Decreased CCA-addition in human mitochondrial tRNAs bearing a pathogenic A4317G or A10044G mutation

Pathogenic point mutations in mitochondrial tRNA genes are known to cause a variety of human mitochondrial diseases. Reports have associated an A4317G mutation in the mitochondrial tRNA^Ile gene with fatal infantile cardiomyopathy and an A10044G mutation in the mitochondrial tRNA^Gly gene with sudden infant death syndrome. Here we demonstrate that both mutations inhibit in vitro CCA-addition to the respective tRNA by the human mitochondrial CCA-adding enzyme. Structures of these two mutant tRNAs were examined by nuclease probing. In the case of the A4317G tRNA^Ile mutant, structural rearrangement of the T-arm region, conferring an aberrantly stable T-arm structure and an increased T_m value, was clearly observed. In the case of the A10044G tRNA^Gly mutant, high nuclease sensitivity in both the T- and D-loops suggested a weakened interaction between the loops. These are the first reported instances of inefficient CCA-addition being one of the apparent molecular pathogeneses caused by pathogenic point mutations in human mitochondrial tRNA genes.