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Cited by 2Fungal and yeast genetics researchRNA and protein synthesis mechanismsRNA Research and Splicing

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Gene Expression Analysis of Yeast Strains with a Nonsense Mutation in the eRF3-Coding Gene Highlights Possible Mechanisms of Adaptation

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Maksiutenko, Evgeniia M., Barbitoff, Yury A., Danilov, Lavrentii G., Matveenko, Andrew G., Zemlyanko, Olga M., Efremova, Elena P., Moskalenko, Svetlana E., Zhouravleva, Galina A.·2024·International Journal of Molecular Sciences
DOI 10.3390/ijms25126308PMC PMC11203930
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Abstract

In yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 (Sup45) and eRF3 (Sup35), which are essential for viability. Previous studies have revealed that presence of nonsense mutations in these genes leads to amplification of mutant alleles (sup35-n and sup45-n), which appears to be necessary for the viability of such cells. However, the mechanism of this phenomenon remained unclear. In this study, we used RNA-Seq and proteome analysis to reveal the complete set of gene expression changes that occur during cellular adaptation to the introduction of the sup35-218 nonsense allele. Our analysis demonstrated significant changes in the transcription of genes that control the cell cycle: decreases in the expression of genes of the anaphase promoting complex APC/C (APC9, CDC23) and their activator CDC20, and increases in the expression of the transcription factor FKH1, the main cell cycle kinase CDC28, and cyclins that induce DNA biosynthesis. We propose a model according to which yeast adaptation to nonsense mutations in the translation termination factor genes occurs as a result of a delayed cell cycle progression beyond the G2-M stage, which leads to an extension of the S and G2 phases and an increase in the number of copies of the mutant sup35-n allele.

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Crossref · 10.3390/ijms25126308

