A deafness-associated tRNA^Aspmutation alters the m¹G37 modification, aminoacylation and stability of tRNA^Aspand mitochondrial function

Abstract

In this report, we investigated the pathogenic mechanism underlying the deafness-associated mitochondrial(mt) tRNAAsp 7551A > G mutation. The m.7551A > G mutation is localized at a highly conserved nucleotide(A37), adjacent (3′) to the anticodon, which is important for the fidelity of codon recognition and stabilization in functional tRNAs. It was anticipated that the m.7551A > G mutation altered the structure and function of mt-tRNAAsp. The primer extension assay demonstrated that the m.7551A > G mutation created the m1G37 modification of mt-tRNAAsp. Using cybrid cell lines generated by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA(mtDNA)-less (ρo) cells, we demonstrated the significant decreases in the efficiency of aminoacylation and steady-state level of mt-tRNAAsp in mutant cybrids, compared with control cybrids. A failure in metabolism of mt-tRNAAsp caused the variable reductions in mtDNA-encoded polypeptides in mutant cybrids. Impaired mitochondrial translation led to the respiratory phenotype in mutant cybrids. The respiratory deficiency lowed mitochondrial adenosine triphosphate production and increased the production of oxidative reactive species in mutant cybrids. Our data demonstrated that mitochondrial dysfunctions caused by the m.7551A > G mutation are associated with deafness. Our findings may provide new insights into the pathophysiology of maternally transmitted deafness that was manifested by altered nucleotide modification of mitochondrial tRNA.

Contribution of the tRNAIle 4317A→G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A→G mutation

“…In addition, impairment of mitochondrial translation resulted in the decreased rates in the basal OCR, or ATP-linked OCR, reserve capacity and maximal OCR among the control and mutant cell lines. These data highlighted that aberrant tRNA metabolism played a critical role in producing their respiration defects, as in the cases of cells carrying deafness-associated m.7511T>C, m.7551A>G and m.14692A>G mutations (6,9,10).…”

“…DIG-labeled oligodeoxynucleotides were generated by using DIG oligonucleotide Tailing kit (Roche). The hybridization and quantification of density in each band were performed as detailed previously (8)(9)(10).…”

“…Western blot analysis -Western blotting analysis was performed as detailed previously (8)(9)(10). Five micrograms of total proteins obtained from lysed mitochondria were denatured and loaded on sodium dodecyl sulfate (SDS) polyacrylamide gels.…”

“…Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was performed as detailed previously (8)(9)(10).…”

“…(C) Quantification of 7 polypeptides. The levels of ND1, ND4, ND5, ND6, CO2, CYTB and ATP8 in mutant and control cell lines were determined as described elsewhere (8)(9)(10). Graph details and symbols are explained in the legend to Figure 2.…”