Def1 regulates codon-dependent mRNA decay. (A) Change in ribosome density over the A-site versus codon optimality. Codons were separated into three bins based on CSC scores: optimal (n = 20), intermediate (n = 21), and nonoptimal (n = 20). Stop codons were not included in this analysis. The change in ribosome density over the A-site obtained in the Ribo-seq experiment is plotted on a log2 scale. P-values were calculated using a Mann–Whitney test. (B) Representative northern blot of codon optimality reporter gene assay. HIS3 ORFs with variable codon optimality (indicated below each panel) were placed under the control of the GPD1 promoter. The endogenous GPD1 mRNA was used as a loading control and to correct for changes in promoter activity in the mutant. The image capture times for each HIS3 derivative varied because of differences in steady-state levels: 150 sec for 0% and 30% OPT, 60 sec for 50% OPT, and 20 sec for 100% OPT (C) Averages and standard deviations of reporter mRNA expression. The signals for the HIS3 mRNA were first normalized to the endogenous GPD1 mRNA, and then the values in the def1Δ cells were divided by the values in DEF1 cells (relative normalized signal) (n = 4). P-values were calculated using a two-tailed, unpaired t-test, and the values of the 0% reporter were compared to those of the other constructs. (****) P < 0.0001.
