25S rRNA depurination alters sucrose gradient profiles and is present in polysome and monosome fractions. (A) Representative sucrose gradient profiles of WT BY4741 cells transformed with cDNAs encoding PAP (red), PAPx (blue), and EV (black) following induction with 2% galactose for 6 h. Cell lysates were separated by a 20%–50% sucrose gradient, and the absorbance of collected fractions was measured at A260. (B) Total RNA (500 ng) was isolated from each sucrose gradient fraction from WT BY4741 cells transformed with PAP, PAPx, and EV cDNAs following induction with 2% galactose for 6 h. RT-qPCR was performed to measure “reference” and “target” cDNA levels as illustrated in Supplemental Figure S1. Fold changes for PAP and PAPx are shown relative to EV. The statistical significance of differences in depurination levels was determined by two-way ANOVA with Tukey's multiple comparisons test. Bars represent means ± SEM of three independent biological replicates (n = 3). The different letters on bars represent statistically significant differences at P < 0.05.
