Figure 2.
Rie1 is important for Ime1 expression in early meiosis. (A) Diagram of Rie1. RRM domains are shown in blue, RNP motifs in green, and IDRs in purple. Phenylalanine residues critical for RRM function that we subsequently mutated are indicated in red. (B) Vegetative growth in various media conditions was determined in diploid wild type (B47, blue) and rie1∆ (B1574, red) strains. Strains were inoculated in YPD and grown overnight and then diluted to 0.2 OD600 the next day in YPD (glucose), BYTA (acetate) SDC (complete synthetic medium with glucose), or YPG (glycerol). Shown are mean values from three biological replicates. Statistical significance was determined by Mann–Whitney test (n.s. = not significant). (C and D) Strains harboring IME1-3HA and rie1∆ (B1653, red), wild type RIE1 (B1662, blue), rie1-F240L (B2403, yellow), rie1-F298L (B2836, pink), and rie1-F604L (B2298, purple) were induced to sporulate at 30°C. (C) Protein levels of Ime1 and Pgk1 (loading) were determined by immunoblot (IB) and mRNA levels of IME1 and rRNA (loading) were determined by Northern blot. (D) Quantification of C showing Ime1 protein abundance corrected for IME1 mRNA levels. Biological replicates = 7. (E and F) Strains harboring N-terminal sfGFP-tagged (sfGFP-IME1) and rie1∆ (B1653, red), wild type RIE1 (B1662, blue), rie1-F240L (B2403, yellow), rie1-F298L (B2836, pink), and rie1-F604L (B2298, purple) were induced to sporulate at 30°C. (E) Single-cell measurements of nuclear sfGFP-Ime1 were determined using fluorescence microscopy. Shown are individual measurements for 50 cells for each time point in each genetic background. Mean is indicated by a black bar and statistical significance (***P < 0.0001) was determined by two-sided student’s t test with Welch’s correction for SD differences. (F) Progression through the meiotic divisions was determined at the indicated times by DAPI staining. Biological replicates = 3 and error bars indicate SEM. (G) Strains harboring NDT80-IN, CLB3-3HA, pRim4-OsTIR1, and either RIE1-6FLAG-AID (B1503) or wild type RIE1 (B1604) were induced to sporulate at 30°C. Cells were treated with auxin at 5 h (or vehicle control), released from G2-arrest at 6 h, and samples were collected at the indicated times. Protein levels of Rie1, Clb3, and Pgk1 were determined by immunoblot, and meiotic progression was determined by DAPI staining. Biological replicates = 3. Source data are available for this figure: SourceData F2.

Rie1 is important for Ime1 expression in early meiosis. (A) Diagram of Rie1. RRM domains are shown in blue, RNP motifs in green, and IDRs in purple. Phenylalanine residues critical for RRM function that we subsequently mutated are indicated in red. (B) Vegetative growth in various media conditions was determined in diploid wild type (B47, blue) and rie1∆ (B1574, red) strains. Strains were inoculated in YPD and grown overnight and then diluted to 0.2 OD600 the next day in YPD (glucose), BYTA (acetate) SDC (complete synthetic medium with glucose), or YPG (glycerol). Shown are mean values from three biological replicates. Statistical significance was determined by Mann–Whitney test (n.s. = not significant). (C and D) Strains harboring IME1-3HA and rie1∆ (B1653, red), wild type RIE1 (B1662, blue), rie1-F240L (B2403, yellow), rie1-F298L (B2836, pink), and rie1-F604L (B2298, purple) were induced to sporulate at 30°C. (C) Protein levels of Ime1 and Pgk1 (loading) were determined by immunoblot (IB) and mRNA levels of IME1 and rRNA (loading) were determined by Northern blot. (D) Quantification of C showing Ime1 protein abundance corrected for IME1 mRNA levels. Biological replicates = 7. (E and F) Strains harboring N-terminal sfGFP-tagged (sfGFP-IME1) and rie1∆ (B1653, red), wild type RIE1 (B1662, blue), rie1-F240L (B2403, yellow), rie1-F298L (B2836, pink), and rie1-F604L (B2298, purple) were induced to sporulate at 30°C. (E) Single-cell measurements of nuclear sfGFP-Ime1 were determined using fluorescence microscopy. Shown are individual measurements for 50 cells for each time point in each genetic background. Mean is indicated by a black bar and statistical significance (***P < 0.0001) was determined by two-sided student’s t test with Welch’s correction for SD differences. (F) Progression through the meiotic divisions was determined at the indicated times by DAPI staining. Biological replicates = 3 and error bars indicate SEM. (G) Strains harboring NDT80-IN, CLB3-3HA, pRim4-OsTIR1, and either RIE1-6FLAG-AID (B1503) or wild type RIE1 (B1604) were induced to sporulate at 30°C. Cells were treated with auxin at 5 h (or vehicle control), released from G2-arrest at 6 h, and samples were collected at the indicated times. Protein levels of Rie1, Clb3, and Pgk1 were determined by immunoblot, and meiotic progression was determined by DAPI staining. Biological replicates = 3. Source data are available for this figure: SourceData F2.

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