophagy. Yet significant gaps persist in the mechanisms governing aggresome formation and elimination in cancer cells. Understanding these mechanisms is crucial, especially considering the involvement of LC3A, a member of the MAP1LC3 family, which plays a unique role in autophagy regulation and has been reported to be epigenetically silenced in many cancers. Herein, we utilized the tetracycline-inducible expression of LC3A to investigate its role in choroid plexus carcinoma cells, which inherently exhibit the presence of aggresomes. Live cell imaging was employed to demonstrate the effect of LC3A expression on aggresome-positive cells, while SILAC-based proteomics identified LC3A-induced protein and pathway alterations. Our findings demonstrated that extended expression of LC3A is associated with cellular senescence. However, the obstruction of lysosomal degradation in this context has a deleterious effect on cellular viability. In response to LC3A-induced autophagy, we observed significant alterations in mitochondrial morphology, reflected by mitochondrial dysfunction and increased ROS production. Furthermore, LC3A expression elicited the activation of the PERK-eIF2α-ATF4 axis of the UPR, underscoring a significant change in the protein quality control network. In conclusion, our results elucidate that LC3A-mediated autophagy alters the protein quality control network, exposing a vulnerability in aggresome-positive cancer cells.

Gene Expression Profiling to Unfolded Proteins Response as a Risk Modulator of Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease. Despite new methods of diagnostics and treatment as well as extensive biological and immunosuppressive treatment, the etiology of RA is not fully understood. Moreover, the problem of diagnosis and treatment of RA patients is still current and affects a large group of patients. It is suggested that endoplasmic reticulum (ER)-related features may impair adaptation to chronic stress, inferring the risk of rheumatoid arthritis. The main goal in this study was evaluation of changes in mRNA translation to determine chronic ER stress conditions in rheumatoid arthritis patients. The study group consist of 86 individuals including a total of 56 rheumatoid arthritis patients and 30 healthy controls. The expression level of mRNA form blood samples of RA patients as well as controls of the unfolded protein response (UPR)-associated genes (p-eIF2, BCL-2, PERK, ATF4, and BAX) were investigated using real-time qPCR. GAPDH expression was used as a standard control. Considering the median, the expression levels of PERK, BCL-2, p-eIF2, ATF4, and BAX were found to be significantly increased in the blood of RA patients compared with the control group. The p-value for the PERK gene was 0.0000000036, the p-value for the BCL-2 gene was 0.000000014, the p-value for the p-eIF2 gene was 0.006948, the p-value for the ATF4 gene was 0.0000056, and the p-value for the BAX gene was 0.00019, respectively. Thus, it can be concluded that the targeting of the components of the PERK-dependent UPR signaling pathway via small-molecule PERK inhibitors may contribute to the development of novel, innovative treatment strategies against rheumatoid arthritis.

Porcine epidemic diarrhea virus E protein induces formation of stress granules and attenuates protein translation through activation of the PERK/eIF2α signaling pathway.

Porcine epidemic diarrhea virus (PEDV) envelope protein (E) has been characterized as an important structural protein that plays critical roles in the interplay with its host to affect the virus life cycle. Stress granules (SGs) are host translationally silent ribonucleoproteins, which are mainly induced by the phosphorylation of eIF2α in the PERK/eIF2α signaling pathway. Our previous study found that PEDV E protein caused endoplasmic reticulum stress response (ERS)-mediated suppression of antiviral proteins' translation. However, the link and the underlying mechanism by which PEDV induces SGs formation and suppresses host translation remain elusive. In this study, our results showed that PEDV E protein significantly elevated the expression of GRP78, CANX, and phosphorylation of PERK and eIF2α, indicating that the PERK/eIF2α branch of ERS was activated. PEDV E protein localized to the ER and aggregated into puncta to reconstruct ER structure, and further induced SGs formation, which has been caused through upregulating the G3BP1 expression level. In addition, a significant global translational stall and endogenous protein translation attenuation were detected in the presence of E protein overexpression, but the global mRNA transcriptional level remained unchanged, suggesting that the shutoff of protein translation was associated with the translation, not with the transcription process. Collectively, this study demonstrates that PERK/eIF2α activation is required for SGs formation and protein translation stall. This study is beneficial for us to better understand the mechanism by which PEDV E suppresses host protein synthesis, and provides us a new insight into the host translation regulation during virus infection.

Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

GCN2-eIF2α signaling pathway negatively regulates the growth of triploid crucian carp.

GCN2-eIF2α signaling pathway plays crucial roles in cell growth,development, and protein synthesis. However, in polyploid fish, the function of this pathway is rarely understood. In this study, genes associated with the GCN2-eIF2α pathway (pkr, pek, gcn2, eif2α) are founded lower expression levels in the triploid crucian carp (3nCC) muscle compared to that of the red crucian carp (RCC). In muscle effect stage embryos of the 3nCC, the mRNA levels of this pathway genes are generally lower than those of RCC, excluding hri and fgf21. Inhibiting gcn2 in 3nCC embryos downregulates downstream gene expression (eif2α, atf4, fgf21), accelerating embryonic development. In contrast, overexpressing of eif2α can alter the expression levels of downstream genes (atf4 and fgf21), and decelerates the embryonic development. These results demonstrate the GCN2-eIF2α pathway's regulatory impact on 3nCC growth, advancing understanding of fish rapid growth genetics and offering useful molecular markers for breeding of excellent strains.

Integrating the response to stressed mitochondria.

Chakrabarty et al.1 demonstrate that phospho-EIF2α (pEIF2α), the translation initiation factor that mediates the integrated stress response (ISR), is necessary and sufficient for the autophagic degradation of mitochondria following the addition of mitochondrial stressors.

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response.

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

Collective cell migration relies on PPP1R15-mediated regulation of the endoplasmic reticulum stress response.

Collective cell migration is integral to many developmental and disease processes. Previously, we discovered that protein phosphatase 1 (Pp1) promotes border cell collective migration in the Drosophila ovary. We now report that the Pp1 phosphatase regulatory subunit dPPP1R15 is a critical regulator of border cell migration. dPPP1R15 is an ortholog of mammalian PPP1R15 proteins that attenuate the endoplasmic reticulum (ER) stress response. We show that, in collectively migrating border cells, dPPP1R15 phosphatase restrains an active physiological protein kinase R-like ER kinase- (PERK)-eIF2α-activating transcription factor 4 (ATF4) stress pathway. RNAi knockdown of dPPP1R15 blocks border cell delamination from the epithelium and subsequent migration, increases eIF2α phosphorylation, reduces translation, and drives expression of the stress response transcription factor ATF4. We observe similar defects upon overexpression of ATF4 or the eIF2α kinase PERK. Furthermore, we show that normal border cells express markers of the PERK-dependent ER stress response and require PERK and ATF4 for efficient migration. In many other cell types, unresolved ER stress induces initiation of apoptosis. In contrast, border cells with chronic RNAi knockdown of dPPP1R15 survive. Together, our results demonstrate that the PERK-eIF2α-ATF4 pathway, regulated by dPPP1R15 activity, counteracts the physiological ER stress that occurs during collective border cell migration. We propose that in vivo collective cell migration is intrinsically "stressful," requiring tight homeostatic control of the ER stress response for collective cell cohesion, dynamics, and movement.

RNA Activators of Stress Kinase PKR within Human Genes That Control Splicing or Translation Create Novel Targets for Hereditary Diseases.

