Fig. 4

Selective loss of 40S subunits from MMS-treated mRNA. A Five out of ten 40S subunits showed statistically significant transiently reduced mRNA binding after MMS treatment, whereas additional four 40S subunits demonstrated very similar, although sub-significantly altered binding pattern (left panel). Such transiently reduced binding was not observed with the 60S subunits (right panel). B 1 h MMS-treatment mediated negligible change in the level of phosphorylated (S52) EIF2S1 within 15 h after treatment (normalized to actin). C Western analysis (left panel) of mRNA-binding proteins after oligo(dT) capture confirmed a marked decrease in the ratio of bound 40S (RPS10) versus 60S (RPL18) subunits in agreement with the SILAC results. Notably, treatment with cycloheximide (10 μg/ml) for 15 min prior to the MMS treatment (CHX) to decrease ribosomal collision, partially or fully restored the RPS10/RPS18 ratio. The right panel shows RPS10/RPL18 ratios bound to mRNA in three independent experiments (given as three individual graphs) after western blotting and probing with IRDye® secondary antibodies and demonstrating the same trend (Spearman rank correlation = 1 between the three series)