Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy.

Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.

Selective degradation of tRNASer(AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease.

Mitochondrial translation is of high significance for cellular energy homeostasis. Aminoacyl-tRNA synthetases (aaRSs) are crucial translational components. Mitochondrial aaRS variants cause various human diseases. However, the pathogenesis of the vast majority of these diseases remains unknown. Here, we identified two novel SARS2 (encoding mitochondrial seryl-tRNA synthetase) variants that cause a multisystem disorder. c.654-14T > A mutation induced mRNA mis-splicing, generating a peptide insertion in the active site; c.1519dupC swapped a critical tRNA-binding motif in the C-terminus due to stop codon readthrough. Both mutants exhibited severely diminished tRNA binding and aminoacylation capacities. A marked reduction in mitochondrial tRNASer(AGY) was observed due to RNA degradation in patient-derived induced pluripotent stem cells (iPSCs), causing impaired translation and comprehensive mitochondrial function deficiencies. These impairments were efficiently rescued by wild-type SARS2 overexpression. Either mutation caused early embryonic fatality in mice. Heterozygous mice displayed reduced muscle tissue-specific levels of tRNASers. Our findings elucidated the biochemical and cellular consequences of impaired translation mediated by SARS2, suggesting that reduced abundance of tRNASer(AGY) is a key determinant for development of SARS2-related diseases.

Elucidating the molecular mechanisms associated with TARS2-related mitochondrial disease.

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. In addition, 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 modeling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.

Nitrosative stress inhibits aminoacylation and editing activities of mitochondrial threonyl-tRNA synthetase by S-nitrosation.

Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.

The G3-U70-independent tRNA recognition by human mitochondrial alanyl-tRNA synthetase.

Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single wobble base pair, G3-U70, of tRNAAla as a major determinant. However, this base pair is divergent in human mitochondrial tRNAAla, but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNAAla, in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNAAla 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, Arg663, 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.

A natural non-Watson-Crick base pair in human mitochondrial tRNAThr causes structural and functional susceptibility to local mutations.

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.