Life without post-transcriptional addition of G-1: two alternatives for tRNAHis identity in Eukarya.

The identity of tRNA(His) is strongly associated with the presence of an additional 5'-guanosine residue (G-1) in all three domains of life. The critical nature of the G-1 residue is underscored by the fact that two entirely distinct mechanisms for its acquisition are observed, with cotranscriptional incorporation observed in Bacteria, while post-transcriptional addition of G-1 occurs in Eukarya. Here, through our investigation of eukaryotes that lack obvious homologs of the post-transcriptional G-1-addition enzyme Thg1, we identify alternative pathways to tRNA(His) identity that controvert these well-established rules. We demonstrate that Trypanosoma brucei, like Acanthamoeba castellanii, lacks the G-1 identity element on tRNA(His) and utilizes a noncanonical G-1-independent histidyl-tRNA synthetase (HisRS). Purified HisRS enzymes from A. castellanii and T. brucei exhibit a mechanism of tRNA(His) recognition that is distinct from canonical G-1-dependent synthetases. Moreover, noncanonical HisRS enzymes genetically complement the loss of THG1 in Saccharomyces cerevisiae, demonstrating the biological relevance of the G-1-independent aminoacylation activity. In contrast, in Caenorhabditis elegans, which is another Thg1-independent eukaryote, the G-1 residue is maintained, but here its acquisition is noncanonical. In this case, the G-1 is encoded and apparently retained after 5' end processing, which has so far only been observed in Bacteria and organelles. Collectively, these observations unearth a widespread and previously unappreciated diversity in eukaryotic tRNA(His) identity mechanisms.

Histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells.

Autoantibodies to histidyl-tRNA synthetase (HisRS) or to alanyl-, asparaginyl-, glycyl-, isoleucyl-, or threonyl-tRNA synthetase occur in approximately 25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl-tRNA synthetases to induce leukocyte migration. HisRS induced CD4(+) and CD8(+) lymphocytes, interleukin (IL)-2-activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH(2)-terminal domain, 1-48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl-tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl-tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl-tRNA and lysyl-tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl-tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Cellular localization of the target structures recognized by the anti-Jo-1 antibody: immunofluorescence studies on cultured human myoblasts.

Antibodies to Jo-1 (alpha Jo-1) are most characteristically detected in patients with the idiopathic inflammatory muscle disease polymyositis (PM). The Jo-1 antigen has previously been identified as histidyl-tRNA synthetase (HRS). In order to clarify the cellular localization of the antigenic targets recognized by the alpha Jo-1 antibody, immunofluorescence (IF) studies were performed with cultured human myoblasts. Incubation with alpha Jo-1 positive sera demonstrated granular cytoplasmic as well as nuclear staining, but only the cytoplasmic fluorescence was specifically inhibited by preabsorbing the sera with recombinant histidyl-tRNA synthetase (rHRS). A polyclonal rabbit anti-rHRS sera demonstrated granular cytoplasmic IF which was also specifically inhibited by preincubation with rHRS protein. Alpha Jo-1 negative healthy control or patient sera demonstrated nonspecific low intensity staining. 35S methionine biosynthetically labelled myoblast cell extracts immunoprecipitated with alpha Jo-1 positive sera and analyzed by SDS-PAGE revealed a specific band of the same molecular weight as the rHRS antigen. Our studies demonstrate that alpha Jo-1 specifically binds to antigen in the cytoplasm of cultured myoblasts. Alpha Jo-1 has been shown to inhibit HRS activity in vitro. Given the importance of aminoacyl tRNA synthetases such as HRS to intracellular protein assembly, intracytoplasmic binding and enzyme inhibition in vivo may potentially contribute to the pathogenesis of autoimmune muscle damage in PM.