DARS2 encodes the mitochondrial aspartate-tRNA ligase (mitochondrial aspartyl-tRNA synthetase), a nuclear-encoded class II aminoacyl-tRNA synthetase of the bacterial AspRS type. The enzyme is imported into mitochondria via an N-terminal arginine-rich targeting sequence and acts as a homodimer: it activates L-aspartate with ATP to form aspartyl-adenylate and transfers the aspartyl group to the 3' end of mitochondrial tRNA(Asp), supplying charged tRNA for synthesis of the 13 mtDNA-encoded respiratory-chain subunits. Each subunit has an N-terminal OB-fold anticodon-binding domain, a class II catalytic domain that binds ATP and aspartate, and a bacterial-type insertion domain that in the human enzyme is rotated to open the active-site groove for the tRNA acceptor arm. In human cells the enzyme is found in both the soluble and membrane-associated fractions of mitochondria. This horse entry (A0A9L0SB67, 616 aa) is the product of an alternative DARS2 transcript that skips one in-frame 108-nt exon, so it lacks 36 residues at the start of the insertion domain (human residues 341-376) while retaining the transit region, the anticodon-binding domain and every ATP- and aspartate-binding residue; whether this shorter isoform is expressed as a stable, active enzyme is unknown. The full-length horse product is A0A3Q2HM61 (652 aa).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000002 | TOO GENERAL | Summary: True but uninformative: the nucleotide bound is ATP, the co-substrate of the aspartate-tRNA ligase reaction, and ATP binding is kept elsewhere in this review. Reason: The class II catalytic domain binds ATP to form aspartyl-adenylate. The ATP-binding residues mapped from human DARS2 (266-268 RDE, E535, 584-587 GLDR) are all retained in A0A9L0SB67, so nucleotide binding is true, but the more specific ATP binding row (GO:0005524) and the aspartate-tRNA ligase activity replacement already entail it. Supporting Evidence: file:HORSE/DARS2/DARS2-bioinformatics/RESULTS-rereview.md All human UniProt substrate-binding features map onto identical residues in A0A9L0SB67 |
| GO:0003676 nucleic acid binding | IEA GO_REF:0000002 | MODIFY | Summary: Correct but too general; the nucleic acid bound by the OB-fold anticodon-binding domain is tRNA(Asp), so tRNA binding is the appropriate term. Reason: The InterPro OB-fold anticodon-binding signature (IPR004365) that drives this mapping is the domain that recognises the tRNA anticodon. In the human DARS2 structure the residues that contact the anticodon and acceptor-end bases of tRNA are conserved with E. coli AspRS, and the tRNA-binding surface is unusually electropositive. The horse gene is a one-to-one ortholog of human DARS2 within PANTHER PTHR22594:SF5, and the anticodon-binding domain is intact in this isoform. tRNA binding (GO:0000049), an is_a descendant of nucleic acid binding, is the supported specific term. It is subsidiary to the ligase activity. Proposed replacements: tRNA binding Supporting Evidence: PMID:23275545 Otherwise, almost all residues making direct and specific interactions with bases at both ends of the Eco tRNA are conserved in the human enzyme PMID:23275545 Electropositive patches render HsaDRS2 more attractive for its RNA partner file:HORSE/DARS2/DARS2-bioinformatics/RESULTS-rereview.md It is the one-to-one ortholog of human DARS2 within SF5. |
| GO:0004812 aminoacyl-tRNA ligase activity | IEA GO_REF:0000002 | MODIFY | Summary: Correct but too general; DARS2 is specifically an aspartate-tRNA ligase. Reason: Human DARS2 aminoacylates tRNA(Asp) with aspartate (direct kinetic measurements), and PAINT curators placed aspartate-tRNA ligase activity at the PTHR22594:SF5 root node PTN000502597, from which the horse DARS2 gene descends. All aspartate- and ATP-binding residues are conserved in this horse isoform, and the HAMAP type-1 AspRS rule (MF_00044) matches it. The amino-acid specificity is therefore supported and GO:0004815 aspartate-tRNA ligase activity is the correct specific term. Aspartate-tRNA(Asn) ligase activity (GO:0050560), which is annotated to human DARS2 from a single in vitro study, was not propagated by PAINT and is not transferred here. Activity of this particular exon-skipping isoform has not been tested; the call is made for the gene product. Proposed replacements: aspartate-tRNA ligase activity Supporting Evidence: PMID:15779907 Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. PMID:23275545 Comparative kinetic properties of HsaDRS2 and EcoDRS in tRNA aspartylation file:HORSE/DARS2/DARS2-bioinformatics/RESULTS-rereview.md So the horse gene sits inside both PAINT IBD nodes used for human DARS2 |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: