Ribosome translational activity decreases upon treatment with menadione or cisplatin in dose-dependent manner. (A) Mid-log wild-type cells BY4741 grown in YPD were treated with 50 µM or 100 µM menadione for 2 h at 30°C or left untreated. Cells were lysed, and ribosomes were precipitated by ultracentrifugation through 20% glycerol cushion as illustrated in Figure 1B. Ribosomal pellet P180 was resuspended in buffer A, and 2 µg of total RNA was analyzed by northern hybridizations with the indicated probes. (B) Ribosomes (9 µg RNA) prepared as described in A were added to translationally active ribosome-free lysate S180 prepared from CFE (Fig. 1A), along with amino acids containing labeled [35S]-Met/Cys. Reactions were incubated at 21°C, 4 µL aliquots were taken at indicated time points, and proteins were precipitated by TCA. Incorporation of [35S]-Met/Cys into nascent peptides was measured by scintillation counting; CPM (count per minute) values were plotted as graphs. (C) Ribosomes (9 µg RNA) prepared as described in A were added to translation reactions containing S180 and 400 ng of capped-mRNA reporter encoding TAP-RLuc (∼56 kDa). Reaction products were analyzed by the Renilla luciferase assay. The luminescent signals were normalized by phosphorimaging units from A and the resulting RLuc/18S rRNA ratios are presented as bar graphs. (D, top) Schematics for Firefly-nano luciferase reporter (∼81 kDa). (Bottom) Mid-log wild-type cells BY4741 grown in YPD were treated with 0, 25 µM, 50 µM, or 100 µM menadione for 2 h. Ribosomes were extracted, solubilized, and added (9 µg RNA) to translation reactions containing 400 ng of the capped dual firefly-nano luciferase reporter mRNA. Reaction products were analyzed by Nano-Glo Dual-Luciferase Reporter assay; the Nano-Luc/FLuc ratio is shown on the right panel. (E) Complete CFE was treated with cisplatin at the concentrations indicated in the figure. Ribosomes were purified from the drug-treated CFE by centrifugation through a glycerol cushion as described for Figure 5A. Ribosomes from the resuspended P180 (9 µg RNA) were added to untreated S180 charged with 300 ng of capped TAP mRNA reporter. Control reactions on the left contained ribosomes only (P180) or ribosome-free supernatant only (S180). TAP and ribosomal protein Rpl3 (control for ribosome amount) were detected by western blotting. In panels C–E, all translation reactions were incubated at 21°C for 90 min. In all graphs, error bars represent standard error of the mean (SEM) of three experiments.
