Mutant A1555G Mitochondrial 12S rRNA and Aminoglycoside Susceptibility

In a recent report published in this journal, Y. Qian and M.-X. Guan reported on their studies on interaction of aminoglycosides with human mitochondrial 12S rRNA carrying the A1555G deafness-associated mutation (15). Using RNA oligonucleotides representing the small subunit's rRNA decoding A site (i.e., the drug binding pocket) and carrying the A1555G mutation, the authors used chemical footprinting by dimethyl sulfate (DMS) to study the interaction of aminoglycosides with 12S rRNA. On the basis of their findings, the authors suggest that the A1555G mutation alters the binding properties of aminoglycosides at the A site of rRNA and leads to conformational changes in 12S rRNA with the A1555G mutation. I agree with the conclusion in general, which is also supported by other available evidence (6, 7). However, I am afraid that the authors' argumentation is somewhat compromised as it is only weakly supported by the experimental design and outcome.

(i) In the authors' footprinting studies, there is no correlation or any relationship between the defined chemical structure of an aminoglycoside compound and the drug-induced change in chemical modification for RNA oligonucleotides carrying the wild-type versus the deafness-associated sequence. This is surprising, as a vast body of evidence at the genetic, biochemical, and structural levels indicates that the interaction of aminoglycosides with the small-subunit rRNA can be categorized along the main structural elements of these compounds: i.e., (i) 4,5 versus 4,6 compounds and (ii) the 6′ OH versus 6′ NH₂ substituent (1, 4, 8, 14).

Hamasaki and Rando are frequently referred to for having demonstrated specific binding of aminoglycosides to a human rRNA construct based on the A1555G polymorphism (5). Similar to Qian and Guan, these investigators made various RNA constructs prepared according to mitochondrial 12S rRNA and bacterial 16S rRNA sequences. It was found that the mitochondrial A1555G mutant construct stoichiometrically bound aminoglycosides, while the wild-type mitochondrial sequence did not bind aminoglycosides at all. However, what is frequently neglected is that an important control was at fault, putting the experimental design into doubt: i.e., a wild-type 16S rRNA bacterial construct bound aminoglycosides stoichiometrically, while a G1491A mutant bacterial 16S rRNA was unable to specifically bind aminoglycosides. The latter observation is at odds with the observation that the G1491A alteration only little affects aminoglycoside susceptibility in complete bacterial ribosomes (9, 10).

(ii) Streptomycin does not bind to the part of the A site rRNA used in Qian's and Guan's study (1). It is thus hard to understand that the authors observe streptomycin-induced changes in DMS reactivity for their RNA oligonucleotides similar to those of aminoglycosides targeting the A site (e.g., paromomycin and tobramycin).

(iii) As pointed out previously (6), there is a frequent misalignment of rRNA residues in the hearing field. This misperception presumably dates back to an early report by Hutchin et al. (12). Mitochondrial 12S rRNA position A1555 is not equivalent to bacterial 16S rRNA position 1491 but is homologous to that of bacterial 16S rRNA position 1490 (13) (Fig. 1). For the sake of the A1555G mutation, reference to studies of aminoglycoside susceptibility in bacterial ribosomes with alterations of the C1409-G1491 interaction (2, 3) is thus misleading. Both, the mitochondrial 12S rRNA wild type and A1555G mutants have a C-C opposition at corresponding E. coli positions 1409 to 1491 (Fig. 1). Investigations on the C1409-G1491 interaction in bacteria can hardly serve as a model for the mitochondrial 12S rRNA A1555G alteration.

FIG. 1.

Secondary-structure comparison of decoding-site rRNA sequences in the small ribosomal subunit. (A) Decoding region of 16S rRNA helix 44 in bacterial ribosomes (M. smegmatis). rRNA nucleotides are numbered according to the bacterial nomenclature (i.e., homologous E. coli 16S rRNA positions). (B) Decoding region of 12S rRNA in human mitochondrial ribosomes. rRNA residues are numbered according to the mitochondrial nomenclature: mitochondrial DNA (mtDNA) positions 1494 and 1555 correspond to E. coli 16S rRNA positions 1410 and 1490, respectively.

In an effort to resolve the problems with mitochondrial 12S rRNA oligonucleotides and their interaction with aminoglycosides, we have recently used a genetic strategy to replace in bacterial ribosomes the small subunit's rRNA decoding A site in H44 (i.e., the aminoglycoside binding site) with that of various eukaryotic ribosomes (6, 7, 11). Toward this end, we constructed hybrid ribosomes in which the bacterial small ribosomal subunit's decoding A site has been replaced with the A site of wild-type mitochondrial ribosomes and with that of mitochondrial ribosomes carrying the A1555G deafness-associated mutation. Subsequent studies demonstrated that the A1555G deafness-associated mutation increased binding of aminoglycosides to the drug binding pocket and resulted in hypersusceptibility to aminoglycoside-induced miscoding. Finally, drug affinity and aminoglycoside susceptibility of the A1555G deafness-associated hybrid ribosomes were found to be similar to those of bacterial ribosomes, with a C1409-C1491 opposition corroborating the alignment given in Fig. 1.