ATP is the co-substrate of the aspartylation reaction and its binding residues are conserved; keep, as a component of the core ligase activity rather than a separate function. Reason: The class II catalytic domain binds ATP to form aspartyl-adenylate. The ATP-binding positions mapped from human DARS2 (266-268, 535, 584-587) are identical in A0A9L0SB67. ATP binding is as specific as a binding term can be here, but it is part of, and entailed in practice by, the aspartate-tRNA ligase activity that is the core function. Supporting Evidence: file:HORSE/DARS2/DARS2-bioinformatics/RESULTS-rereview.md All human UniProt substrate-binding features map onto identical residues in A0A9L0SB67 |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | MODIFY | Summary: Correct but too general; DARS2 is a mitochondrial enzyme, and mitochondrion is part of the cytoplasm in GO. Reason: The horse isoform retains the arginine-rich N-terminal extension corresponding to the human mitochondrial transit peptide. Human DARS2 is found in the soluble and membrane fractions of purified mitochondria. PAINT placed mitochondrion (GO:0005739) at node PTN000502598, which the horse gene descends from; the curators did not propagate the finer matrix or membrane terms, so mitochondrion is the replacement used here. The cytoplasm term itself derives from a generic class II aaRS rule and does not distinguish cytosolic from mitochondrial members. Proposed replacements: mitochondrion Supporting Evidence: PMID:30006346 This experiment reveals that the mt-AspRS is distributed in both the soluble and membrane fractions file:HORSE/DARS2/DARS2-bioinformatics/RESULTS-rereview.md The N-terminal mitochondrial transit region (human 1-47) is retained |
| GO:0006418 tRNA aminoacylation for protein translation | IEA GO_REF:0000002 | MODIFY | Summary: Correct but too general; DARS2 charges tRNA(Asp) for mitochondrial translation, so the mitochondrial compartment term is the supported refinement. Reason: DARS2 itself catalyses the aminoacylation step, so participation is direct. PAINT propagated aspartyl-tRNA aminoacylation (GO:0006422) from PTN000502597 and mitochondrion from PTN000502598, both of which are ancestors of horse DARS2. The conjunction of those two facts used to be expressed as mitochondrial aspartyl-tRNA aminoacylation (GO:0070146), but GO has obsoleted the amino-acid-specific aminoacylation processes, GO:0006422 and GO:0070146 among them, because the paired molecular function already carries the amino acid. Only the compartment half of the conjunction is still a process, so the refinement is GO:0070127 tRNA aminoacylation for mitochondrial protein translation, an is_a descendant of the annotated GO:0006418; aspartate specificity is carried by GO:0004815 on the MF row, which this review proposes separately. Proposed replacements: tRNA aminoacylation for mitochondrial protein translation Supporting Evidence: PMID:15779907 Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. file:HORSE/DARS2/DARS2-bioinformatics/RESULTS-rereview.md So the horse gene sits inside both PAINT IBD nodes used for human DARS2 |
| GO:0016874 ligase activity | IEA GO_REF:0000002 | MODIFY | Summary: Correct but very general; the specific activity is aspartate-tRNA ligase activity. Reason: The mapping comes from the type-1 AspRS signature (IPR004524). As for the aminoacyl-tRNA ligase row, the specific supported activity is GO:0004815 aspartate-tRNA ligase activity, placed by PAINT at PTN000502597 above horse DARS2 and measured for the human ortholog. Proposed replacements: aspartate-tRNA ligase activity Supporting Evidence: PMID:15779907 Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. file:HORSE/DARS2/DARS2-bioinformatics/RESULTS-rereview.md So the horse gene sits inside both PAINT IBD nodes used for human DARS2 |
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Download this section (compressed HTML)Q: Is the DARS2 transcript that skips the 108-nt exon (ENSECAT00000125808, encoding A0A9L0SB67) supported by horse RNA-seq or long-read data, and is the same exon skip seen in other mammals?
Q: Does removal of residues equivalent to human 341-376, at the start of the bacterial-type insertion domain, affect folding, dimerisation or tRNA(Asp) aminoacylation of mitochondrial AspRS?
Experiment: Express the full-length (A0A3Q2HM61) and exon-skipped (A0A9L0SB67) horse DARS2 products, compare tRNA(Asp) aminoacylation kinetics and oligomeric state, and test whether the short isoform rescues mitochondrial translation in DARS2-deficient cells.
Type: in vitro aminoacylation assay and complementation
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