A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism

In this report, we investigated the molecular mechanism underlying a deafness-associated m.5783C > T mutation that affects the canonical C50-G63 base-pairing of T psi C stem of tRNA(Cys) and immediately adjacent to 5 ' end of light-strand origin of mitochondrial DNA (mtDNA) replication (OriL). Two dimensional agarose gel electrophoresis revealed marked decreases in the replication intermediates including ascending arm of Y-fork arcs spanning OriL in the mutant cybrids bearing m.5783C > T mutation. mtDNA replication alterations were further evidenced by decreased levels of Pol gamma A, Twinkle and SSBP1, newly synthesized mtDNA and mtDNA contents in the mutant cybrids. The m.5783C > T mutation altered tRNA(Cys) structure and function, including decreased melting temperature, conformational changes, instability and deficient aminoacylation of mutated tRNA(Cys). The m.5783C > T mutation impaired the 5 ' end processing efficiency of tRNA(Cys) precursors and reduced the levels of tRNA(Cys) and downstream tRNA(Tyr). The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced effects in the polypeptides harboring higher numbers of cysteine and tyrosine codons. These alterations led to deficient oxidative phosphorylation including instability and reduced activities of the respiratory chain enzyme complexes I, III, IV and intact supercomplexes overall. Our findings highlight the impact of mitochondrial dysfunction on deafness arising from defects in mitochondrial DNA replication and tRNA metabolism.