Specific sequences within RNA encoded by human genes essential for survival possess the ability to activate the RNA-dependent stress kinase PKR, resulting in phosphorylation of its substrate, eukaryotic translation initiation factor-2α (eIF2α), either to curb their mRNA translation or to enhance mRNA splicing. Thus, interferon-γ (IFNG) mRNA activates PKR through a 5'-terminal 203-nucleotide pseudoknot structure, thereby strongly downregulating its own translation and preventing a harmful hyper-inflammatory response. Tumor necrosis factor-α (TNF) pre-mRNA encodes within the 3'-untranslated region (3'-UTR) a 104-nucleotide RNA pseudoknot that activates PKR to enhance its splicing by an order of magnitude while leaving mRNA translation intact, thereby promoting effective TNF protein expression. Adult and fetal globin genes encode pre-mRNA structures that strongly activate PKR, leading to eIF2α phosphorylation that greatly enhances spliceosome assembly and splicing, yet also structures that silence PKR activation upon splicing to allow for unabated globin mRNA translation essential for life. Regulatory circuits resulting in each case from PKR activation were reviewed previously. Here, we analyze mutations within these genes created to delineate the RNA structures that activate PKR and to deconvolute their folding. Given the critical role of intragenic RNA activators of PKR in gene regulation, such mutations reveal novel potential RNA targets for human disease.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

Multiple mechanisms activate GCN2 eIF2 kinase in response to diverse stress conditions.

Diverse environmental insults induce the integrated stress response (ISR), which features eIF2 phosphorylation and translational control that serves to restore protein homeostasis. The eIF2 kinase GCN2 is a first responder in the ISR that is activated by amino acid depletion and other stresses not directly related to nutrients. Two mechanisms are suggested to trigger an ordered process of GCN2 activation during stress: GCN2 monitoring stress via accumulating uncharged tRNAs or by stalled and colliding ribosomes. Our results suggest that while ribosomal collisions are indeed essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 via its direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. GCN2 activation by UV irradiation features lowered amino acids and increased uncharged tRNAs and UV-induced ribosome collisions are suggested to be dispensable. We conclude that there are multiple mechanisms that activate GCN2 during diverse stresses.

CHIP suppresses the proliferation and migration of A549 cells by mediating the ubiquitination of eIF2α and upregulation of tumor suppressor RBM5.

The protein kinase RNA-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α) pathway plays an essential role in endoplasmic reticulum (ER) stress. When the PERK-eIF2α pathway is activated, PERK phosphorylates eIF2α (p-eIF2α) at Ser51 and quenches global protein synthesis. In this study, we verified eIF2α as a bona fide substrate of the E3 ubiquitin ligase carboxyl terminus of the HSC70-interaction protein (CHIP) both in vitro and in cells. CHIP mediated the ubiquitination and degradation of nonphosphorylated eIF2α in a chaperone-independent manner and promoted the upregulation of the cyclic AMP-dependent transcription factor under endoplasmic reticulum stress conditions. Cyclic AMP-dependent transcription factor induced the transcriptional enhancement of the tumor suppressor genes PTEN and RBM5. Although transcription was enhanced, the PTEN protein was subsequently degraded by CHIP, but the expression of the RBM5 protein was upregulated, thereby suppressing the proliferation and migration of A549 cells. Overall, our study established a new mechanism that deepened the understanding of the PERK-eIF2α pathway through the ubiquitination and degradation of eIF2α. The crosstalk between the phosphorylation and ubiquitination of eIF2α shed light on a new perspective for tumor progression.

Spatially distinct molecular patterns of gene expression in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a heterogeneous disease that is pathologically characterized by areas of normal-appearing lung parenchyma, active fibrosis (transition zones including fibroblastic foci) and dense fibrosis. Defining transcriptional differences between these pathologically heterogeneous regions of the IPF lung is critical to understanding the distribution and extent of fibrotic lung disease and identifying potential therapeutic targets. Application of a spatial transcriptomics platform would provide more detailed spatial resolution of transcriptional signals compared to previous single cell or bulk RNA-Seq studies. METHODS: We performed spatial transcriptomics using GeoMx Nanostring Digital Spatial Profiling on formalin-fixed paraffin-embedded (FFPE) tissue from 32 IPF and 12 control subjects and identified 231 regions of interest (ROIs). We compared normal-appearing lung parenchyma and airways between IPF and controls with histologically normal lung tissue, as well as histologically distinct regions within IPF (normal-appearing lung parenchyma, transition zones containing fibroblastic foci, areas of dense fibrosis, and honeycomb epithelium metaplasia). RESULTS: We identified 254 differentially expressed genes (DEGs) between IPF and controls in histologically normal-appearing regions of lung parenchyma; pathway analysis identified disease processes such as EIF2 signaling (important for cap-dependent mRNA translation), epithelial adherens junction signaling, HIF1α signaling, and integrin signaling. Within IPF, we identified 173 DEGs between transition and normal-appearing lung parenchyma and 198 DEGs between dense fibrosis and normal lung parenchyma; pathways dysregulated in both transition and dense fibrotic areas include EIF2 signaling pathway activation (upstream of endoplasmic reticulum (ER) stress proteins ATF4 and CHOP) and wound healing signaling pathway deactivation. Through cell deconvolution of transcriptome data and immunofluorescence staining, we confirmed loss of alveolar parenchymal signals (AGER, SFTPB, SFTPC), gain of secretory cell markers (SCGB3A2, MUC5B) as well as dysregulation of the upstream regulator ATF4, in histologically normal-appearing tissue in IPF. CONCLUSIONS: Our findings demonstrate that histologically normal-appearing regions from the IPF lung are transcriptionally distinct when compared to similar lung tissue from controls with histologically normal lung tissue, and that transition zones and areas of dense fibrosis within the IPF lung demonstrate activation of ER stress and deactivation of wound healing pathways.

