Abstract Aminoacyl-tRNA Synthetases Research Articles

Isolation of bacterial compartments to track movement of protein synthesis factors

Aminoacyl-tRNA synthetases (AARSs) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARSs also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities. Cell fractionation included isolation of the cytoplasm, periplasm, membrane, outer membrane vesicles, and extracellular media. Specific endogenous proteins and RNAs were probed respectively within each fraction via Western blots using antibodies and by Northern blots with primers to unique regions of the nucleic acid.

MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.

EMBRYONIC FACTOR 31 encodes a tyrosyl-tRNA synthetase that is essential for seed development

Aminoacyl-tRNA synthetases (AARSs) involve the process of catalyzing the ligation of specific amino acids to their cognate tRNAs. Here we identified an Arabidopsis mutant embryonic factor 31 (fac31), its embryos arrested at development from one cell to globular stage. The FAC31 gene was identified by positional cloning and confirmed by a genetic complementation test with two independent T-DNA insertion lines and transgenic rescue with full-length genomic DNA. FAC31 encodes a Tyrosyl-tRNA synthetase and localize to mitochondria and cytoplasm. Fac31 mutants contain a point mutation from CAA to a stop codon TAA which may lead to a truncated protein. The phenotype of fac31 mutants are very similar to the T-DNA insertion lines Salk_016722 and Salk_045570 displayed smaller embryo sac contains only less number of endosperm nucleolus. Genetic analysis showed that the FAC31 gene had no parental effects through the transmission of mutated FAC31 gene by gametes. FAC31 is a high-conserved protein among animals and plants. RT-PCR analysis and promoter-GUS expression showed that it is expressed in nearly all tissues tested, strongly expressed in meristem of seedlings, the primordium of lateral root, young inflorescences, mature pollen, germinated pollen tubes and embryo sacs before heart stage. Our findings suggest that FAC31 is essential for the seed development through regulation the expanding of embryo sac and proliferation of endosperm nucleolus.

TRNA Synthetase as a Cytokine

Aminoacyl-tRNA synthetases (ARSs) have diverse biological functions beyond that of protein synthesis. Park et al. cloned the 17 human ARSs into human embryonic kidney 293 (HEK293) cells and screened the conditioned medium from the 11 ARS clones that were highly expressed. Lysyl-tRNA synthetase (KRS) was detectable in the medium, and analysis of various cell lines showed that tumor necrosis factor α (TNF-α) stimulated KRS secretion of between 0.4 and 1% of total cellular KRS in a subset of these cells, including HCT116 colon cancer cells. When added to immune cell lines, KRS exhibited saturable binding to the cell surface. Addition of KRS, but not tryptophanyl tRNA synthetase (WRS), to the RAW264.7 macrophage cell line inhibited cell proliferation, stimulated TNF-α secretion, stimulated metalloproteinase 9 activity, and stimulated chemokinesis. Pharmacological inhibitors were used to show that Gα i and the mitogen-activated protein kinase (MAPK) cascades involving p38 and ERK were involved in mediating the effects of KRS on the RAW264.7 cells. Thus, KRS joins the list of ARSs that have noncanonical signaling functions. S. G. Park, H. J. Kim, Y. H. Min, E.-C. Choi, Y. K. Shin, B.-J. Park, S. W. Lee, S. Kim, Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response. Proc. Nat. Acad. Sci. U.S.A. 102 , 6356-6361 (2005). [Abstract] [Full Text]