Figure 2. Aminoglycosides stimulate genome-wide stop codon readthrough.

(A) Average gene plot showing normalized ribosome densities relative to the distance, in nucleotides, from the stop codon at position 0. Ribosome densities from untreated cells (black), or cells treated for 24 hr with G418 (orange, 0.5 mg/mL), gentamicin (red, 0.5 mg/mL), paromomycin (green, 3 mg/mL), neomycin (blue, 2 mg/mL), tobramycin (yellow, 1 mg/mL), and amikacin (pink, 2 mg/mL) cells are overlaid. Arrows demonstrate the height of peaks at stop codons for Untr, G418, and paromomycin to facilitate comparison. (B) Magnified view of the 3′UTR showing increased densities of ribosomes in this region for AG treated cells. (C) Densities of ribosomes in 3′UTRs (from +5 to +100) are plotted relative to densities of ribosomes in the coding sequence (positions −147 to −16) for each AG. Each replicate is displayed, along with the mean value. (D) RRTS values are displayed for all genes in pooled replicates using box and whisker plots. Median values are represented with the notch indicating the 95% confidence interval, and whiskers representing 1.5 times the interquartile range. Outliers are not shown.

Figure 2—figure supplement 1. Ribosome profiling sample replicates are well correlated.

CDS densities are shown comparing replicates for each AG treatment. Densities were sorted into histograms with 20 bins each, and plotted on the corresponding axis for each sample. In addition to the samples analyzed in Figure 2, a higher concentration of G418 (2.0 mg/mL, light blue) and a 10-minute G418 treatment (0.5 mg/mL, orange) were also tested. Pearson correlations of log-transformed CDS densities show very strong correlations (R > 0.99) as expected.

Figure 2—figure supplement 2. RRTS values are well correlated among replicates.

(A) Average gene plot 3′UTR regions as in Figure 2B of untreated cells (black) or cells treated with G418 (24 hr – 0.5 mg/mL – orange solid line, 24 hr – 2.0 mg/mL – cyan, and 10 min – 0.5 mg/mL – orange dashed line). (B) Ribosome ReadThrough Scores were calculated for all genes by computing the density of ribosomes in the 3′UTR between the NTC and the first in-frame stop codon and dividing this value by the density of ribosomes in the coding sequence. (C) Scatterplots showing log-transformed RRTS values were compared between replicates. Transcripts with RRTS values of zero were assigned the arbitrarily small value of 2⁻¹⁵ and not included when calculating correlations. Histograms were generated for each sample by sorting RRTS values into 20 bins. Pearson correlations reveal strong correlation of this metric between biological replicates (R > 0.7).

Figure 2—figure supplement 3. Aminoglycosides perturb translation initiation and elongation.

(A) Average gene plot showing normalized ribosome densities relative to the distance of the start codon for untreated cells (black), or cells treated with G418 (orange - 0.5 mg/mL, cyan - 2.0 mg/mL, dashed orange – 0.5 mg/mL - 10 min), gentamicin (red - 0.5 mg/mL), paromomycin (green - 3 mg/mL), neomycin (blue - 2 mg/mL), tobramycin (yellow - 1 mg/mL), and amikacin (pink - 2 mg/mL). All cells were treated for 24 hr with the exception of the 10-minute G418 treatment. Each trace is shifted to the right by six nucleotides (two codons) to facilitate comparison of start codon peaks. (B) Codon occupancies are plotted for aminoglycoside treatments (y-axis) compared to untreated cells (x-axis). The average occupancy between two biological replicates is plotted for all 61 sense codons with error bars indicating the standard deviation of these measurements. Glycine (orange), aspartic acid (blue) and isoleucine (green) codons are highlighted, and Pearson correlations are displayed for each treatment.