"Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS)."

"HmtAlaRS misactivated Ser with a discriminator factor of 1/569 (Table 1), which is significantly larger than 1/3300, the proposed threshold for frequency of errors in protein synthesis (31), suggesting that mtAlaRS requires editing function to prevent Ala-to-Ser misincorporation in mitochondria."

"In vitro aminoacylation assay indicated that the mutants C749A and V760E displayed only slightly decreased tRNAAla charging activity compared with wild type mtAlaRS (Figure 1D). Determination of post-transfer editing of mischarged Ser-tRNAAla showed that the C749A mutant was severely defective in editing while the initial velocity of the post-transfer editing activity of the V760E mutant was only slightly reduced (Figure 1E and F)."

"In this study, AARS2 overexpression did not result in a detectable increase of the mutated mt-tRNAAla but caused an increase incharged mt-tRNAAla in mutant cybrids, leading to enhanced mitochondrial translation. This strongly suggested that AARS2 improved the aminoacylation activity rather than preventing the degradation of mt-tRNAAla 5655A>G."

"Taken together, our data suggest that the p.Arg580Trp variant impacts on stability of mt-AlaRS protein but not aminoacylation or editing activities and that the β-barrel subdomain has a critical role in protein folding and stability."

"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."

"However, it synthesized more mischarged Gly-tRNAAla than did the wild-type hmtAlaRS, comparable with our previously constructed editing-defective C749A (42) (Figure 5D and E), supporting its defect in editing. Indeed, the result of the post-transfer editing assay showed that hmtAlaRS-R663E was unable to hydrolyze Gly-tRNAAla (Figure 5F)."

"Hs mt AlaRS charges cognate mt tRNAAla but not mt tRNAs specific for Asp, Leu, or Ser."

"The appended C-Ala domain, which plays an auxiliary role in prokaryotic AlaRSs and is dispensable for aminoacylation in the human cytoplasmic AlaRS system43,44, plays a central role as a folding aid for the intrinsically flexible mt tRNA elbow. Fine-tuned induced-fit adaptation selects mt tRNAAla and rejects non-cognate mt tRNAs (Supplementary Fig. 7g)."

"AARS2 lactylated the synthetic K336-containing PDHA1 peptide as well as the K457/8-containing CPT2 peptide (Fig. 5b; Supplementary information, Fig. S6b) via lactate- and ATP-dependent and pyrophosphate-inhibitable mechanisms."

"Treatment with 10 mM Me-Lac increased the Lac-K336 and Lac-K457/8 levels of ectopically expressed PDHA1 (Fig. 5d) and CPT2 (Fig. 5e) in C2C12 and HL-1 cells (Supplementary information, Fig. S6g). However, the lactylation was not observed when Aars2 was knocked out (Fig. 5d, e)."

"Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular L-lactate sensors required for L-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to L-lactate with micromolar affinity and they directly catalyse L-lactate for ATP-dependent lactylation on the lysine acceptor end."

"In response to L-lactate, AARS2 associates with cyclic GMP-AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice."

"To compare the relative contribution of AARS enzymes towards global basal levels of Kla in cells, we used siRNA to knockdown AARS1 and AARS2. However, the knockdown of either enzyme did not alter Kla levels (Fig. S6, B–E), suggesting the AARS pathway is also not required for global lysine lactylation."

"AARS2 deletion abrogated PDHA1 K336 and CPT2 K457/8 lactylation in the human granulosa cell line COV343 [38], and R199C exhibited a higher ability to lactylate PDHA1 K336 and CPT2 K457/8 in AARS2−/− COV343 cells (Fig. 3A, B)."

"The pathogenicity of the 2 AARS2 variants of P1 was proven in a recombinant yeast model."

"We found that mutations of either PCBP1 site 1 or site 2, or canonical splice donor or acceptor sites, are sufficient to cause exclusion of AARS2 exon-16 (Fig. 1l,m). Knockdown of human PCBP1 in both systems shows a similar pattern of AARS2 exon-16 exclusion (Fig. 1l,m and Extended Data Fig. 1c)."

"AARS2 (mitochondrial alanyl tRNA synthetase) catalyzes the reaction of alanine, mitochondrial tRNA(Ala), and ATP to form Ala-tRNA(Ala), AMP, and pyrophosphate."

"CC -!- FUNCTION: Catalyzes the attachment of alanine to tRNA(Ala) in a two- CC step reaction: alanine is first activated by ATP to form Ala-AMP and CC then transferred to the acceptor end of tRNA(Ala). Also edits CC incorrectly charged tRNA(Ala) via its editing domain (PubMed:21549344)."

"CC -!- SUBCELLULAR LOCATION: Mitochondrion {ECO:0000255|HAMAP-Rule:MF_03133, CC ECO:0000269|PubMed:21549344}."

"CC -!- CAUTION: Given that AARS2 is a mitochondrial protein, it is unclear how CC it can mediate lactylation of CGAS, which localizes in the cytosol and CC nucleus. {ECO:0000269|PubMed:39322678}."