Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNA Ala as a major determinant. However, this base pair is divergent in human mitochondrial tRNA Ala , but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNA Ala , in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNA Ala in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg 663 , likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.
TARS2 encodes human mitochondrial threonyl tRNA-synthetase that is responsible for generating mitochondrial Thr-tRNAThr and clearing mischarged Ser-tRNAThr during mitochondrial translation. Pathogenic variants in TARS2 have hitherto been reported in a pair of siblings and an unrelated patient with an early onset mitochondrial encephalomyopathy and a combined respiratory chain enzyme deficiency in muscle. We here report five additional unrelated patients with TARS2-related mitochondrial diseases, expanding the clinical phenotype to also include epilepsy, dystonia, hyperhidrosis and severe hearing impairment. Additionally, we document seven novel TARS2 variants—one nonsense variant and six missense variants—that we demonstrate are pathogenic and causal of the disease presentation based on population frequency, homology modelling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.
Six pathogenic mutations have been reported in human mitochondrial tRNAThr (hmtRNAThr); however, the pathogenic molecular mechanism remains unclear. Previously, we established an activity assay system for human mitochondrial threonyl-tRNA synthetase (hmThrRS). In the present study, we surveyed the structural and enzymatic effects of pathogenic mutations in hmtRNAThr and then focused on m.15915 G > A (G30A) and m.15923A > G (A38G). The harmful evolutionary gain of non-Watson–Crick base pair A29/C41 caused hmtRNAThr to be highly susceptible to mutations disrupting the G30–C40 base pair in various ways; for example, structural integrity maintenance, modification and aminoacylation of tRNAThr, and editing mischarged tRNAThr. A similar phenomenon was observed for hmtRNATrp with an A29/C41 non-Watson–Crick base pair, but not in bovine mtRNAThr with a natural G29–C41 base pair. The A38G mutation caused a severe reduction in Thr-acceptance and editing of hmThrRS. Importantly, A38 is a nucleotide determinant for the t6A modification at A37, which is essential for the coding properties of hmtRNAThr. In summary, our results revealed the crucial role of the G30–C40 base pair in maintaining the proper structure and function of hmtRNAThr because of A29/C41 non-Watson–Crick base pair and explained the molecular outcome of pathogenic G30A and A38G mutations.
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