Abstract
The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable.
Highlights
Translation is terminated by a stop codon entering the A site of the ribosome, inducing the release of the polypeptide chain from the peptidyl-t-RNA [1]
The rules governing readthrough efficiency are far from clear, but readthrough levels have been shown to depend on the type of stop codon, with UAA being a better terminator than UAG and UGA, and even more strongly on the surrounding nucleotide context [3,4,5,6]
We demonstrated that the nucleotide immediately upstream from the stop codon was a major determinant of gentamicin response and that this effect was mediated by the nucleotide itself, rather than by the nature of the last amino acid or the tRNA present in the ribosomal P-site
Summary
Translation is terminated by a stop codon entering the A site of the ribosome, inducing the release of the polypeptide chain from the peptidyl-t-RNA [1]. Readthrough can be stimulated by aminoglycoside antibiotics, such as gentamicin, making it possible to generate a full-length protein from genes carrying a nonsense mutation [9,10]. These antibiotics interact with the highly conserved decoding center of ribosomal RNA, promoting the recognition of the stop codon by a near-cognate tRNA [11,12]. A number of studies and clinical trials have investigated the possible use of this antibiotic for the treatment of human diseases resulting from the presence of a premature termination codon (PTC) in a particular gene (for review, see [13,14]).
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