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. 2023 Mar 2;13(8):jkad049. doi: 10.1093/g3journal/jkad049

Fig. 7.

Fig. 7.

The rrp4-M68T mpp6Δ double mutant cells exhibit impaired growth that is exacerbated on drugs that impact RNA processing. a) Cartoon schematic of the budding yeast nuclear RNA exosome in complex with nuclear cofactors Mpp6 (turquoise, labeled "Mpp6") and Rrp6/47 (light green/dark green, labeled "Rrp6" and "Rrp47") (Schuller et al. 2018). Serial dilution growth assays of double mutant (B) rrp4-M68T mpp6Δ or (C) rrp4-M68T rrp47Δ cells at 30°C and 37°C. The double mutant cells (rrp4Δ with mpp6Δ or rrp47Δ) containing control RRP4 or rrp4 variants rrp4-M68T and rrp4-G226D plasmids were serially diluted, spotted onto selective solid media, and grown at the indicated temperatures for two days. The double mutant cells rrp4-G226D mpp6Δ and rrp4-G226D rrp47Δ were included as a comparative control and show growth defects as described previously (Sterrett et al. 2021). Data shown are representative of three independent assays (n = 3). d) and e) Double mutant cells containing rrp4-G226D and mpp6Δ exhibit a statistically significant increase in doubling time in liquid culture. Double mutant cells (rrp4Δ mpp6Δ or rrp4Δ rrp47Δ) containing control RRP4 or rrp4-M68T plasmids were diluted in selective media and grown at 37°C with optical density measurements used to assess cell density over time. Data shown is collected from four independent samples (n = 4). e) Doubling time for each sample was quantified and normalized to the growth rate of control RRP4 cells. All calculations were performed as described in Materials and Methods. Full liquid growth curves of both rrp4-M68T mpp6Δ and rrp4-M68T rrp47Δ mutant cells are shown in Supplementary Fig. 5. f) Double mutant cells rrp4-M68T mpp6Δ exhibit impaired growth on solid media containing drugs impacting RNA processing. The rrp4Δ mpp6Δ cells expressing RRP4, rrp4-M68T, or rrp4-G226D were serially diluted, spotted onto solid YEPD media containing 3% formamide or selective media containing 25 µM fluorouracil (5-FU) and grown at 30°C for three days. The rrp4-M68T mpp6Δ cells show impaired growth when compared to RRP4 mpp6Δ cells. The rrp4-G226D mpp6Δ cells show exacerbated growth defects on 3% formamide and 25 µM 5-FU at 30°C. Data shown are representative of three independent assays (n = 3).