ABSTRACT
Eukaryotic cells possess special mechanism of the degradation of mRNAs containing premature termination codons (PTCs)--nonsense-mediated mRNA decay (NMD) pathway. In yeast Saccharomyces cerevisiae the activity of this pathway depends on the recognition of the PTC by the translational machinery and interaction of translation termination factors eRF1 (Sup45) and eRF3 (Sup35) with Upfl, Upf2 and Upf3 proteins. Previously we have shown that decreasing of eRF1 amount causes an impairment of NMD. Here we show that sup35 nonsense and missense mutations lead to accumulation of PTC-containing transcripts such as his7-1 mRNA and CYH2 pre-mRNA. Thus sup35 mutations do not only decrease translation fidelity but also influence mRNA stability. Remarkably, deletion of either UPF1 or UPF2 increased viability of sup35 mutants, while UPF3 deletion leads to decreased viability of sup35 mutants.
Subject(s)
Peptide Termination Factors/genetics , RNA Stability/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Aminohydrolases/genetics , Codon, Nonsense/genetics , Gene Deletion , Mutation, Missense , Peptide Termination Factors/metabolism , Protein Biosynthesis/genetics , RNA Helicases/genetics , Saccharomyces cerevisiae Proteins/metabolism , Suppression, GeneticABSTRACT
Earlier we have characterized strains bearing mutations in essential genes SUP45 and SUP35 of yeast S. cerevisiae, encoding translation termination factors eRF1 and eRF3 respectively. In the present work nonsense-mutants on genes SUP45 and SUP35 have been compared by a level of eight tRNA: tRNATyr, tRNAGln, tRNATrp, tRNALeu and tRNAArg (previously described as potentially suppressor tRNA), and also tRNAPro, tRNAHis and tRNAGly. We have not revealed preferable increase in amount of natural suppressor tRNA. The majority of the investigated mutations leads to increase in a level of all investigated tRNA. The mechanisms providing viability of nonsense-mutants on essential genes SUP45 and SUP35 are discussed.
Subject(s)
Peptide Termination Factors/metabolism , RNA, Transfer/metabolism , Saccharomyces cerevisiae/metabolism , Alleles , Codon, Nonsense , Codon, Terminator , Peptide Termination Factors/genetics , Prions/genetics , Prions/metabolism , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Transfer/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1-eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.
