Dominant-negative NARS1 R534* mutation causes wild-type subunit poisoning and heterodimer predominance in cells.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNA with their cognate amino acids, an essential step in protein biosynthesis. While biallelic mutations in aaRSs often result in severe multi-organ dysfunction accompanied by developmental delays, monoallelic mutations typically cause milder, tissue-specific symptoms. However, a de novo monoallelic nonsense mutation (R534*) in the asparaginyl-tRNA synthetase (AsnRS)-resulting in a premature stop codon and 15-residue C-terminal truncation-has been identified in multiple families and is associated with severe neurodevelopmental symptoms. Here, we find that patient-derived lymphoblasts express similar amounts of wild-type (WT) and mutant (R534*) AsnRS and exhibit a severe proliferation defect. Like most aaRS family members, AsnRS functions as a homodimer. Structural analysis indicates that the region deleted in AsnRS (R534-P548) contributes to dimerization, tRNA binding, and stabilization of the catalytic site architecture. Indeed, AsnRSforms a weaker homodimer than AsnRS, displays impaired tRNA binding, along with a severe loss of enzymatic activity. Nevertheless, when exogenously expressed in human cells, AsnRS shows a much stronger tendency than AsnRS to dimerize with the endogenous WT enzyme, driving R534*/WT heterodimer predominance in the cell. Notably, the heterodimer is severely defective in enzymatic function, comparable to the AsnRShomodimer, indicating that AsnRSexerts a dominant-negative loss-of-function effect on the WT subunit through heterodimerization. These findings provide a mechanistic explanation for how a monoallelic AsnRS mutation can lead to profound cellular dysfunction and contribute to severe neurodevelopmental disease, offering new insights into aaRS-associated pathologies and potential therapeutic strategies.