Fig. 3: Ribosome profiling supports induction of translational readthrough by FUr.

A, Schematic overview of the Ribo-seq experiments. B, Line plots of normalised Ribo-seq read coverage across the entire TP53 CDS (top) and a zoomed view of the region between R213X and the canonical TP53 stop codon (bottom). Locations of start and stop positions and premature termination codon R213X are annotated. C, Segment plots of relative Ribo-seq read coverage of TP53 between the premature termination codon R213X and the canonical stop codon. Positive and negative values indicate coverage changes in H1299-R213X cells upon treatment with either FUr or G418 in comparison to untreated cells. D, Box plot of premature R213X stop codon readthrough ratios observed by Ribo-seq. Ratios were calculated by taking the sum of all in-frame reads after the R213X premature stop until the canonical stop codon and dividing it with the sum of all in-frame reads from the canonical start position until the premature stop codon, normalized for CDS codon length (see Methods), N = 4. Statistical analyses were performed comparing all conditions to each other using Friedman test followed by Dunn’s multiple comparisons test as data did not fit normal distribution (ns = not significant). E, Metagene bar plot showing footprint coverage around the stop codons of annotated reading frames of all translated protein-coding genes. F, Box plot of stop codon readthrough ratios on a metagene level, as determined by Ribo-seq per treatment condition. Ratios were calculated by taking the sums of in-frame reads after the stop codon and dividing these with the sums of in-frame reads before the stop codon, both normalised for feature length. Replicate experiments in H1299-EV and H1299-R213X cells were merged per treatment type for this visualization, N = 8. Statistical analyses were performed comparing all conditions to each other using repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). In D and F, each dot represents a replicate experiment. Data are represented as median ± 95% confidence interval, box limits represent the upper and lower quartiles.