The environmental stress response causes ribosome loss in aneuploid yeast cells

Abstract

Aneuploidy, a condition characterized by whole chromosome gains and losses, is often associated with significant cellular stress and decreased fitness. However, how cells respond to the aneuploid state has remained controversial. In aneuploid budding yeast, two opposing gene-expression patterns have been reported: the "environmental stress response" (ESR) and the "common aneuploidy gene-expression" (CAGE) signature, in which many ESR genes are oppositely regulated. Here, we investigate this controversy. We show that the CAGE signature is not an aneuploidy-specific gene-expression signature but the result of normalizing the gene-expression profile of actively proliferating aneuploid cells to that of euploid cells grown into stationary phase. Because growth into stationary phase is among the strongest inducers of the ESR, the ESR in aneuploid cells was masked when stationary phase euploid cells were used for normalization in transcriptomic studies. When exponentially growing euploid cells are used in gene-expression comparisons with aneuploid cells, the CAGE signature is no longer evident in aneuploid cells. Instead, aneuploid cells exhibit the ESR. We further show that the ESR causes selective ribosome loss in aneuploid cells, providing an explanation for the decreased cellular density of aneuploid cells. We conclude that aneuploid budding yeast cells mount the ESR, rather than the CAGE signature, in response to aneuploidy-induced cellular stresses, resulting in selective ribosome loss. We propose that the ESR serves two purposes in aneuploid cells: protecting cells from aneuploidy-induced cellular stresses and preventing excessive cellular enlargement during slowed cell cycles by down-regulating translation capacity.

Keywords: CAGE; ESR; aneuploidy; ribosome loss.

Fig. 1.

Reanalysis of published aneuploid transcription data from Tsai et al. (9). Transcription data of haploid strain RLY4388 and euploid and aneuploid cell populations obtained from tetrad dissection (tetrad) or MATa selection (MATa selection) were reanalyzed with the RSEM processing method [Tsai et al. (9), accession no. GSE107997]. Raw TPM values were calculated for euploid cell populations, aneuploid cell populations, the haploid strain RLY4388 and exponentially growing haploid strain A2050. (A) Row centered log₂(TPM) values for each gene-expression set (CAGE up-regulated, CAGE down-regulated, iESR, and rESR). Each gene set was row centered individually and has a separate maximum (red) and minimum (blue) value, noted underneath. (B) CAGE up-regulated and down-regulated ssGSEA projection values for the haploid strains A2050 and RLY4388, and euploid and aneuploid cell populations (tetrad and MATa selection). (C) iESR and rESR ssGSEA projection values for the haploid strains A2050 and RLY4388, and euploid and aneuploid cell populations (tetrad and MATa selection). The horizontal lines represent the iESR and rESR ssGSEA projection values for W303 wild-type cells (A2587) treated with 500 mM NaCl for 40 min, a positive control for the ESR induction. Error bars represent SD from the mean of technical replicates.

Fig. 2.

Effects of culture density on ESR strength in aneuploid cell populations. (A) iESR (red) and rESR (blue) ssGSEA projection values were determined at the indicated OD(600 nm) for S288C wild-type haploid cells (A2050) grown in YEPD over 28 h. Vertical lines represent the OD(600 nm) values of pooled euploid and aneuploid cell populations generated by tetrad dissection. Error bars represent SD from the mean of technical replicates. (B and C) Tetrads of sporulated S288C diploid and triploid cells (A40877 and A40878) were dissected to produce heterogeneous haploid and aneuploid cell populations, respectively. A total of 144 individual haploid colonies and 432 aneuploid colonies were inoculated and grown overnight in 200 μL YEPD. The next morning 300 μL YEPD were added to cultures and grown for an additional 5 h. Individual euploid and aneuploid cultures were then pooled and their transcriptomes analyzed. An exponentially growing haploid strain (A2050) was included as a control. Gene-expression data were analyzed by calculating ssGSEA projection values for the (B) iESR and rESR and (C) CAGE up-regulated and down-regulated genes. Error bars represent SD from the mean of technical replicates; one-way two-tailed ANOVA test with multiple comparisons and Bonferroni correction, P < 0.0001 (****), P = 0.0021 (**). For additional statistical analysis see SI Appendix, Fig. S4. (D and E) Tetrads of sporulated S288C diploid and triploid cells (A40877 and A40878) were dissected to produce heterogeneous haploid and aneuploid cell populations, respectively. A total of 144 individual haploid colonies and 432 aneuploid colonies were inoculated and grown overnight in 200 μL YEPD. The next morning cultures were diluted 1:20 and grown for an additional 5 h. Colonies were then pooled, further diluted to approximately OD(600 nm) = 0.3, and grown for 2 additional hours. Transcriptomes of pooled euploid and aneuploid populations and an exponentially growing haploid strain (A2050) were analyzed with RNA-Seq, and ssGSEA projection values were calculated for (D) iESR and rESR and (E) CAGE up-regulated and down-regulated genes. Error bars represent SD from the mean of technical replicates; one-way two-tailed ANOVA test with multiple comparisons and Bonferroni correction, P < 0.0001 (****), P = 0.1234 (ns, no statistical significance). For additional statistical analysis see SI Appendix, Fig. S4.

