Rie1 affects Ime2 and Ime4 levels and binds IME1 mRNA. (A) Strains harboring IME2-3HA and rie1∆ (B1701, red), or wild type RIE1 (B1704, blue), or IME4-3HA and rie1∆ (B1656, red), or wild type RIE1 (B1698, blue) were induced to sporulate at 30°C. Protein levels of Ime2, Ime4, and Pgk1 (loading) protein levels were determined by immunoblot (IB) and mRNA levels of IME2, IME4, and rRNA (loading) were determined by Northern blot. Quantifications of Ime2 and Ime4 protein levels (corrected for mRNA levels) are shown below. (B and C) Rie1 binds IME1 mRNA. (B) Coomassie-stained SDS-PAGE of recombinant Rie1 purified from E. coli. (C) 5 pmol of in vitro transcribed IME1 mRNA (including UTRs) was incubated with purified Rie1 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 which were stained with SYBR Gold. Binding caused IME1 mRNA to shift upwards. Percent bound IME1 mRNA is plotted (y axis) vs. concentration of Rie1 (x axis). Source data are available for this figure: SourceData FS2.