Figure 6. Rcm1 mediates changes in ribosome profiles and stop codon read-through.

Ribosomal profiles provide a snapshot of the translational status and are obtained by UV260-tracing of polysomes, single 80S ribosomes and free LSU and SSU populations, respectively, separated on a continuous sucrose gradient. (a) Loss of Rcm1 does not alter the ribosomal profile representing bulk translation. (b) Both in wt and Rcm1-deficient cells, 30 min H₂O₂ stress induces a shift in ribosomal profile subpopulations, with decrease of polysomes and increase of 80S. (c) After 90 min of H₂O₂ exposure, the decrease in polysome population remains, with the 80S peak recovering to a wt-like recording. (d,e) Loss of Rcm1 (d) as well as oxidative stress (e) significantly promote reading through a PTC mutation within a FF reporter construct (N=6, two-sided Student’s t-test **P<0.01, ***P<0.001, ****P<0.0001). FF luminescence, representing PTC read-through, was normalized to REN luminescence, representing transfection efficiency. Reporter luminescence without a PTC and normalized as above is also shown (no PTC). In this case, Rcm1 deletion and oxidative stress decrease normalized reporter luminescence.