Oridonin Promotes Apoptosis in Rheumatoid Arthritis Fibroblast-like Synoviocytes Through PERK/eIF2α/CHOP of Endoplasmic Reticulum Stress Pathway.

Oridonin (ORI), derived from Chinese herbs Rabdosia rubescens, has anti-inflammatory, proapoptotic, anticancer effects. Previous studies have found that ORI induces apoptosis in rheumatoid arthritis fibroblast synovial cells (RA-FLSs), but this mechanism is not clear. We will investigate the apoptosis mechanism of ORI on RA-FLSs. RA-FLSs were treated with various concentrations of ORI (0, 5, 10, 15, 20, 25, and 30 µM) for 24 h. CCK8, LDH, and hochest/PI assay determined the viability, cytotoxicity, and death of ORI on RA-FLSs. The endoplasmic reticulum probe was used to observe structural changes of endoplasmic reticulum in RA-FLSs. RNA expression was detected with RNA sequencing analysis and quantitative real-time PCR. The PERK/eIF2α/CHOP pathway protein of the endoplasmic reticulum was verified with Western Blot. Our results show that ORI induced the apoptosis of RA-FLSs from CCK8, LDH, and Hochest/PI. The endoplasmic reticulum distribution was altered in RA-FLSs after being treated with ORI. Bioinformatics analysis of RNA sequencing data found that 1453 genes were elevated. The PERK/eIF2α/CHOP pathway of the endoplasmic reticulum was regulated from the Gene ontology and KEGG analysis. The results of quantitative real-time PCR and Western blot analysis verified the regulation of PERK/eIF2α/CHOP pathway in RA-FLSs. Our data imply that the endoplasmic reticulum's PERK/eIF2α/CHOP signaling pathway is certainly implicated in the induction of RA-FLS apoptosis by ORI. This study has important implications for the pharmacological effects of ORI and the treatment of RA.

A highly efficient human cell-free translation system.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

eIF3d controls the persistent integrated stress response.

Cells respond to intrinsic and extrinsic stresses by reducing global protein synthesis and activating gene programs necessary for survival. Here, we show that the integrated stress response (ISR) is driven by the non-canonical cap-binding protein eIF3d that acts as a critical effector to control core stress response orchestrators, the translation factor eIF2α and the transcription factor ATF4. We find that during persistent stress, eIF3d activates the translation of the kinase GCN2, inducing eIF2α phosphorylation and inhibiting general protein synthesis. In parallel, eIF3d upregulates the m6A demethylase ALKBH5 to drive 5' UTR-specific demethylation of stress response genes, including ATF4. Ultimately, this cascade converges on ATF4 expression by increasing mRNA engagement of translation machinery and enhancing ribosome bypass of upstream open reading frames (uORFs). Our results reveal that eIF3d acts in a life-or-death decision point during chronic stress and uncover a synergistic signaling mechanism in which translational cascades complement transcriptional amplification to control essential cellular processes.