Gene expression profile at Ptpmt1 cardiomyocytes-specific Knockout mcie, Eif2a mutant mice, Gcn2 Knockout mice, and Hri Knockout mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE201042 | GEO | 2022/04/21
REPOSITORIES: GEO
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Project description:Gene expression profile at Ptpmt1 cardiomyocytes-specific Knockout mcie, Eif2a mutant mice, Gcn2 Knockout mice, and Hri Knockout mice
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Project description:The major heat shock protein Hsp70 has been shown to form a complex with a scaffold protein Bag3, linking it to multiple signaling pathways. Via these interactions, the Hsp70-Bag3 module functions as a proteotoxicity sensor that controls cell signaling. Here, as a tool to identify signaling pathways regulated by this complex, we utilized JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3. Gene expression profiling followed by the pathway analysis indicated that a set of signaling pathways including the unfolded protein response (UPR) was activated by JG-98. Surprisingly, only the translation initiation factor eIF2a-associated branch of the UPR was activated under these conditions, while other UPR branches mediating induction of ER chaperones were not induced, suggesting that the response was not related to ER proteotoxicity and thus to ER-associated kinase PERK1. Indeed, induction of the UPR genes under these conditions was dependent on activation of a distinct cytoplasmic eIF2a kinase, HRI. We demonstrated that the Hsp70-Bag3 complex directly interacted with HRI and regulated phosphorylation of eIF2a upon induction of cytoplasmic proteotoxicity. Therefore, we uncovered a novel signaling response, which regulates cell death upon the buildup of abnormal protein species in cytoplasm via an Hsp70-Bag3-HRI-eIF2a axis.
2021-04-14 | GSE171440 | GEO
Project description:Cytoplasmic proteotoxicity regulates HRI-dependent phosphorylation of eIF2a via the Hsp70-Bag3 module.
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Project description:Iron and heme play central roles in red blood cell production. However, the mechanisms by which iron and heme levels coordinate erythropoiesis remain incompletely understood. HRI is a heme-regulated kinase that controls translation by phosphorylating eIF2a. Here, we investigate the global impact of iron, heme and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. By defining the underlying changes in translation during iron and HRI deficiencies, we validate known regulators of this process, including Atf4, and identify novel pathways such as co-regulation of ribosomal protein mRNA translation. Surprisingly, we found that heme and HRI pathways, but not iron-regulated pathways, mediate the major protein translational and transcriptional responses to iron deficiency in erythroblasts in vivo and thereby identify previously unappreciated regulators of erythropoiesis. Our genome-wide study uncovers the major impact of the HRI-mediated integrated stress response for the adaptation to iron deficiency anemia.
2018-09-03 | GSE119365 | GEO
Project description:Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNAMeti) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNAMeti binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNAMeti to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.
| S-EPMC5134716 | biostudies-literature
Project description:Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A-knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A-KO and wild-type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.
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Project description:Many positive-strand RNA viruses, including all known coronaviruses, employ programmed –1 ribosomal frameshifting (–1 PRF) to regulate the translation of polycistronic viral RNAs. However, only a few host factors have been shown to regulate –1 PRF. Through a reporter-based genome-wide CRISPR/Cas9 knockout screen, we identified several host factors that either suppressed or enhanced –1 PRF of SARS-CoV-2. One of these factors is eukaryotic translation initiation factor 2A (eIF2A), which specifically and directly enhanced –1 PRF in vitro and in cells. Consistent with the crucial role of efficient –1 PRF in transcriptase/replicase expression, loss of eIF2A reduced SARS-CoV-2 replication in cells. Transcriptome-wide analysis of eIF2A-interacting RNAs showed that eIF2A primarily interacted with 18S ribosomal RNA near the contacts between the SARS-CoV-2 frameshift-stimulatory element (FSE) and the ribosome. Thus, our results revealed an unexpected role for eIF2A in modulating the translation of specific RNAs independent of its previously described role during initiation.
2024-07-11 | GSE223690 | GEO
Project description:We apply ribosome profiling here to assess the role of eIF2A in translation initiation. For this we test the change in translation efficiency between HeLa control and eIF2A-KO cells, however we do not find any transcript to depend on eIF2A. Since eIF2A is thought to take over the function of eIF2 when eIF2 is inhibited, we also test conditions where the integrated stress response is activated, thereby leading to eIF2 inactivation. In none of our assays, however, could we detect a role of eIF2A in translation initiation.
2024-11-29 | GSE282509 | GEO
Project description:Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1) as a novel HbF regulator. Since HRI is an erythroid-specific protein kinase it presents a potential target for pharmacologic intervention. We find that maximal HbF induction requires >80-85% HRI depletion. As it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF inducing activity at 10-fold lower concentrations, at which condition there were minimal observed detrimental effects on erythroid cell maturation and viability as well as fewer alterations in the erythroid transcriptome. In sum, this study provides a foundation for the exploration of combining future small molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.
2020-09-21 | GSE150341 | GEO
Project description:Earlier investigations have associated mammalian eIF2A with Met-tRNAi binding to the 40S subunit and its recruitment to specialized mRNAs in a GTP-independent manner. Additionally, eIF2A has been implicated in non-AUG start codon initiation, particularly under conditions where eIF2 function is attenuated by phosphorylation of its α-subunit during stress or starvation. However, the precise role of eIF2A in vivo translation remains unclear. Moreover, it's uncertain if the conserved ortholog in budding yeast can functionally substitute for eIF2 during stress. To address these questions, we conducted ribosome profiling on a yeast deletion mutant lacking eIF2A, alongside isogenic wild-type (WT) cells, both in the presence or absence of eIF2α phosphorylation induced by amino acid starvation.
2024-01-03 | GSE241473 | GEO