Redox-sensitive mTOR-eIF4A signaling promotes selective P-glycoprotein translation.

The GCN2-ATF4 signaling axis orchestrates the hepatic transcriptional stress response to short-term sulfur amino acid deprivation.

Mammals rely on the integrated stress response (ISR) to maintain essential amino acid (EAA) homeostasis. The kinase GCN2 is a key ISR sensor that is rapidly activated by uncharged tRNAs during EAA deprivation, leading to eIF2α phosphorylation and selective translation of ATF4. ATF4 subsequently orchestrates a transcriptional program regulating amino acid metabolism, redox balance, and autophagy. In this study, we investigated the role of GCN2 in the early hepatic transcriptional response to dietary sulfur amino acids (SAA; methionine and cysteine) deprivation. Using ATF4-luciferase reporter mice, we demonstrate that short-term SAA deprivation rapidly activates the eIF2α-ATF4 pathway within 3 hours, with activation primarily localized to the liver. Complementary in vivo and ex vivo approaches revealed that genetic deletion or pharmacological inhibition of GCN2 abolishes early eIF2α phosphorylation and induction of ATF4 target gene, while PERK is dispensable for this response. Furthermore, GCN2 controls the induction of multiple adaptive transcriptional programs involved in amino acid transport, aminoacyl-tRNA synthesis, autophagy, serine biosynthesis, one-carbon metabolism and glutathione degradation highlighting a coordinated adaptive response to acute SAA deprivation. These findings establish GCN2 as a major sensor mediating the early hepatic response to SAA deprivation, and define a transcriptional program essential for maintaining amino acid homeostasis. In contrast, Fgf21 induction occurs independently of GCN2, indicating the existence of parallel adaptive mechanisms. Collectively, this work provides new insight into the early dynamics and molecular specificity of ISR activation in response to acute dietary SAA deprivation.

hY3 RNA binds and inhibits ribosomes and modulates the starvation-induced integrated stress response

Abstract Cells adapt to metabolic stress by orchestrating gene expression to mitigate cellular damage, sustain homeostasis, and promote survival. Within this framework, translational control provides a rapid and efficient layer of regulation. Non-coding RNAs have recently emerged as effective modulators of translation, partly by targeting the ribosome. The contribution of ribosome-associated non-coding RNAs (rancRNAs) to translation regulation, however, remains largely unexplored in human cells. Here, we identified the human Y3 (hY3) RNA as a rancRNA that inhibits protein synthesis and attenuates cellular metabolism. hY3 function was particularly critical under nutrient deprivation, where it promoted adaptive stress responses. In this context, depletion of hY3 disrupted the delicate balance between survival and apoptosis by reducing the expression of pro-survival factors and impairing the activation of the integrated stress response (ISR). Loss of hY3 reduced starvation-dependent phosphorylation of eukaryotic translation initiation factor 2α, thereby attenuating ISR signalling, which results in non-physiologically elevated global translation rates during nutrient deprivation. Together, our findings establish hY3 as a ribosome-bound regulator of translation and stress responses, positioning it as a determinant of cell fate under metabolic stress.

Stress induces DCP2 translation via a stalling-dependent mechanism

The integrated stress response (ISR) globally suppresses protein synthesis while selectively permitting translation of a small subset of stress-responsive mRNAs, many of which contain upstream or overlapping open reading frames (uORFs/oORFs). Although translational induction of transcripts such as ATF4 has classically been attributed to delayed re-initiation caused by reduced ternary complex availability, the mechanisms by which uORFs and oORFs allow ISR-selective translation remain incompletely understood. Here, using ribosome profiling during early ISR activation combined with reporter assays, we identify DCP2, encoding a major mRNA decapping enzyme, as a previously unrecognized ISR-induced transcript. We show that translational induction of DCP2 depends on an overlapping ORF whose conserved 3′ region, corresponding to a ribosome pausing site, acts as a potent inhibitory element. Both the DCP2 oORF and main ORF increase in translation during stress, indicating that stress relieves repression by this inhibitory element. This reveals a mode of ISR- dependent gene regulation in which inhibition by a nascent peptide or stalling element embedded either in a uORF or an oORF is relieved upon stress to induce translation.

The integrated stress response kinase GCN2 prevents ZAKα-Dependent inflammatory hyperactivation in macrophages.

Macrophages orchestrate inflammation through rapid and extensive proteome remodeling, yet the translational programs governing macrophage activation remain poorly defined. Here, we show that classically activated macrophages (LPS+IFNγ-treated) and alternatively activated macrophages (IL-4-treated) engage fundamentally distinct translational trajectories. Whereas alternatively activated macrophages sustain elevated protein synthesis, classically activated macrophages undergo a rapid but transient increase in translation that is subsequently restrained by the integrated stress response (ISR) kinase General Control Nonderepressible 2 (GCN2). Using puromycin incorporation, polysome profiling, and quantitative proteomics, we demonstrate that GCN2-mediated phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) limits global translation and constrains the pro-inflammatory response. Genetic loss of GCN2 results in excessive translation and hyperinflammation driven by the ribosome-associated stress sensor ZAKα (MAP3K20). Importantly, pharmacological inhibition of ZAKα in GCN2-deficient macrophages selectively normalizes tumor necrosis factor α (TNFα) secretion, establishing a functional regulatory axis in which GCN2 suppresses ZAKα-dependent inflammatory signaling. Together, these findings redefine translational control as a central checkpoint in macrophage activation, revealing how GCN2 mitigates ribosomal stress to prevent inflammatory hyperactivation, with potential therapeutic implications for TNFα-driven inflammatory diseases.

m6A pathway suppression reprograms gene expression during the integrated stress response

eIF3A Promotes the Warburg Effect in Cervical Cancer through m6A-dependent Translational Regulation of ENO1.

Eukaryotic translation initiation factors (eIFs) play a crucial role in tumor progression; however, which eIFs are most significant in cervical cancer (CC) remains unclear. In this study, eIF3A and eIF4E were found to be highly expressed in CC and associated with poor prognosis. Silencing either factor inhibited CC cell proliferation, induced apoptosis, and reduced glycolytic activity. Quantitative proteomic analysis and subsequent functional validation identified α-enolase (ENO1) as a common downstream effector of eIF3A and eIF4E, through which they promoted aerobic glycolysis and CC progression. Notably, eIF3A consistently exhibited a more pronounced functional effect than eIF4E. Further analyses demonstrated that eIF3A associated with N6-methyladenosine (m6A)-modified ENO1 mRNA and promoted its translation. eIF3A depletion reduced the abundance of ENO1 mRNA in actively translating polysomes and decreased the ENO1 protein-to-mRNA ratio without affecting total ENO1 mRNA abundance. Mutation of a key m6A site A359 in ENO1 mRNA similarly weakened its association with eIF3A and reduced ENO1 translation. Moreover, pharmacological inhibition of m6A-related regulation partially attenuated eIF3A-induced ENO1 expression, glycolytic activation, and tumor growth. Collectively, our results identify eIF3A as a key regulator of CC progression in an m6A-dependent ENO1 translation and suggest that targeting the eIF3A-m6A-ENO1 may have therapeutic potential for CC.