Reciprocal regulation of TORC signaling and tRNA modifications by Elongator enforces nutrient-dependent cell fate.
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ABSTRACT:
SUBMITTER: Candiracci J
PROVIDER: S-EPMC6584457 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5/ncm5 groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5/ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2-thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications.
| S-EPMC6738719 | biostudies-literature
Project description:In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only are previously established modifications observable by mass spectrometry in strains with the most conserved genes of each step deleted, but also additional modifications are detected, indicating a certain plasticity of the pathway in the amoeba. Unlike described for yeast, D. discoideum allows for an unconditional deletion of the single tQCUG gene, as long as the Elongator-dependent modification pathway is intact. In gene deletion strains of the modification pathway, protein amounts are significantly reduced as shown by flow cytometry and Western blotting, using strains expressing different glutamine leader constructs fused to GFP. Most dramatic are these effects, when the tQCUG gene is deleted, or Elp3, the catalytic component of the Elongator complex is missing. In addition, Elp3 is the most strongly conserved protein of the modification pathway, as our phylogenetic analysis reveals. The implications of this observation are discussed with respect to the evolutionary age of the components acting in the Elongator-dependent modification pathway.
| S-EPMC7430636 | biostudies-literature
Project description:Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.
| S-EPMC7072221 | biostudies-literature
Project description:Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure and function of its regulatory factor Kti12 have remained elusive. Here, we present the crystal structure of Kti12's nucleotide hydrolase domain trapped in a transition state of ATP hydrolysis. The structure reveals striking similarities to an O-phosphoseryl-tRNA kinase involved in the selenocysteine pathway. Both proteins employ similar mechanisms of tRNA binding and show tRNASec-dependent ATPase activity. In addition, we demonstrate that Kti12 binds directly to Elongator and that ATP hydrolysis is crucial for Elongator to maintain proper tRNA anticodon modification levels in vivo. In summary, our data reveal a hitherto uncharacterized link between two translational control pathways that regulate selenocysteine incorporation and affect ribosomal tRNA selection via specific tRNA modifications.
| S-EPMC6511879 | biostudies-literature
Project description:Elp3, the catalytic subunit of the eukaryotic Elongator complex, is a lysine acetyltransferase that acetylates the C5 position of wobble-base uridines (U34) in transfer RNAs (tRNAs). This Elongator-dependent RNA acetylation of anticodon bases affects the ribosomal translation elongation rates and directly links acetyl-CoA metabolism to both protein synthesis rates and the proteome integrity. Of note, several human diseases, including various cancers and neurodegenerative disorders, correlate with the dysregulation of Elongator's tRNA modification activity. In this review, we focus on recent findings regarding the structure of Elp3 and the role of acetyl-CoA during its unique modification reaction.
| S-EPMC7663514 | biostudies-literature
Project description:More than a decade ago, the purification of the form of the RNA polymerase II (PolII) engaged in elongation led to the discovery of an associated, multi-subunit (Elp1-6) complex named "Elongator" by the Svejstrup lab. Although further evidence supported the original notion that Elongator is involved in transcription, Elongator lacked some of the expected features for a regulator of the elongating PolII. The discovery by the Byström lab, based on genetic dissection, that Elongator is pivotal for tRNA modifications, and that all the reported phenotypes of Elongator mutants are suppressed by the overexpression of two tRNAs added to the confusion. The increasing range of both potential substrates and biological processes regulated by Elongator in higher eukaryotes indicates that the major challenge of the field is to determine the biologically relevant function of Elongator. Our recent proteome-wide study in fission yeast supports a coordinated codon-dependent regulation of translation by Elongator. Here we provide additional analyses extending this hypothesis to budding yeast and worm.
| S-EPMC3562296 | biostudies-literature
Project description:Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.
| S-EPMC4287497 | biostudies-literature
Project description:A signaling pathway involving ZAP-70, LAT, and SLP76 has been regarded as essential for receptor-driven T cell development and activation. Consistent with this model, mice deficient in SLP76 have a complete block at the double negative 3 stage of T cell development. Recently, however, it has been reported that inactivation of Cbl, a ubiquitin-protein isopeptide ligase, partially rescues T cell development in SLP76-deficient mice. To probe the influence of Cbl on domain-specific SLP76 functions, we reconstituted SLP76(-/-) Cbl(-/-) mice with Slp76 transgenes bearing mutations in each of three functional domains of SLP76 as follows: Y3F, in which the amino-terminal tyrosine residues of SLP76 were mutated, eliminating sites of SLP76 interaction with Vav, Nck, and Itk; Delta20, in which 20 amino acids in the proline-rich region of SLP76 were deleted, removing a binding site for Gads; and RK, in which arginine 448 of SLP76 was replaced by lysine, abolishing function of the Src homology 2 domain. Although each of these transgenes has been shown to partially rescue T cell development in SLP76(-/-) mice, we report here that Cbl inactivation completely reverses the severe double negative 3 developmental block that occurs in SLP76-deficient mice expressing the Y3F transgene (Y3F mice) and partially rescues the defect in positive selection in T cell receptor transgenic Y3F mice, but in contrast fails to rescue thymic development of SLP76-deficient mice expressing the Delta20 or RK transgene. Rescue in SLP76(-/-)Cbl(-/-)Y3F double-positive thymocytes is associated with enhanced tyrosine phosphorylation of signaling molecules, including Lck, Vav, PLC-gamma1, and ERKs, but not Itk, in response to T cell receptor stimulation. Thus, our data demonstrate that Cbl suppresses activation of a bypass signaling pathway and thereby enforces SLP76 dependence of early T cell development.
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Project description:Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, ?-adrenergic receptor-mediated activation of GTP-Rac-bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA-boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how ?-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling.
| S-EPMC3666698 | biostudies-literature
Project description:BackgroundBy studying genome-wide expression patterns in healthy and diseased tissues across a wide range of pathophysiological conditions, DNA microarrays have revealed unique insights into complex diseases. However, the high-dimensionality of microarray data makes interpretation of heterogeneous gene expression studies inherently difficult.ResultsUsing a large-scale analysis of more than 40 microarray studies encompassing ~2400 mammalian tissue samples, we identified a common theme across heterogeneous microarray studies evident by a robust genome-wide inverse regulation of metabolic and cell signaling pathways: We found that upregulation of cell signaling pathways was invariably accompanied by downregulation of cell metabolic transcriptional activity (and vice versa). Several findings suggest that this characteristic gene expression pattern represents a new principle of mammalian transcriptional regulation. First, this coordinated transcriptional pattern occurred in a wide variety of physiological and pathophysiological conditions and was identified across all 20 human and animal tissue types examined. Second, the differences in metabolic gene expression predicted the magnitude of differences for signaling and all other pathways, i.e. tissue samples with similar expression levels of metabolic transcripts did not show any differences in gene expression for all other pathways. Third, this transcriptional pattern predicted a profound effect on the proteome, evident by differences in structure, stability and post-translational modifications of proteins belonging to signaling and metabolic pathways, respectively.ConclusionsOur data suggest that in a wide range of physiological and pathophysiological conditions, gene expression changes exhibit a recurring pattern along a transcriptional axis, characterized by an inverse regulation of major metabolic and cell signaling pathways. Given its widespread occurrence and its predicted effects on protein structure, protein stability and post-translational modifications, we propose a new principle for transcriptional regulation in mammalian biology.
| S-EPMC2861677 | biostudies-literature