Figure S5.
Rie1 and Sgn1 work together to facilitate timely meiotic entry. (A) Strains harboring rie1∆ (B1574) or wild type RIE1 (B47) were induced to sporulate at 30°C and samples were collected at 2 h. IME1 poly(A) tail length was assessed by oligo(dT) Northern blot assay. Differences in band sizes between samples with and without oligo(dT) indicate size of the poly(A) tail. Biological replicates = 2. (B) Strains harboring N-terminally tagged sfGFP-IME1 and rie1∆ (B2430 ), wild type RIE1 and SGN1 (B2459), sgn1∆ (B3375), or rie1∆ sgn1∆ (B3378) were induced to sporulate at 30°C. Meiotic progression was determined by DAPI staining. Biological replicates = 3 and error bars indicate SEM. (C and D) Strains harboring NDT80-IN, CLB3-3HA, and either rie1∆ (B1418), sgn1∆ (B2606), or wild type RIE1 SGN1 (B48) were induced to sporulate at 30°C. (C) Protein levels of Clb3 and Pgk1 (loading) protein levels were determined by immunoblot (IB), and mRNA levels of CLB3 and rRNA (loading) were determined by Northern blot. Biological replicates = 2. (D) Clb3 quantification (loading corrected) are shown. (E and F) Sgn1 binds IME1 mRNA. (E) Coomassie stained SDS-PAGE of recombinant Sgn1 purified from E. coli. (F) 5 pmol of in vitro transcribed IME1 mRNA (including UTRs) was incubated with purified Sgn1 or BSA (gradient from 100, 50, 10, 5, 1, 0.5, 0.1 pmol) for 45 min at 30°C. Samples were separated on agarose gels that were stained with SYBR Gold. Binding caused IME1 mRNA to shift upwards. Percent bound IME1 mRNA is plotted (y axis) vs. concentration of Sgn1 (x axis). (G) Strains harboring N-terminal sfGFP-tagged (sfGFP-IME1) and rie1∆ (B1653, red), wild type RIE1 (B1662, blue), or rie1-D429A, S433A, S435A (B3562, yellow) were induced to sporulate at 30°C. Meiotic progression was determined by DAPI. (H) Cell sizes of diploid wild type (B47, blue), rie1∆ (B1574, red), sgn1∆ (B3088, orange), and rie1∆ sgn1∆ (B3342, gray) strains were determined after overnight growth in BYTA (pre-meiotic) medium. Mean is indicated by a black bar and statistical significance (P < 0.05) was determined by student’s t test.

Rie1 and Sgn1 work together to facilitate timely meiotic entry. (A) Strains harboring rie1∆ (B1574) or wild type RIE1 (B47) were induced to sporulate at 30°C and samples were collected at 2 h. IME1 poly(A) tail length was assessed by oligo(dT) Northern blot assay. Differences in band sizes between samples with and without oligo(dT) indicate size of the poly(A) tail. Biological replicates = 2. (B) Strains harboring N-terminally tagged sfGFP-IME1 and rie1∆ (B2430 ), wild type RIE1 and SGN1 (B2459), sgn1∆ (B3375), or rie1∆ sgn1∆ (B3378) were induced to sporulate at 30°C. Meiotic progression was determined by DAPI staining. Biological replicates = 3 and error bars indicate SEM. (C and D) Strains harboring NDT80-IN, CLB3-3HA, and either rie1∆ (B1418), sgn1∆ (B2606), or wild type RIE1 SGN1 (B48) were induced to sporulate at 30°C. (C) Protein levels of Clb3 and Pgk1 (loading) protein levels were determined by immunoblot (IB), and mRNA levels of CLB3 and rRNA (loading) were determined by Northern blot. Biological replicates = 2. (D) Clb3 quantification (loading corrected) are shown. (E and F) Sgn1 binds IME1 mRNA. (E) Coomassie stained SDS-PAGE of recombinant Sgn1 purified from E. coli. (F) 5 pmol of in vitro transcribed IME1 mRNA (including UTRs) was incubated with purified Sgn1 or BSA (gradient from 100, 50, 10, 5, 1, 0.5, 0.1 pmol) for 45 min at 30°C. Samples were separated on agarose gels that were stained with SYBR Gold. Binding caused IME1 mRNA to shift upwards. Percent bound IME1 mRNA is plotted (y axis) vs. concentration of Sgn1 (x axis). (G) Strains harboring N-terminal sfGFP-tagged (sfGFP-IME1) and rie1∆ (B1653, red), wild type RIE1 (B1662, blue), or rie1-D429A, S433A, S435A (B3562, yellow) were induced to sporulate at 30°C. Meiotic progression was determined by DAPI. (H) Cell sizes of diploid wild type (B47, blue), rie1∆ (B1574, red), sgn1∆ (B3088, orange), and rie1∆ sgn1∆ (B3342, gray) strains were determined after overnight growth in BYTA (pre-meiotic) medium. Mean is indicated by a black bar and statistical significance (P < 0.05) was determined by student’s t test.

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