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Figures

Figure 1 Transcriptome profile of strains harboring nonsense-mutation sup35-218. (A) Scheme illustrating the experimental design used to study yeast adaptation to nonsense mutations in release factor genes. (B) Scatterplot representing principal component analysis of 10 yeast transcriptomes carrying different combinations of alleles of the SUP35 gene. (C) Comparative analysis of the number of differentially expressed genes, depending on the log2FC cutoff and set of controls used. (D) Analysis of differential gene expression in cells at the 3rd stage of the experiment, carrying the sup35-218 allele compared to 3rd stage harboring the SUP35 wild-type allele. Significantly differentially expressed genes (FDR < 0.05) are represented by red dots (upregulated) and blue dots (downregulated). Grey dots represent genes with unaltered expression.
Figure 1 Transcriptome profile of strains harboring nonsense-mutation sup35-218. (A) Scheme illustrating the experimental design used to study yeast adaptation to nonsense mutations in release factor genes. (B) Scatterplot representing principal component analysis of 10 yeast transcriptomes carrying different combinations of alleles of the SUP35 gene. (C) Comparative analysis of the number of differentially expressed genes, depending on the log2FC cutoff and set of controls used. (D) Analysis of differential gene expression in cells at the 3rd stage of the experiment, carrying the sup35-218 allele compared to 3rd stage harboring the SUP35 wild-type allele. Significantly differentially expressed genes (FDR < 0.05) are represented by red dots (upregulated) and blue dots (downregulated). Grey dots represent genes with unaltered expression.CC BY · PubMed Central
Figure 2 Results of gene set enrichment analysis using Biological Process (BP) and Cellular Component (CC) terms from Gene Ontology. The analysis was performed using the clusterProfiler package for R. The dot size is proportional to the enrichment ratio, and the color gradient represents the adjusted significance level. (A) Genes with increased expression in cells bearing sup35-218 (biological processes). (B) Genes with increased expression in cells bearing sup35-218 (cellular component). (C) Genes with decreased expression in cells bearing (biological processes).
Figure 2 Results of gene set enrichment analysis using Biological Process (BP) and Cellular Component (CC) terms from Gene Ontology. The analysis was performed using the clusterProfiler package for R. The dot size is proportional to the enrichment ratio, and the color gradient represents the adjusted significance level. (A) Genes with increased expression in cells bearing sup35-218 (biological processes). (B) Genes with increased expression in cells bearing sup35-218 (cellular component). (C) Genes with decreased expression in cells bearing (biological processes).CC BY · PubMed Central
Figure 3 A diagram showing the changes in the expression of genes controlling the yeast cell cycle progression. Visualization of the relationship of genes involved in cell cycle control using KEGG PATHWAY. Genes with increased expression in the presence of the sup35-218 allele are marked in red; genes with reduced expression are marked in blue. Color contrast reflects the degree of increase or decrease in expression. Cases where two genes with similar functions have inconsistent expression changes are highlighted with two colors (Yox1/Yhp1, Clb1/2, Dbf2/20). Genes where no significant changes in expression were found are highlighted in green.
Figure 3 A diagram showing the changes in the expression of genes controlling the yeast cell cycle progression. Visualization of the relationship of genes involved in cell cycle control using KEGG PATHWAY. Genes with increased expression in the presence of the sup35-218 allele are marked in red; genes with reduced expression are marked in blue. Color contrast reflects the degree of increase or decrease in expression. Cases where two genes with similar functions have inconsistent expression changes are highlighted with two colors (Yox1/Yhp1, Clb1/2, Dbf2/20). Genes where no significant changes in expression were found are highlighted in green.CC BY · PubMed Central
Figure 4 Validation of changes in expression of DEGs identified in RNA-Seq analysis using qPCR. Shown are boxplots of the relative expression levels calculated using the ΔΔCt method (see Section 4 for more details). ns—not significant, ** p-value < 0.05, *** p-value < 0.001 according to Wilcoxon–Mann–Whitney rank sum test. At least seven biological replicates were used for each group.
Figure 4 Validation of changes in expression of DEGs identified in RNA-Seq analysis using qPCR. Shown are boxplots of the relative expression levels calculated using the ΔΔCt method (see Section 4 for more details). ns—not significant, ** p-value < 0.05, *** p-value < 0.001 according to Wilcoxon–Mann–Whitney rank sum test. At least seven biological replicates were used for each group.CC BY · PubMed Central
Figure 5 Proteome profile of strains harboring nonsense-mutation sup35-218. (A) Analysis of differential protein production in cells at the 3rd stage of the experiment, carrying the sup35-218 allele compared to cells harboring SUP35 wild-type allele. Differentially produced proteins (FDR < 0.05) are represented by red dots (upregulated) and blue dots (downregulated). Grey dots represent proteins with unaltered production. (B) Evaluation of the correlation of gene and protein expression levels in strains carrying the wild-type SUP35 allele or mutant sup35-218 allele (left and right plots, respectively).
Figure 5 Proteome profile of strains harboring nonsense-mutation sup35-218. (A) Analysis of differential protein production in cells at the 3rd stage of the experiment, carrying the sup35-218 allele compared to cells harboring SUP35 wild-type allele. Differentially produced proteins (FDR < 0.05) are represented by red dots (upregulated) and blue dots (downregulated). Grey dots represent proteins with unaltered production. (B) Evaluation of the correlation of gene and protein expression levels in strains carrying the wild-type SUP35 allele or mutant sup35-218 allele (left and right plots, respectively).CC BY · PubMed Central
Figure 6 Cell cycle analysis by flow cytometry, illustrating the DNA content of yeast cultures of wild-type strain (L-14-D1690) or sup35-218 mutant cells (218L-14-D1690). Ten independent yeast cultures were grown and then prepared for flow cytometry as described in Section 4. Curves representing independent clones are shown in different colors. Locations of peaks of the 1C and 2C cell populations are indicated by arrowheads.
Figure 6 Cell cycle analysis by flow cytometry, illustrating the DNA content of yeast cultures of wild-type strain (L-14-D1690) or sup35-218 mutant cells (218L-14-D1690). Ten independent yeast cultures were grown and then prepared for flow cytometry as described in Section 4. Curves representing independent clones are shown in different colors. Locations of peaks of the 1C and 2C cell populations are indicated by arrowheads.CC BY · PubMed Central
Figure 7 A schematic representation of the possible changes in the cell cycle in cells carrying the sup35-218 mutation. (A) Changes in expression of genes responsible for the progression of cell cycle phases in cells carrying the sup35-218 allele (right) compared to cells carrying the SUP35 allele (left). Purple asterisks indicate checkpoints in cell cycle regulation. Genes with increased expression are depicted in red (as in Figure 3) and are marked with an up arrow; genes with decreased expression are depicted in blue and are marked with a down arrow. (B) Scheme of changes occurring during plasmid replication and segregation in cells carrying the sup35-218 allele (right) compared to cells carrying the SUP35 allele (left). Plasmids with wild-type SUP35 are shown in green, plasmids with sup35-218 allele are shown in blue. Genes with changes in expression validated using qPCR are underlined.
Figure 7 A schematic representation of the possible changes in the cell cycle in cells carrying the sup35-218 mutation. (A) Changes in expression of genes responsible for the progression of cell cycle phases in cells carrying the sup35-218 allele (right) compared to cells carrying the SUP35 allele (left). Purple asterisks indicate checkpoints in cell cycle regulation. Genes with increased expression are depicted in red (as in Figure 3) and are marked with an up arrow; genes with decreased expression are depicted in blue and are marked with a down arrow. (B) Scheme of changes occurring during plasmid replication and segregation in cells carrying the sup35-218 allele (right) compared to cells carrying the SUP35 allele (left). Plasmids with wild-type SUP35 are shown in green, plasmids with sup35-218 allele are shown in blue. Genes with changes in expression validated using qPCR are underlined.CC BY · PubMed Central

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Research works printed but not linked: 1

  1. Kaiser, C., Michaelis, S., and Mitchell, A. (1994). Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press.

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Metadata

Authors
Maksiutenko, E. M., Barbitoff, Y. A., Danilov, L. G., Matveenko, A. G., Zemlyanko, O. M., Efremova, E. P., Moskalenko, S. E., & Zhouravleva, G. A.
Affiliations
  • Russian Academy of Sciences (RU)
  • St Petersburg University (RU)
Corresponding author
Zhouravleva, Galina A.
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2024-06-07
Volume
25
Issue
12
Pages
6308
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms25126308
PMC
PMC11203930
OpenAlex
W4399434292
Cited by
2
References
59
Citations by year
2025: 1 · 2024: 1
FWCI
0.27
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Russian Science Foundation (23-14-00063)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
eRF3, SUP45, translation termination factor, cellular adaptation, gene expression changes, cell cycle regulation, anaphase-promoting complex, CDC20, Fkh1, Cdc28, cyclins, DNA synthesis, Saccharomyces cerevisiae
MeSH terms
Adaptation, Physiological, Cell Cycle, Peptide Termination Factors, Saccharomyces cerevisiae, Gene Expression Regulation, Fungal, Codon, Nonsense, Saccharomyces cerevisiae Proteins

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