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Interaction of aminoglycosides with human mitochondrial 12S rRNA carrying the deafness-associated mutation - PubMed

Interaction of aminoglycosides with human mitochondrial 12S rRNA carrying the deafness-associated mutation

Yaping Qian et al. Antimicrob Agents Chemother. 2009 Nov.

Abstract

The mitochondrial 12S rRNA A1555G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we employed an RNA-directed chemical-modification approach to understanding the pathogenesis of aminoglycoside-induced hearing loss. The patterns of chemical modification of RNA oligonucleotides carrying the A1555G mutation by dimethyl sulfate (DMS) were distinct from those of the RNA oligonucleotides carrying wild-type sequence in the presence of aminoglycosides. In the RNA analogue carrying the A1555G mutation, reduced reactivity to DMS occurred in base G1555 as well as in bases C1556 and A1553 in the presence of paromomycin, neomycin, gentamicin, kanamycin, tobramycin, or streptomycin. In particular, base G1555 exhibited marked but similar levels of protection in the presence of 0.1 microM to 100 microM neomycin, gentamicin, or kanamycin. In contrast, the levels of protection in base G1555 appeared to be correlated with the concentration of paromycin, tobramycin, or streptomycin. Furthermore, increasing reactivities to DMS in the presence of these aminoglycosides were observed for bases A1492, C1493, C1494, and A1557 in the RNA analogue carrying the A1555G mutation. These data suggested that the A1555G mutation altered the binding properties of aminoglycosides at the A site of 12S rRNA and led to local conformational changes in 12S rRNA carrying the A1555G mutation. The interaction between aminoglycosides and 12S rRNA carrying the A1555G mutation provides new insight into the pathogenesis of aminoglycoside ototoxicity.

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Figures

FIG. 1.
FIG. 1.

Decoding-region analogues of human mitochondrial 12S rRNA with nucleotides either protected from DMS modification or exhibiting enhanced reactivity with DMS in the presence of paromomycin, neomycin, gentamicin, kanamycin, tobramycin, or streptomycin. (A) Secondary structure of E. coli 16S rRNA (with the A site boxed) and corresponding regions of oligoribonucleotides with portions of wild-type (WT) human mitochondrial 12S rRNA sequence or human mitochondrial 12S rRNA sequence containing the A1555G or the C1494U mutation. The analogue contains 12S rRNA decoding-region sequences from base 1488 to 1498 on the proximal side and base 1551 to 1562 on the distal side. (B) Protection (filled diamonds) and enhanced reactivity (filled circles) are indicated. Small or large symbols represent weak or strong reactivity, respectively.

FIG. 2.
FIG. 2.

Experimental schema for chemical modification.

FIG. 3.
FIG. 3.

Autoradiographs of DMS probing reactions with human mitochondrial 12S rRNA oligonucleotides with the wild-type (WT) sequence or human mitochondrial 12S rRNA oligonucleotides carrying the A1555G or C1494T mutation. (A) Lanes a, b, c, and d show U, C, G, and A dideoxy sequencing reactions. (B) Lanes e and g, primer extension reactions using unmodified RNA analogues carrying base A1555 (WT) or G1555; lanes f and h, primer extension reactions using modified RNA analogues carrying base A1555 (WT) or G1555. (C) DMS probing reactions with RNA analogues in the presence of 0.1, 1, 10, and 100 μM paromomycin are shown in lanes 1 to 4 (WT), lanes 5 to 8 (A1555G mutation), and lanes 9 to 12 (C1494U mutation), respectively. The positions of bands corresponding to nucleotides of 12S rRNA are indicated to the right and left of the gels. An arrow indicates the nucleotide at position 1555.

FIG. 4.
FIG. 4.

Interactions of aminoglycosides with human mitochondrial 12S rRNA analogues carrying the wild-type sequence (WT) or the A1555G mutation (MT). DMS probing reactions with RNA analogues carrying base A1555 (WT) or G1555 (MT) in the presence of 0.1, 1, 10, and 100 μM paromomycin, neomycin, gentamicin, kanamycin, tobramycin, or streptomycin are shown in lanes 1 to 4 and lanes 5 to 8, respectively. The positions of bands corresponding to nucleotides of 12S rRNA are indicated to the right of each gel. An arrow indicates the nucleotide at position 1555.

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