Fig. 4

Rpl22 enhances IME1 mRNA translation. a Incorporation of ³⁵S methionine and cysteine into bulk protein was monitored by scintillation spectroscopy in wild type (RSY333) and rpl22∆ double mutant (RSY1483) at the indicated times (min) following addition of radioactive amino acids to starved cultures. Error bars represent SEM from two experiments with two independent isolates each. b Wild type (RSY333) and rpl22Δ (RSY1561) double mutant cells were induced to enter meiosis and cycloheximide was added at 9 h following transfer to SPM. Timepoints were taken after addition of CHX at the times indicated. Ime1-3HA specific chemiluminescence signals were detected by Western blot analysis and quantified by CCD camera. Graph shows the averaged results from two independent experiments. Error bars indicate SEM. c Polysomes were isolated from wild type and rpl22∆ double mutant cultures 9 h following transfer to SPM. Total RNA isolated from the indicated fractions was subjected to Northern blot analysis with the indicated probes. The position of the ribosomal subunits and monosomes are indicated. Total RNA lane indicates samples prepared directly from harvested cells. Input represents total RNA isolated from polysome extracts prior to fractionation. d The relative amounts of IME1 mRNA in total RNA preparations from wild type (RSY333) and rpl22Δ (RSY1561) double mutant cells were determined by qRT-PCR. The graph shows the results and SEM from three technical replicates from one experiment