Impact of Ecm2 on splicing of ACT1–CUP1 reporter pre-mRNAs. (A) Schematic of the ACT1–CUP1 reporter pre-mRNA with nonconsensus substitutions noted. (B) ACT1–CUP1 assay results. Representative images of yeast growth after 3 days at 30°C on agar plates made with -leu -trp dropout media containing 0 or 0.7 mM Cu2+ are shown above the bar graph. Each value in the graph represents the highest concentration of Cu2+ at which growth was observed on plates containing defined concentrations of Cu2+ and are representative of results obtained in replicate assays. (C) Schematic of the modified ACT1–CUP1 reporter containing a competing, cryptic 5′ SS. (D) Primer extension assay of cryptic 5′ SS usage using the reporter shown in panel C. Primer extension of the U6 snRNA was included as a control. The percentages of cryptic products (ratios of cryptic products/total products) are shown below the gel and are the averages of three replicate experiments ± the standard deviation.
