Gcn2 eIF2a kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in the mRNA 5’ leader regions
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PROVIDER: PRJNA599945 | ENA |
REPOSITORIES: ENA
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Project description:Cellular signaling controls translation through cis regulatory elements found in mRNAs. Gcn2 is the master regulator of translation during nutrient limitation in normal and cancer cells. Activated Gcn2 phosphorylates eIF2a, thereby repressing general translation while activating translation of specific mRNAs with upstream ORFs (uORFs) in its leader regions. Here we performed genome-wide measurement of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysomal microarray hybridization experiments identified a group of 1779 genes whose translation is up-regulated in Gcn2-dependent manner. We find that translation is reprogrammed to enhance organellar synthesis and transcription and repress ribosome synthesis. The 1779 genes included gcn5 and hri2 shown to promote growth under histidine starvation. They encode histone acetyl transferase and heme-binding eIF2a kinase activated by oxidative stress, and their 5’-leader contains 3 or 4 uORFs, respectively. Our reporter studies show that uORFs in gcn5 and hri2 operate similarly to those found in S. cerevisiae GCN4, the founding case for uORF-dependent regulation. Moreover, motif analysis identified 5’-UGA(C/G)GG-3’ as a motif promoting translation during starvation. Using hrd1 5’UTR with such a motif, we demonstrated its requirement in Gcn2-dependent translational control. To our knowledge, this is the first nucleotide motif found to be responsible for translational control by Gcn2. We propose that Gcn2 mediates translational control of more specific mRNAs than previously anticipated
2020-09-23 | GSE143299 | GEO
Project description:To study the mitochondrial stress response HRI-eIF2a-ATF4 signaling pathway in mitochondrial cardiomyopathy , we generated Ptpmt1 cardiomyocytes-specific Knockout mcie, Eif2a mutant mcie, Gcn2 Knockout mcie, and Hri Knockout mcie
2022-04-21 | GSE201042 | GEO
Project description:General translational repression is predicted as a key process to reduce energy consumption under hypoxia. We have previously showed that mRNA loading onto polysome is reduced in Arabidopsis under submergence. Here, we showed that plant stress activated GCN2 (general control nonderepressible 2) can phosphorylate eIF2a (Eukaryotic Initiation Factor 2a) in Arabidopsis under submergence, and this process is reversible after desubmergence. Compared to the wild-type, the reduction in polysome loading during submergence was less severe in the gcn2 mutant. Transgenic lines overexpressing GCN2 had more ATP and conferred better tolerance under submergence, suggesting that GCN2 might modulate the dynamics of translation to adjust the energy homeostasis under hypoxia. Interestingly, GCN2-eIF2a signaling was activated by ethylene under submergence. However, GCN2 activity was not affected in ein2-5 and eil1ein3 under submergence, suggesting that GCN2 activity was regulated by noncanonical ethylene signaling. In addition, the polysome loading was retained in both ein2-5 and etr1-1 under submergence, implying that ethylene modulated the dynamic translation under submergence via EIN2 and GCN2. Notably, our NGS analysis also demonstrated that EIN2 and GCN2 regulated the translation of 23 core hypoxia genes as well as 53% translational repressed genes under submergence. On the other hand, EIN2 and GCN2 also affected the expression of genes involved in hypoxic response, ethylene response, biotic stress and negative regulation of cytokinin signaling. Taken together, these demonstrated that entrapped ethylene triggers GCN2 and EIN2 to ensure the translation of stress required proteins under submergence and also provide a step stone for future investigation how eukaryotic cells modulate the translation to response for the changeable environments.
2022-06-08 | GSE149414 | GEO
Project description: Not available
| S-EPMC7498311 | biostudies-literature
Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Gcn2 wild-type or knockout mouse liver were perfused with complete amino acids media or media lacking methionine for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Gcn2+/+, +Met or -Met; total and polysome fraction of Gcn2-/-, +Met or -Met. Each array analysis was done in duplicate.
2009-11-23 | E-GEOD-11496 | biostudies-arrayexpress
Project description:Residual cell-intrinsic innate immunity in cancer cells hampers infection with oncolytic viruses. Control of mRNA translation is an important feature of innate immunity, yet the identity of mRNA substrates that participate in host defences remain ill-defined. We characterized the translatome of resistant murine “4T1” breast cancer cells infected with three of the most clinically advanced oncolytic viruses: Herpes Simplex virus 1, Reovirus and Vaccinia virus. Common among all three infections were translationally de-repressed mRNAs including Inpp5e, encoding an inositol 5-phosphatase that modifies lipid second messenger signalling. We found that viral infection induced expression of an Inpp5e mRNA variant that lacks repressive upstream open reading frames (uORFs) within its 5’ leader and is efficiently translated. Furthermore, we show that INPP5E contributes to antiviral immunity by altering virus attachment. These findings uncover a role for translational control through alternative 5’ leader expression and assign an antiviral function to the ciliopathy gene INPP5E.
2019-11-30 | GSE137757 | GEO
Project description:General control nonderepressible 2 (GCN2) phosphorylates eIF2α, regulating translation in response to nutritional stress. Here, we show that mammalian ribosomes are potent GCN2 activators. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) showed GCN2 interacting with domain II of the uL10 P-stalk protein. The P-stalk is a uL10/P12/P22 pentameric complex that is part of the ribosomal GTPase-associated center. Recombinant human P-stalk greatly stimulates GCN2. Both domain II of uL10 and the C-terminal tails of P1 and P2 are necessary for maximal GCN2 activation. On actively translating ribosomes, the C-terminal tails of P1 and P2 are sequestered by elongation factors, suggesting P-stalk availability could link translational stress to GCN2 activation.
2019-05-24 | PXD013051 | Pride
Project description:Carcinoma dissemination can occur when heterogeneous tumor and tumor stromal cells clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and interact with the microenvironment to direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system that mimics native breast tumor microenvironment, we developed 3D computational models which hypothesize that leader cells generate high protrusive forces and overcome extracellular matrix (ECM) resistance. Using single cell sequencing, we reveal leader cells are heterogeneous, and we identify and isolate a unique Cadherin-3 (Cdh3) positive leader cell subpopulation necessary and sufficient to lead migration. Cdh3 controls leader cell protrusion dynamics and local production of Laminin-332 which is required for integrin/focal adhesion function. Our findings highlight how a subset of leader cells interact with the microenvironment to direct collective migration.
2022-11-25 | GSE171203 | GEO
Project description:Compared protein synthesis in Gcn2 WT vs Gcn2 KO murine PDAC cells (KRPC) in amino acid-replete or leucine-free media.
2022-10-13 | PXD028143 | Pride
Project description:When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. We examine how yeast growth and tRNA charging or aminoacylation is affected during amino acid depletion in the presence and absence of GCN2. tRNA charging is measured using a microarray technique which allows for simultaneous measurement of all cytosolic tRNAs. A fully prototrophic and its isogenic GCN2 deletion strain were used. We measured relative tRNA charging levels in yeast strains with an intact and deleted GCN2.
2010-08-01 | E-GEOD-22452 | biostudies-arrayexpress