Fig. 3.

Complex aneuploid yeast strains exhibit the ESR. (A–C) Aneuploid yeast strains harboring aneuploidies ranging from 2N to 3N were grown to log phase in YEPD. For each strain, degree of aneuploidy was calculated as the fraction of base pairs in the aneuploid strain/base pairs in a haploid control strain. Doubling times were calculated from growth curves generated by measuring OD(600 nm) in 20-min intervals over 5 h in a plate reader. (A) Correlation between doubling time and degree of aneuploidy (Spearman, ρ² = 0.7620, P < 0.0001). Transcriptomes of the complex aneuploid strains were analyzed by RNA-Seq, and ssGSEA projection values were calculated for iESR and rESR genes. Correlations between iESR ssGSEA projections and mean doubling time (Spearman, ρ² = 0.3144, P = 0.0066) and rESR ssGSEA projections and mean doubling time (Spearman, ρ² = 0.4942, P = 0.0003) are shown in B. Correlations between iESR ssGSEA projections and degree of aneuploidy (Spearman, ρ² = 0.2864, P = 0.0103) and rESR ssGSEA projections and degree of aneuploidy (Spearman, ρ² = 0.4707, P = 0.0004) are shown in C. Error bars represent SD from the mean. (D) Select complex aneuploid strains were grown in a phosphate-limiting chemostat until steady state was reached. Transcriptomes of harvested cells were analyzed by RNA-Seq. ssGSEA projection values were calculated for iESR and rESR genes. Correlations between iESR ssGSEA projections and degree of aneuploidy (Spearman, ρ² = 0.1912, P = 0.2066) and rESR ssGSEA projections and degree of aneuploidy (Spearman, ρ² = 0.0107, P = 0.7850) are shown. Error bars represent SD from the mean of experimental replicates. The data point circled in red represents a complex aneuploid strain that does not mount the ESR.

Fig. 4.

ESR induction causes ribosome loss in aneuploid strains. (A and B) Euploid and aneuploid cell populations were grown in YEPD with the 1:20 dilution protocol, and Slt2 Thr202/Tyr204 phosphorylation was determined. Wild-type euploid (A2050) and slt2Δ cells (A41265) treated with 5 μg/mL Calcofluor White for 2 h served as positive and negative controls, respectively, in immunoblots (A). Pgk1 served as a loading control. Quantifications of Slt2 Thr202/Tyr204 phosphorylation are shown in B. Slt2/Pgk1 values were normalized to the wild-type cells treated with Calcofluor White. Error bars represent SD from the mean of experimental replicates; one-way ANOVA test with multiple comparisons and Bonferroni correction, P < 0.0001 (****), P = 0.0021 (**). All other comparisons between samples had a significant difference of P < 0.0001 (****) with the exception of the euploid populations and slt2Δ + Calcofluor, which had a significant difference of P = 0.0288. (C) The fraction of ribosome in total protein extracts ([ribosome]/[protein]) was determined in euploid and aneuploid cell populations grown with the 1:20 dilution protocol. [ribosome]/[protein] in aneuploid cell populations was normalized to that in euploid cell populations. Error bars represent SD from the mean of technical replicates; unpaired two-tailed t test test, P = 0.0332 (*). (D) Aneuploid yeast strains harboring aneuploidies ranging from 2N to 3N were grown to log phase in YEPD and the fraction of ribosomes in total protein extracts ([ribosome]/[protein]) was determined. Correlation between [ribosome]/[protein] and degree of aneuploidy (ρ² = 0.6158, P = 0.0001, Spearman) is shown. The calculated values were normalized to the [ribosome]/[protein] of a diploid control. (E) Aneuploid yeast strains harboring aneuploidies ranging from 2N to 3N were grown in a phosphate-limited chemostat and the fraction of ribosome in total protein extracts ([ribosome]/[protein]) was determined. Correlation between [ribosome]/[protein] and degree of aneuploidy (ρ² = 0.2780, P = 0.1231, Spearman) is shown. The calculated values were normalized to the [ribosome]/[protein] of a diploid control. Error bars represent SD from the mean of experimental replicates.