Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance

Abstract

Hypoxia is a widely occurring condition experienced by diverse organisms under numerous physiological and disease conditions. To probe the molecular mechanisms underlying hypoxia responses and tolerance, we performed a genome-wide screen to identify mutants with enhanced hypoxia tolerance in the model eukaryote, the yeast Saccharomyces cerevisiae. Yeast provides an excellent model for genomic and proteomic studies of hypoxia. We identified five genes whose deletion significantly enhanced hypoxia tolerance. They are RAI1, NSR1, BUD21, RPL20A, and RSM22, all of which encode functions involved in ribosome biogenesis. Further analysis of the deletion mutants showed that they minimized hypoxia-induced changes in polyribosome profiles and protein synthesis. Strikingly, proteomic analysis by using the iTRAQ profiling technology showed that a substantially fewer number of proteins were changed in response to hypoxia in the deletion mutants, compared with the parent strain. Computational analysis of the iTRAQ data indicated that the activities of a group of regulators were regulated by hypoxia in the wild-type parent cells, but such regulation appeared to be diminished in the deletion strains. These results show that the deletion of one of the genes involved in ribosome biogenesis leads to the reversal of hypoxia-induced changes in gene expression and related regulators. They suggest that modifying ribosomal function is an effective mechanism to minimize hypoxia-induced specific protein changes and to confer hypoxia tolerance. These results may have broad implications in understanding hypoxia responses and tolerance in diverse eukaryotes ranging from yeast to humans.

Fig. 1.

The comparison of cell growth in air and under hypoxia between the parent BY4741 and Δrai1 (A), Δrpl20a (B), Δrsm22 (C), Δnsr1 (D), or Δbud21 (E) knockout strains. Yeast cells were grown in air or under hypoxia in synthetic complete medium containing glucose. Cells were collected at the indicated time points, and absorbance at 600 nm was measured. The data plotted here are averaged from at least 3 independent cultures. Welch 2-sample t-tests were performed to determine the statistical significance of the difference between the growth of hypoxic and normoxic wild-type or mutant cells. The P values were calculated by using the R program. The P values for the data at time points greater than 15 h were <0.005.

Fig. 2.

A comparison of the levels of total protein synthesis in the parent BY4741 and knockout strains. Yeast cells were grown in air or under hypoxia for 24 h, to an A₆₀₀ of ∼0.6. Then, the cells were labeled with l-[³⁵S]-methionine for 90 min. Cells were collected and lysed. Proteins were collected by TCA precipitation. Radiolabeled proteins were detected by using a scintillation counter. Data plotted here are averages from 5 independent replicates. Welch 2-sample t-tests were performed to compare hypoxic with normoxic wild-type or mutant cells. The P values were calculated by using the R program. *P values of < 0.005; **P values of < 0.001.

Fig. 3.

The polyribosome profiles of the wild-type parent and deletion mutant cells. Polyribosome preparation was performed by using wild-type BY4741 (A), Δnsr1 (B), Δrpl20a (C), Δbud21 (D), Δrai1 (E), and Δrsm22 (F) cells, grown under normoxic and hypoxic conditions, respectively. Then polyribosome profiles were analyzed by using 7–50% sucrose gradients.

Fig. 4.

The effect of hypoxia on the activity of unfolded protein response element (UPRE)-driven reporters in the parent BY4741 and knockout strains. A: the effect of hypoxia on the activity of the UPRE-1-driven reporter. B: the effect of hypoxia on the activity of the UPRE-2-driven reporter. C: the effect of hypoxia on the activity of the UPRE-3-driven reporter. The parent BY4741, Δrai1, Δrpl20a, Δrsm22, Δnsr1, and Δbud21 cells bearing a UPRE-driven reporter were grown in air or in a hypoxia chamber. Cells were collected, and β-galactosidase activities (in Miller units) were measured and plotted here. Data shown here are averages of data from at least three independent cultures. Welch 2-sample t-tests were performed to compare hypoxic with normoxic wild-type or mutant cells. The P values were calculated by using the R program. *P values of < 0.005; **P values of < 0.001.

Fig. 5.

The effect of tunicamycin (Tm) on the activity of UPRE-driven reporters in the parent BY4741 and knockout strains. A: the effect of Tm on the activity of the UPRE-1-driven reporter in normoxic cells. B: the effect of Tm on the activity of the UPRE-2-driven reporter in normoxic cells. C: the effect of Tm on the activity of the UPRE-1-driven reporter in hypoxic cells. The parent BY4741, Δrai1, Δrpl20a, Δrsm22, Δnsr1, and Δbud21 knockout cells bearing a UPRE-driven reporter were grown in the presence (+) or absence (−) of Tm. Cells were collected, and β-galactosidase activities (in Miller units) were measured and plotted here. Data shown here are averages of data from at least three independent cultures. Welch 2-sample t-tests were performed to compare Tm-treated with untreated wild-type or mutant cells. The P values were calculated by using the R program. *P values of < 0.005; **P values of < 0.001.

Fig. 6.

The effects of deletion of GCN4 and IRE1 on the doubling times of the wild-type parent and knockout strains with enhanced hypoxia tolerance. A: a comparison of the effect of deletion of GCN4 and IRE1 on the doubling times of the wild-type parent and Δrsm22 strains. B: a comparison of the effect of deletion of GCN4 and IRE1 on the doubling times of the wild-type parent and Δrai1 strains. C: a comparison of the effect of deletion of GCN4 and IRE1 on the doubling times of the wild-type parent and Δrpl20a strains. D: a comparison of the effect of deletion of GCN4 and IRE1 on the doubling times of the wild-type parent and Δnsr1 strains. E: a comparison of the effect of deletion of GCN4 and IRE1 on the doubling times of the wild-type parent and Δbud21 strains. The data plotted here are averages from at least 3 independent cultures. Welch 2-sample t-tests were performed to compare hypoxic with normoxic wild-type or mutant cells. The P values were calculated by using the R program. *P values of < 0.005; **P values of < 0.001.

Fig. 7.

The ratio of total protein levels per rRNA unit in hypoxic cells to those in normoxic cells. Cell extracts containing proteins and ribosomes were prepared from the indicated hypoxic and normoxic cells. Extracts containing 800 μg of proteins were fractionated by using 7–50% sucrose gradient as described in materials and methods. Fractions containing ribosomes were pooled. The rRNA levels were detected by measuring A₂₆₀. The rRNA levels in the fractions should reflect the amounts of ribosomes. Ribosome efficiency in hypoxic cells compared with normoxic cells was estimated by dividing the total protein levels per rRNA unit in hypoxic parent or mutant cells with those in the corresponding normoxic cells. Welch 2-sample t-tests were performed to compare hypoxic with normoxic wild-type or mutant cells. The P values were calculated by using the R program. **P values of < 0.001.

Fig. 8.

The cell growth and regulatory network formed by regulators whose targets were altered by hypoxia. The network was constructed by using Pathway Studio (version 7.1, Ariadne Genomics). Shown here are the regulators that exhibit interactions and connections with other regulators or cell growth. Each line or arrow indicates a previously identified biochemical or genetic interaction. “+” indicates a positive effect, while a stop line indicates a negative effect.