RIE1 affects global translation and transcript levels of the UME6 regulon. (A–C) Strains harboring rie1∆ (B1574, red) or wild type RIE1 (B47, blue) were (A) induced to sporulate at 30°C or (B) grown to mid-log phase in rich medium (YPD) and samples were collected at the indicated times. Lysates were fractionated on 10–50% sucrose density gradients with continuous monitoring at 260 nm. Positions of the RNP, 40S, 60S, 80S, and polysomal ribosome peaks are indicated. (C) Lysates were treated with 25 mM EDTA to disrupt ribosomes prior to fractionation. Biological replicates = 2. IB, immunoblot. (D) Strains harboring rie1∆ (B1574, red) or wild type RIE1 (B47) were induced to sporulate at 30°C and total RNA samples were collected at 0 and 4 h for RNA-seq analysis. Shown is a volcano plot of log₂ fold change plotted against −log₁₀ P value of RNA-seq reads for wild type and rie1∆. Values represent a ratio of expression between the wild type and rie1∆ cells between the 0 and 4 h time points. The further right the value, the more enriched the mRNA in the wild type strain. Genes with a log₂ fold change less than −0.75 are colored red and genes with a log₂ fold change >0.75 are colored blue. Genes within the UME6 regulon (putative IME1 targets; from Williams et al., 2002) are labeled. UME6 regulon genes (mean enrichment 0.561) are significantly enriched in wild type (two-tailed t test of 10,000 data randomizations: P value <0.0001). IME1 mRNA is highlighted in red (fold change = 0.368; P value 0.67). Source data are available for this figure: SourceData FS4.