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^Glu 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^Glu 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^Glu The destabilization of base-pairing (18A-Ψ55) caused by the m.14692A→G mutation perturbed the conformation and stability of tRNA^Glu An approximately 65% decrease in the steady-state level of tRNA^Glu was observed in mutant cells compared with control cells. A failure in tRNA^Glu 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^Glu.

Keywords: diabetes; hearing; mitochondrial disease; mtDNA; mutant; pathogenesis; posttranslational modification (PTM); tRNA.

FIGURE 1.

Pseudouridine sequencing of mitochondrial tRNA^Glu. A, schematic of pseudouridine sequencing shown in the cloverleaf structures of the human mitochondrial tRNA^Glu (WT and mutant (MT). Solid lines represent the DIG-labeled oligonucleotide probe specific for tRNA^Glu. Broken lines represent the potential stops of reverse transcription reaction caused by base modification, such as CMC-pseudouridine and m¹A. B, 2 μg of mitochondrial RNA from control (C3 and C4) and mutant (III-6 and III-9) cells was incubated with CMCT for CMC modification of Ψ residues. Reverse transcription was carried out to identify the stops caused by CMC-pseudouridine. The tRNA^Glu transcript was used as a control without base modification. Marker, DIG-labeled oligonucleotides of variable length.

FIGURE 2.

In vitro angiogenin cleavage assays. A, angiogenin digestion pattern of in vitro transcripts of wild-type (55U) and 14692A→G (55C) mutant tRNA^Glu. 2 μg of purified tRNA transcripts was used for the ANG cleavage reaction at various lengths (from 0–45 min). Cleavage products of tRNA transcripts were electrophoresed through a denaturing polyacrylamide gel and stained with methylene blue. Broken squares indicate the differences between wild-type (U55) and mutant tRNA^Glu (C55). B, angiogenin digestion pattern of mt-tRNA^Glu purified from the control cell line (C3) and mutant cell line (III-9). 2 μg of human mitochondrial RNAs was used for the ANG cleavage reaction at various lengths (from 0–36 h). Cleavage products of mitochondrial tRNAs were resolved in 15% denaturating polyacrilamide gels with 8 m urea, electroblotted, and hybridized with a DIG-labeled oligonucleotide probe specific for tRNA^Glu. Broken squares indicate the differences between RNAs from control (C3) and mutant (III-9) cells, respectively.

FIGURE 3.

Analysis of the conformation and stability of tRNA^Glu. A, cloverleaf structure of mutant human mitochondrial tRNA^Glu. Broken lines represent the anticipated tertiary base pairings such as A18:U55. B, schematic of the tertiary structure of tRNA^Glu derived from Suzuki et al. (27). The broken square represents the elbow region (D- and T-loops) of tRNA. C, assessment of conformation changes by PAGE analysis under denaturing and native conditions. The transcripts of wild-type and mutant tRNAs^Glu, tRNA^Phe (74 nt), tRNA^Asp, and tRNA^Thr were electrophoresed through native or denaturing polyacrylamide gel stained with ethidium bromide. D, Northern blotting analysis of mitochondrial tRNA under native conditions. 2 μg of total mitochondrial RNA from various cell lines was electrophoresed through native polyacrylamide gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probes specific for tRNA^Glu and tRNA^His, respectively.

FIGURE 4.

Northern blotting analysis of mitochondrial tRNA under a denaturing condition. A, equal amounts (2 μg) of total mitochondrial RNA from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probes specific for tRNA^Glu, tRNA^Ala, tRNA^His, tRNA^Ile, tRNA^Thr, and 5S rRNA. B, quantification of mitochondrial tRNA levels. Shown is the average relative tRNA content per cell normalized to the average content per cell of 5S rRNA in cells derived from two affected subjects (WZD81-III-6 and III-9) carrying the m.14692A→G mutation and two Chinese controls belonging to the same haplogroup (C3 and C4) lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. The error bars indicate 2 standard errors of the means. p indicates the significance, according to a t test, of the differences between mutant and control cell lines. The horizontal dashed lines represent the average value for each group.

FIGURE 5.

Western blotting analysis of mitochondrial proteins. A, 20 μg of total cellular proteins from various cell lines was electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with seven respiratory complex subunits in mutant and control cells with GAPDH as a loading control. B, quantification of mitochondrial protein levels. The levels of mitochondrial proteins in two mutant cell lines and two control cell lines were determined as described elsewhere (26). C, quantification of seven respiratory complex subunits. The levels of ND1, ND4, ND5, ND6, CO2, CYTB, and ATP6 in two mutant cell lines and two control cell lines were determined as described elsewhere (26). The graph details and symbols are explained in the legend for Fig. 4.

FIGURE 6.

Enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were investigated by enzymatic assay on complexes I, II, III, and IV in mitochondria isolated from various cell lines. The calculations were based on four independent determinations. The graph details and symbols are explained in the legend for Fig. 4.

FIGURE 7.

Respiration assays. A, an analysis of O₂ consumption in the various cell lines using different inhibitors. The OCRs were first measured on 2 × 10⁴ cells of each cell line under basal condition and then sequentially added to oligomycin (1.5 μm), FCCP (0.5 μm), rotenone (1 μm), and antimycin A (1 μm) at the indicated times to determine different parameters of mitochondrial functions. B, the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and non-mitochondrial OCR in mutant and control cell lines. The non-mitochondrial OCR was determined as the OCR after rotenone/antimycin A treatment. The basal OCR was determined as the OCR before oligomycin minus OCR after rotenone/antimycin A. The ATP-lined OCR was determined as the OCR before oligomycin minus OCR after oligomycin. Proton leak was determined as basal OCR minus ATP-linked OCR. The maximal OCR was determined as the OCR after FCCP minus non-mitochondrial OCR. The reserve capacity was defined as the difference between maximal OCR after FCCP minus basal OCR. The average values of five determinations for each cell line are shown. The horizontal dashed lines represent the average value for each group. The graph details and symbols are explained in the legend for Fig. 4.

FIGURE 8.

Measurement of cellular and mitochondrial ATP levels using a bioluminescence assay. Cells were incubated with 10 mm glucose or 5 mm 2-deoxy-d-glucose plus 5 mm pyruvate to determine ATP generation under mitochondrial ATP synthesis. Average rates of ATP level per cell line and are shown. A, ATP level in total cells. B, ATP level in mitochondria. Four to five determinations were made for each cell line. The graph details and symbols are explained in the legend for Fig. 4.

FIGURE 9.

Mitochondrial membrane potential analysis. The mitochondrial membrane potential was measured in two mutant and two control cell lines using a fluorescence probe JC-10 assay system. The ratio of fluorescence intensities Ex/Em = 490/590 and 490/530 nm (FL590/FL530) were recorded to delineate the ΔΨm level of each sample. The relative ratios of FL590/FL530 geometric mean between mutant and control cell lines were calculated to reflect the level of ΔΨm. Shown is the relative ratio of JC-10 fluorescence intensities at Ex/Em = 490/530 and 490/590 nm in the absence (A) and presence (B) of 10 μm FCCP. The average of four to five determinations for each cell line is shown. The graph details and symbols are explained in the legend for Fig. 4.

FIGURE 10.

Measurement of ROS. The rates of production in ROS from two affected matrilineal relatives and two control individuals were analyzed by a BD-LSR II flow cytometer system with or without H₂O₂ stimulation. The relative ratio of intensity (stimulated versus unstimulated with H₂O₂) was calculated. The average of three determinations for each cell line is shown. The graph details and symbols are explained in the legend for Fig. 4.