An MRPS12 mutation modifies aminoglycoside sensitivity caused by 12S rRNA mutations

Abstract

Several homoplasmic pathologic mutations in mitochondrial DNA, such as those causing Leber hereditary optic neuropathy or non-syndromic hearing loss, show incomplete penetrance. Therefore, other elements must modify their pathogenicity. Discovery of these modifying factors is not an easy task because in multifactorial diseases conventional genetic approaches may not always be informative. Here, we have taken an evolutionary approach to unmask putative modifying factors for a particular homoplasmic pathologic mutation causing aminoglycoside-induced and non-syndromic hearing loss, the m.1494C>T transition in the mitochondrial DNA. The mutation is located in the decoding site of the mitochondrial ribosomal RNA. We first looked at mammalian species that had fixed the human pathologic mutation. These mutations are called compensated pathogenic deviations because an organism carrying one must also have another that suppresses the deleterious effect of the first. We found that species from the primate family Cercopithecidae (old world monkeys) harbor the m.1494T allele even if their auditory function is normal. In humans the m.1494T allele increases the susceptibility to aminoglycosides. However, in primary fibroblasts from a Cercopithecidae species, aminoglycosides do not impair cell growth, respiratory complex IV activity and quantity or the mitochondrial protein synthesis. Interestingly, this species also carries a fixed mutation in the mitochondrial ribosomal protein S12. We show that the expression of this variant in a human m.1494T cell line reduces its susceptibility to aminoglycosides. Because several mutations in this human protein have been described, they may possibly explain the absence of pathologic phenotype in some pedigree members with the most frequent pathologic mutations in mitochondrial ribosomal RNA.

Keywords: aminoglycoside; compensatory mutation; evolutionary approaches; incomplete penetrance; mitochondrial ribosomal RNA; mitochondrial ribosomal protein S12; pathologic mutation.

Figure 1

Electropherograms of MT-RNR1 and MRPS12 partial sequences. (A) MT-RNR1 from Macaca sp. (B) MRPS12 from Macaca sp. and Chlorocebus aethiops.

Figure 2

Paromomycin effect on macaque cells. Representative images of macaque fibroblasts growing with different amount of paromomycin (paro) and growth quantification. The number of cells after 3 days growing without aminoglycosides has been considered 100%.

Figure 3

Paromomycin effect on OXPHOS complexes. (A) CIV specific activity and quantity after treatment with paromomycin 2 mg/ml. Dots line represents values in the absence of the antibiotic. (B) p.MT-CO1 and actin levels from macaque cells treated with paromomycin 0, 2, or 4 mg/ml. (C) Semiquantitative determination, by gel scanning, of p.MT-CO1/actin ratios from macaque cells treated with paromomycin 0 (dot line), 2 or 4 mg/ml. (D) Representative image of p.MT-CO1 and SDHA levels from macaque cells treated with paromomycin 0, 2 or 4 mg/ml. (E) Quantitative determination, by ELISA, of p.MT-CO1/SDHA ratios from macaque cells treated with paromomycin 0, 2 or 4 mg/ml. (F,G) Representative images of mitochondrial translation products, and loading controls, from macaque cells treated with paromomycin 0, 2, or 4 mg/ml.

Figure 4

Mitochondrial fraction antibiogram. TOM20 is a mitochondrial protein. CF, CF+P, MF, and MF+P code for cell fraction, cell fraction from cells treated with paromomycin 2 mg/ml, mitochondrial fraction and mitochondrial fraction from cells treated with paromomycin 2 mg/ml, respectively.

Figure 5

Helix 39 of the 12S rRNA decoding site from Human and Cercopithecidae. Blue number denotes helix numbering (Springer and Douzery, 1996). The m.1494C>T transition, in the yellow-encircled nucleotide position, is a human pathologic mutation (m). Red nucleotides denote differences vs. the human sequence. Green numbers indicate the absolute frequency of that nucleotide in 90 Cercopithecidae species. Several mutations have been compensated in this helix 39.

Figure 6

MRPS12′s amino acid variation. (A) Protein alignment. MRPS12 sequences from 4 Cercopithecidae species, Homo sapiens and Thermus thermophilus. Red amino acids denote differences between human and Cercopithecidae species. Yellow background indicates human polymorphic positions. Underlined sequence is the mitochondrial targeting sequence. (B,C) Molecular view of the interaction between the small subunit rRNA (ss rRNA) and the ribosomal protein S12 in the Thermus thermophilus ribosome. The mitochondrial 12S rRNA nucleotides (1494 and 1555, red and green, respectively) and the MRPS12 amino acid variation between human and macaque sequences (64, 68, 101, 106, 107, and 124) or the human MRPS12 polymorphic amino acids (38, 44, 52, 55, 67, 68, 84, 115, and 125) have been located in this bacterial structure.

Figure 7

MRPS12 expression. (A) MRPS12 mRNA levels. The levels of non-transfected cells are considered 100%. (B) MRPS12 codon 68. Leu and Arg code for leucine and arginine, respectively. (C) Relative amount of MRPS12 and Actin proteins.

Figure 8

Paromomycin effect on OXPHOS proteins of MRPS12 transfected cells. (A) Mitochondrial translation products (right panel) and loading controls (left panel). AR, AL, GR, GL, ARp, ALp, GRp, and GLp code for 12S rRNA m.1555A-MRPS12^R68R, 12S rRNA m.1555A-MRPS12^R68L, 12S rRNA m.1555G-MRPS12^R68R, 12S rRNA m.1555G-MRPS12^R68L cells grown without or with paromomycin (p), respectively. (B) CIV/CS ratio of wild-type (m.1555A 12S rRNA) and mutant (m.1555G 12S rRNA) osteosarcoma 143B cybrids exposed or not to paromomycin. (C) CIV/CS ratio of wild-type (m.1555A 12S rRNA) and mutant (m.1555G 12S rRNA) osteosarcoma 143B cybrids that overexpress the R68 or L68 MRPS12 variants exposed or not to paromomycin. Asterisks denote P ≤ 0.0414.

Figure 9

Primates' phylogeny. Numbers indicate million of years. This tree is based in Figure 2 from Perelman et al. (2011).