RNA immunoprecipitation and sequencing of aminoacyl-tRNA synthetase-specific genes
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE120182 | GEO | 2019/02/05
REPOSITORIES: GEO
Similar Datasets
Project description:RNA immunoprecipitation and sequencing of aminoacyl-tRNA synthetase-specific genes
| PRJNA492296 | ENA
Project description:Identification of mRNAs enriched in G3BP immunoprecipitation
| PRJNA122685 | ENA
Project description:A phosphorylation-dependent endoribonuclease G3BP localizes in cytoplasmic stress granules (SGs). G3BP promotes SG assembly, oligomerizes, binds mRNA, and regulates levels of certain mRNAs. Transcripts bound to G3BP were identified through expression profiling of a total of 12,135 genes by immunoprecipitation (IP) with anti-G3BP or control IgG from S2-013 cells, converting the immunoprecipitated transcripts to cDNA by reverse transcription, and hybridizing these products to cDNA microarrays. Five G3BP-associated transcripts were identified.
2009-07-23 | GSE17056 | GEO
Project description:Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme's anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.
| S-EPMC5321558 | biostudies-literature
Project description:Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K(M) of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA(Gln) by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA(Gln) by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA(Gln) complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.
| S-EPMC2396676 | biostudies-literature
Project description:IGF2BP3-bound transcripts were identified by RNA immunoprecipitation and sequencing (RIP-Seq) and bioinformatics analysis. A pancreatic cancer cell line S2-013 was cultured on fibronectin. We performed RIP of IGF2BP3 using anti-IGF2BP3 antibody from the fibronectin-stimulated S2-013 extracts, and identified the associated mRNAs using a next generation sequencer.
2016-11-09 | GSE47597 | GEO
Project description:About 1 billion years ago, in a single-celled holozoan ancestor of all animals, a gene fusion of two tRNA synthetases formed the bifunctional enzyme, glutamyl-prolyl-tRNA synthetase (EPRS). We propose here that a confluence of metabolic, biochemical, and environmental factors contributed to the specific fusion of glutamyl- (ERS) and prolyl- (PRS) tRNA synthetases. To test this idea, we developed a mathematical model that centers on the precursor-product relationship of glutamic acid and proline, as well as metabolic constraints on free glutamic acid availability near the time of the fusion event. Our findings indicate that proline content increased in the proteome during the emergence of animals, thereby increasing demand for free proline. Together, these constraints contributed to a marked cellular depletion of glutamic acid and its products, with potentially catastrophic consequences. In response, an ancient organism invented an elegant solution in which genes encoding ERS and PRS fused to form EPRS, forcing coexpression of the two enzymes and preventing lethal dysregulation. The substantial evolutionary advantage of this coregulatory mechanism is evidenced by the persistence of EPRS in nearly all extant animals.
| S-EPMC6295713 | biostudies-literature
Project description:Aminoacyl-tRNA synthetases (aaRSs) are enzymes that catalyze the transfer of amino acids to their cognate tRNA. They play a pivotal role in protein synthesis and are essential for cell growth and survival. The aaRSs are one of the leading targets for development of antibiotic agents. In this review, we mainly focused on aaRS inhibitor discovery and development using in silico methods including virtual screening and structure-based drug design. These computational methods are relatively fast and cheap, and are proving to be of great benefit for the rational development of more potent aaRS inhibitors and other pharmaceutical agents that may usher in a much needed generation of new antibiotics.
| S-EPMC3907873 | biostudies-literature
Project description:Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the Methanosarcina barkeri monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA. M. barkeri encodes a special amber suppressor tRNA (tRNA(Pyl)) that presumably recognizes this UAG codon. It was reported that Lys-tRNA(Pyl) can be formed by the aminoacyl-tRNA synthetase-like M. barkeri protein PylS [Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) Science 296, 1459-1462], whereas a later article showed that Lys-tRNA(Pyl) is synthesized by the combined action of LysRS1 and LysRS2, the two different M. barkeri lysyl-tRNA synthetases. Pyrrolysyl-tRNA(Pyl) formation was presumed to result from subsequent modification of lysine attached to tRNA(Pyl). To investigate whether pyrrolysine can be directly attached to tRNA(Pyl) we chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-tRNA synthetase for charging pyrrolysine to tRNA(Pyl); lysine and tRNA(Lys) are not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNA(Pyl) with lysine. These in vitro data suggest that Methanosarcina cells have two pathways for acylating the suppressor tRNA(Pyl). This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon.
| S-EPMC515082 | biostudies-literature
Project description:Summary Aminoacyl-tRNA synthetases (aaRSs) serve a dual role in charging tRNAs. Their enzymatic activities both provide protein synthesis flux and reduce uncharged tRNA levels. Although uncharged tRNAs can negatively impact bacterial growth, substantial concentrations of tRNAs remain deacylated even under nutrient-rich conditions. Here we show that tRNA charging in Bacillus subtilis is not maximized due to optimization of aaRS production during rapid growth, which prioritizes demands in protein synthesis over charging levels. The presence of uncharged tRNAs is alleviated by precisely tuned translation kinetics and the stringent response, both insensitive to aaRS overproduction but sharply responsive to underproduction, allowing for just-enough aaRS production atop a “fitness cliff”. Notably, we find that the stringent response mitigates fitness defects at all aaRS underproduction levels even without external starvation. Thus, adherence to minimal, flux-satisfying protein production drives limited tRNA charging and provides a basis for the sensitivity and setpoints of an integrated growth-control network.
2020-07-20 | GSE141389 | GEO