A Deafness- and Diabetes-associated tRNA Mutation Causes Deficient Pseudouridinylation at Position 55 in tRNAGlu and Mitochondrial Dysfunction

Abstract

Several mitochondrial tRNA mutations have been associated with maternally inherited diabetes and deafness. However, the pathophysiology of these tRNA mutations remains poorly understood. In this report, we identified the novel homoplasmic 14692A→G mutation in the mitochondrial tRNA gene among three Han Chinese families with maternally inherited diabetes and deafness. The m.14692A→G mutation affected a highly conserved uridine at position 55 of the TΨC loop of tRNA The uridine is modified to pseudouridine (Ψ55), which plays an important role in the structure and function of this tRNA. Using lymphoblastoid cell lines derived from a Chinese family, we demonstrated that the m.14692A→G mutation caused loss of Ψ55 modification and increased angiogenin-mediated endonucleolytic cleavage in mutant tRNA The destabilization of base-pairing (18A-Ψ55) caused by the m.14692A→G mutation perturbed the conformation and stability of tRNA An approximately 65% decrease in the steady-state level of tRNA was observed in mutant cells compared with control cells. A failure in tRNA metabolism impaired mitochondrial translation, especially for polypeptides with a high proportion of glutamic acid codons such as ND1, ND6, and CO2 in mutant cells. An impairment of mitochondrial translation caused defective respiratory capacity, especially reducing the activities of complexes I and IV. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increasing production of reactive oxygen species in the mutant cells. Our findings may provide new insights into the pathophysiology of maternally inherited diabetes and deafness, which is primarily manifested by the deficient 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).…”

“…As a result, cochlear hair cells bearing both m.1555A>G and m.4317A>G mutations may be particularly sensitive to increased ATP demand (1,2,58). Furthermore, the deficient activities of respiratory chain complexes often impaired mitochondrial membrane potentials, which is a key indicator of cellular viability (8,10). Indeed, mitochondrial membrane potentials reflect the pumping of hydrogen ions across the inner membrane during the process of electron transport and oxidative phosphorylation (49,59).…”

“…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).…”