Nonsense mutations in the essential gene SUP35 of Saccharomyces cerevisiae are non-lethal

Abstract

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.

Regulation of release factor expression using a translational negative feedback loop: A systems analysis

“…Similar nonlethal nonsense alleles of the eRF3 gene (SUP35) have also been identified in yeast (Chabelskaya et al 2004). The mutant SUP45 phenotypes exhibit increased nonsense suppression, which promote tRNA-mediated readthrough of the early stop codon and hence production of some reduced level of full-length release factor (Stansfield et al 1996).…”

Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation

“…Of course, we cannot rule out the possibility that eRF3a forms containing mutations in the eRF1-binding site were misfolded and targeted to ubiquitin-proteasomedependent proteolysis. However, in yeast, eRF3 variants harboring C-terminal truncation that included the eRF1-binding site were expressed at almost the same level as that of wild-type eRF3, suggesting that these truncated proteins did not have increased sensitivity to proteolysis (Chabelskaya et al 2004). Moreover, we also observed polyubiquitinated forms of GFP-3a in MG132-treated cells, although at a lower level than that of GFP-3a-FRAA (Fig.…”

“…Depletion of eRF3a, which is likely to be the main factor acting in translation termination, causes a significant reduction of 1 the eRF1 level by decreasing its stability (Chauvin et al 2005). In budding yeast, some reports showed that a reduced level of either eRF1 or eRF3 did not affect the level of its partner (Chabelskaya et al 2004;Salas-Marco and Bedwell 2004), whereas Valouev et al (2002) reported that depletion of either of the release factors is accompanied by a reduction in the level of the other.…”

Unassigned Codons, Nonsense Suppression, and Anticodon Modifications in the Evolution of the Genetic Code

“…A significant body of data is available regarding the translational fate of mRNA molecules containing nonsense mutations (Beier and Grimm 2001;Chabelskaya et al 2004;Doronina and Brown 2006;Lao et al 2009). These studies have revealed that a significant level of translational readthrough across stop codons occurs.…”