Pathology-related substitutions in human mitochondrial tRNA(Ile) reduce precursor 3' end processing efficiency in vitro

The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA^Ile precursor 3′ end maturation in vitro by 3′-tRNase. Strikingly, four pathogenic tRNA^Ile mutations reduce 3′-tRNase processing efficiency (V_max / K_M) to ~10-fold below that of wild-type, principally due to lower V_max. The structural impact of mutations was sought by secondary structure probing and wild-type tRNA^Ile precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA^Ile precursors with the greatest 3′ end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA^Ile precursor 3′ end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.