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Abstract
Protein synthesis (translation) is one of the fundamental processes occurring in the cells of living organisms. Translation can be divided into three key steps: initiation, elongation, and termination. In the yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 and eRF3. These factors are encoded by the SUP45 and SUP35 genes, which are essential; deletion of any of them leads to the death of yeast cells. However, viable strains with nonsense mutations in both the SUP35 and SUP45 genes were previously obtained in several groups. The survival of such mutants clearly involves feedback control of premature stop codon readthrough; however, the exact molecular basis of such feedback control remain unclear. To investigate the genetic factors supporting the viability of these SUP35 and SUP45 nonsense mutants, we performed whole-genome sequencing of strains carrying mutant sup35-n and sup45-n alleles; while no common SNPs or indels were found in these genomes, we discovered a systematic increase in the copy number of the plasmids carrying mutant sup35-n and sup45-n alleles. We used the qPCR method which confirmed the differences in the relative number of SUP35 and SUP45 gene copies between strains carrying wild-type or mutant alleles of SUP35 and SUP45 genes. Moreover, we compare the number of copies of the SUP35 and SUP45 genes in strains carrying different nonsense mutant variants of these genes as a single chromosomal copy. qPCR results indicate that the number of mutant gene copies is increased compared to the wild-type control. In case of several sup45-n alleles, this was due to a disomy of the entire chromosome II, while for the sup35-218 mutation we observed a local duplication of a segment of chromosome IV containing the SUP35 gene. Taken together, our results indicate that gene amplification is a common mechanism of adaptation to nonsense mutations in release factor genes in yeast.
Highlights
The very existence of viable nonsense mutations in the essential release factor genes is unexpected; it is important that yeast cells are capable of adapting to these mutations
If the wild-type allele is reintroduced into the same cells and the plasmid loss procedure is repeated, a plasmid with a wild-type allele is lost at the same rate in cells with either another wild-type or mutant allele. This means that a certain amount of stable genetic or epigenetic changes accumulate in the yeast strains during the first round of plasmid shuffling, conferring adaptation to mutations in the release factor genes
244-1B-D1606 and 260-1B-D1606 strains bore a disomy for chromosome XIII (Figure 4B). These results suggest that adaptation to nonsense mutations in the release factor genes in 1B-D1606 strains involves both amplification of the corresponding allele via local duplication or disomy and duplication of other chromosomes
Summary
Nonsense mutations are point mutations which lead to the appearance of premature termination codons (PTC) that cause untimely translation termination and prevent normal full-length protein synthesis. There is a natural mechanism neutralizing the consequences of nonsense mutations by allowing stop codon readthrough. It is called nonsense suppression and takes place in all living cells, from bacteria to mammals [6]. The efficiency of nonsense suppression depends on the functioning of various cellular components. Components of the translational apparatus, tRNAs, ribosomes, protein elongation and termination factors, make the biggest contribution to the nonsense suppression
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