Mutation in TRMU related to transfer RNA modification modulates the phenotypic expression of the deafness-associated mitochondrial 12S ribosomal RNA mutations

Abstract

The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.

Figure 1.

Mutation analysis of TRMU. A, Partial sequence chromatograms of TRMU from affected individual V-3 and normal-hearing individual V-4 of the Arab-Israeli family and control #3. The arrow indicates the location of the nucleotide changes at position 28. B, RFLP PCR for the G28T mutation in several members of the Arab-Israeli family. C, Sequence alignment of the N-terminal sequence of human TRMU with its homologs. The arrow indicates the position of the A10S mutation.

Figure 2.

Eight pedigrees with nonsyndromic deafness. The TRMU G28T mutation was initially identified, by direct sequencing of PCR fragments spanning coding regions, with use of genomic DNA derived from members of an Arab-Israeli kindred as template and subsequently screened by RFLP PCR. Members marked with an asterisk (*) were examined for the TRMU mutation by direct sequencing of PCR fragments spanning coding regions. Hearing-impaired individuals are indicated by blackened symbols. Individuals who harbored homozygous (−/−), heterozygous (+/−), or wild type (+/+) mutations are indicated.

Figure 3.

Import into mitochondria and processing of human TRMU precursor. SDS-PAGE analysis of the in vitro transcribed/translated products synthesized in the presence of [³⁵S]methionine, with use of cDNA carrying human wild-type and mutant (A10S) TRMU or mTERF as template, analyzed directly or after incubation with isolated HeLa cell mitochondria for 30 min at 37°C under various conditions, detailed in the “Material and Methods” section.

Figure 4.

Quantification of 2-thiouridine modification in mt tRNA^Lys, tRNA^Glu, and tRNA^Gln. A, The 3′ end-labeled mt tRNA^Lys from each lymphoblastoid cell line, analyzed by 10% polyacrylamide gel electrophoresis with (+) or without (−) APM. The retarded bands of 2-thiolated tRNAs and nonretarded bands of tRNA without thiolation are marked with arrows. B, The 3′ end-labeled mt tRNA^Lys, tRNA^Glu, and tRNA^Gln from mutant lymphoblastoid cell lines carrying the A10S mutation, analyzed by 10% polyacrylamide gel electrophoresis with (+) or without (−) APM. C, Proportion in vivo of the 2-thiouridine modification levels of mt tRNAs. The calculations were based on three independent determinations of each mt tRNA in each cell line. The error bars indicate 2 SEMs; P indicates the significance, according to Student's t test, of the difference between mutant and control values for each mt tRNA.

Figure 5.

A, Northern-blot analysis of mt tRNA. Equal amounts (5 μg) of total mtRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, were electroblotted, and were hybridized with DIG-labeled oligonucleotide probes specific for the mt tRNA^Lys, tRNA^Glu, tRNA^Leu(UUR), tRNA^Ser(UCN), tRNA^His, and tRNA^Met; the blots were then stripped and rehybridized with DIG-labeled 5S rRNA probe as a control. B, Quantification of the levels of mt tRNA. Average relative tRNA^Lys, tRNA^Glu, tRNA^Leu(UUR), tRNA^Ser(UCN), tRNA^His, and tRNA^Met content per cell, was normalized to the average content per cell of 5S RNA in the two control cell lines and in the six mutant cell lines. 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 of each mt tRNA in each cell line. The error bars indicate 2 SEMs; P indicates the significance, according to Student's t test, of the difference between mutant and control values for each mt tRNA.

Figure 6.

Mitochondrial protein–labeling analysis. A, Electrophoretic patterns of the mitochondrial translation products. The lymphoblastoid cell lines and of 143B.TK⁻ cells were labeled for 30 min with [³⁵S]methionine in the presence of 100 μg of emetine per ml, an inhibitor for cytosolic protein synthesis. Samples containing equal amounts of protein (30 μg) were run in SDS/polyacrylamide gradient gels. COI, COII, and COIII, subunits I, II, and III of cytochrome c oxidase; ND1, ND2, ND3, ND4, ND4L, ND5, and ND6, subunits 1, 2, 3, 4, 4L, 5, and 6 of the respiratory-chain NADH dehydrogenase; A6 and A8, subunits 6 and 8 of the H⁺-ATPase; CYTb, apocytochrome b. B, Quantification of the rates of labeling of the mitochondrial translation products in lymphoblastoid cell lines. The rates of mitochondrial-protein labeling, determined as described elsewhere, are expressed as percentages of the value for 143B.TK⁻ in each gel, with error bars indicating 2 SEMs. Three independent labeling experiments and three or four electrophoretic analyses of each labeled preparation were performed on each lymphoblastoid cell line. The horizontal dashed lines represent the average value for each group. P indicates the significance, according to Student's t test, of the difference between mutant and control values for each mt tRNA.