Crosstalk between eIF2α and eEF2 phosphorylation pathways optimizes translational arrest in response to oxidative stress
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE137409 | GEO | 2019/09/14
REPOSITORIES: GEO
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Project description:Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which results in the increased phosphorylation of the eukaryotic initiation factor, eIF2a, and widespread translational repression. Protein synthesis is subsequently restored following the stress-induced transcriptional upregulation of GADD34 (growth arrest and DNA damage transcript 34) protein, a regulator of an eIF2a phosphatase. Genome-wide ribosome foot-printing in WT and GADD34-/- MEFs established that GADD34 mRNA is translated in unstressed cells and identified numerous mRNAs, whose translation was dependent on GADD34 even in the absence of ER stress. Following UPR activation, temporal analyses showed that the translational profile in GADD34-/- MEFs was stalled, displaying a pattern that mirrors the early response to UPR in WT MEFs. Basal GADD34 expression is also required for de-repression of translation and displacement of ER-bound polysomes that occur in early UPR. Thus, the overall UPR response is delayed in the GADD34-/- MEFs, gradually recovering as CReP expression increased. These studies reveal a critical role for basal GADD34 in the propagation of UPR signals in MEFs and mice and suggest that delayed UPR signaling protects GADD34-/- mice from tunicamycin-induced renal toxicity.
2015-10-01 | GSE69800 | GEO
Project description:Crosstalk between eIF2α and eEF2 phosphorylation pathways optimizes translational arrest in response to oxidative stress
| PRJNA565395 | ENA
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115161 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115160 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115159 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115158 | GEO
Project description:Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide b cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in b cells is unknown. EIF2A abundance is high in human and mouse islets relative to other tissues, and both thapsigargin and palmitate significantly increased EIF2A mRNA and EIF2A protein levels in MIN6 cells, mouse islets and human islets. Knockdowns of EIF2A, the related factor EIF2D, or both EIF2A and EIF2D, were not sufficient to cause apoptosis. On the other hand, transient or stable EIF2A over-expression protected MIN6 cells, primary mouse islets, and human islets from ER stress-induced, caspase-3-dependent apoptosis. Mechanistically, EIF2A overexpression decreased ERN1 (also known as IRE1a) expression in thapsigargin-treated MIN6 cells or human islets. In vivo, b cell specific EIF2A viral overexpression reduced ER stress, improved insulin signaling, and abrogated hyperglycemia in Ins2Akita/WT mice. EIF2A overexpression significantly increased expression of genes involved protein translation and reduced expression of pro-apoptotic genes (e.g. ALDH1A3). Remarkably, the decrease in global protein synthesis during UPR was prevented by EIF2A, despite ER stress-induced EIF2S1 phosphorylation. The protective effects of EIF2A were additive to those of ISRIB, a drug that counteracts the effects of EIF2S1 phosphorylation. Cells overexpressing EIF2A showed higher expression of translation factor EIF2B5, which may contribute to the lack of translational inhibition in these cells. We conclude that EIF2A is a novel target for b cell protection and the circumvention of EIF2S1-mediated translational repression.
2022-08-31 | GSE166829 | GEO
Project description:Cells and organisms frequently experience starvation. To adapt and survive, they mount an evolutionarily conserved stress response. A vital component in the mammalian starvation response is eukaryotic elongation factor 2 (eEF2) kinase (eEF2K), which responds to starvation by phosphorylating and inactivating the translation elongation driver eEF2, thus shutting down translation and facilitating survival. C. elegans efk-1/eEF2K phosphorylates EEF-2/eEF2 on a conserved residue and is required for starvation survival, but how it promotes survival remains unclear. Surprisingly, we found that eEF2 phosphorylation is unchanged in starved C. elegans, suggesting that efk-1 promotes survival via a noncanonical pathway. We show that efk-1 upregulates transcription of the DNA repair pathways, nucleotide excision repair (NER) and base excision repair (BER), to promote starvation survival. Furthermore, efk-1 suppresses oxygen consumption and ROS production in starvation to prevent oxidative stress. Thus, efk-1 enables starvation survival by protecting animals from starvation-induced oxidative damage through a translation-independent pathway.
2025-01-06 | GSE259223 | GEO
Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2α, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2α/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2α S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2α phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2α, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.
2016-07-03 | E-GEOD-68265 | biostudies-arrayexpress
Project description:Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal -1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.
2022-08-11 | PXD034873 | Pride