S. cerevisiae Rrp4 variants that model EXOSC2 variants identified in patients show impaired function. S. cerevisiae cell expressing Rrp4 variants that model pathogenic amino acid changes found in EXOSC2 were generated as described in Materials and Methods. (A) Although cell growth is comparable for all mutants that contain a wild-type RRP4 maintenance plasmid (Ura− Leu−), rrp4-G58V mutant cells are not viable on plates containing 5-FOA where the maintenance plasmid is not present. The rrp4-G226D cells show temperature-sensitive growth on 5-FOA relative to control RRP4 cells. The cells were grown at the indicated temperatures. (B,C) The rrp4-G226D cells exhibit profoundly impaired growth compared to control RRP4 cells at 37°C as assessed by (B) serial dilution growth assay on plates or (C) growth in liquid media. (B) The rrp4Δ cells expressing only RRP4 or rrp4-G226D and rrp40Δ cells expressing only RRP40 or rrp40-W195R were serially diluted, spotted onto solid media grown at the indicated temperatures or (C) grown in liquid media at 37°C with optical density measurement used to assess cell density over time. The growth of rrp40-W195R cells, previously reported to be moderately impaired at 37°C (Fasken et al. 2017; Gillespie et al. 2017), was included as a comparative control. (D) The steady-state level of the Rrp4 G226D protein variant is modestly decreased at 37°C. Lysates of rrp4Δ cells solely expressing Myc-tagged wild-type Rrp4 or rrp4-G226D grown at 30°C or 37°C were analyzed by immunoblotting with an anti-Myc antibody to detect Rrp4-Myc and an anti-Pgk1 antibody to detect 3-phosphoglycerate kinase (Pgk1) as a loading control. (E) The Rrp4-G58V protein variant is expressed and the steady-state level of the Rrp4 G226D protein variant is decreased in cells coexpressing wild-type Rrp4. Lysates of rrp4Δ cells coexpressing untagged wild-type Rrp4 and Myc-tagged wild-type Rrp4, Rrp4 G58V, or Rrp4 G226D grown at 30°C were analyzed by immunoblotting with an anti-Myc antibody to detect Rrp4-Myc and anti-Pgk1 antibody to detect 3-phosphoglycerate kinase (Pgk1) as loading control. (F) Quantitation of the percentage of Rrp4 or Rrp4 G226D protein detected in lysates of rrp4Δ cells solely expressing Myc-tagged Rrp4 or Rrp4 G226D grown at 30°C or 37°C. Graph shows the mean percentage of Rrp4-Myc protein from three independent experiments (n = 3). Error bars represent standard error of mean. Statistical significance is denoted by asterisk (*P-value ≤0.05). (G) Quantitation of the percentage of Rrp4, Rrp-G58V and Rrp4-G226D protein detected in lysates of rrp4Δ cells expressing Myc-tagged Rrp4 or Rrp4 variants grown at 30°C or 37°C. Graph shows the mean percentage of Rrp4-Myc from three independent experiments (n = 3). Error bars represent standard error of mean. Statistical significance is denoted by asterisk (*P-value ≤0.05). Quantitation of immunoblots in F and G was performed as described in Materials and Methods.
