Identification of polysome-associated translational regulators during oxidative stress and amino acid starvation

To identify RBPs with previously unappreciated roles in translational regulation, we performed polysome proteomics (polysome profiling and label-free mass spectrometry) to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and changes in association during two acute stresses that induce the integrated stress response (ISR): oxidative stress (0.45 mM H2O2 treatment for 15 min) and amino acid starvation (10 mM 3-AT treatment for 15 min). Five fractions were isolated from sucrose gradients prepared from each extract, corresponding to monosomes (F1) and polysomes with increasing numbers of ribosomes per mRNA (F2-F5). Unfractionated cyotplasmic lysates (totals) were analysed for each sample. Four replicate extracts were prepared and analysed for each condition.