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. 2020 Jan 23;9:e52611. doi: 10.7554/eLife.52611

Figure 5. Surrounding sequence context influences stop codon readthrough genome-wide.

(A) Within a sequence window corresponding to the footprint of a translating ribosome at the NTC (15 nt upstream to 12 nt downstream), the likelihood of each nucleotide increasing or decreasing RRTS is plotted with positive values indicating more readthrough and negative values indicating less readthrough. Each nucleotide was tested using one-sided t-tests against all other nucleotides at each position for untreated (top) and G418-treated (bottom) cells. P values were adjusted using the Benjamini-Hochberg correction. Letters are scaled in proportion to the adjusted P value. (B) The frequencies of each nucleotide are plotted for all positions 40 nt upstream to 60 nt downstream of the stop codon. Nucleotides are plotted in order of increasing frequencies. (C) As in (B), nucleotide frequencies are plotted for first in-frame 3′TCs, 12 nt upstream to 12 nt downstream of the 3′TC. (D) Using normalized ribosome densities in a window 30 nt upstream to 30 nt downstream of stop codons, ribosome density downstream of stop codons was calculated for NTCs and the first in-frame 3′TCs in G418-treated cells. A paired t-test was performed revealing significant differences between levels of SCR between 3′TCs and NTCs (p=0.02).

Figure 5—source data 1. Source data from ribosome profiling analysis used in Figure 5.

Figure 5.

Figure 5—figure supplement 1. Linear regression analysis of stop codon readthrough.

Figure 5—figure supplement 1.

(A) A magnified view of Figure 5A (bottom) is shown to facilitate comparison between untreated and G418-treated cells. (B) Ridge regressions were performed to calculate regression coefficients for each nucleotide in the same sequence window as in Figure 5A (15 nt upstream to 12 nt downstream of the NTC). The three nucleotides of the stop codon were encoded by a single position. Positive values indicate increased likelihood of readthrough while negative values indicate decreased likelihood of readthrough. Letters are scaled to the size of the regression coefficient. Regression coefficients were calculated for untreated (top) and G418-treated cells (bottom).
Figure 5—figure supplement 2. Metagene plots for readthrough at NTCs and 3′TCs.

Figure 5—figure supplement 2.

(A–B) Normalized ribosome densities were calculated in a window 30 nt upstream to 30 nt downstream of NTCs (A) and 3′TCs (B). Average ribosome densities upstream and downstream of the stop codons were calculated, and are indicated by dashed lines for these regions. These densities were used for calculations in Figure 5D. The codons before and after the stop codon were excluded from this analysis.