Fig. 5: Similar ribosome occupancy at the termination codon of NMD-sensitive and NMD-insensitive mRNAs.

a Scheme of in vitro synthesized RLuc reporter mRNAs. with (+pA) or without (−pA) a 80 nts long poly(A) tail. TC: orange dot. b Toeprint analysis with reporter mRNAs depicted in a. In vitro translation was performed in the presence (+) or absence (−) of puromycin. Sanger sequencing reactions were run in parallel (G,T,C,A). The TC position and the toeprint band corresponding to the ribosomes at the TC (+18) are indicated. c Relative quantification of +18 translation-dependent band from three individual experiments versus translation-independent mRNA signals normalized to 200 + pA conditions. Bars denote average values, dots depict the measurements of the three independent experiments, bars represent SD (Unpaired, two-sided statistical t-tests: 200-pA/p200 + A p value = 0.983, 1400-pA/1400 + pA p value = 0.701 1400 + pA/200 + pA p value = 0,078). d (Top) Metagene analysis of ribosome-protected footprints from HeLa cells from three independent ribosome profiling experiments. Transcripts were aligned to the stop codon (0) and the mapped reads from 300 nucleotides upstream to 100 nucleotides downstream of the stop codon are shown. (Bottom) Heatmap of the ribosome-protected reads of all 60’000 transcripts considered for the metagene analysis. Transcripts were ordered according to their ribosome occupancy at the termination codon (transcripts with the highest number of reads at the stop codon at the top). NMD-sensitive transcripts are shown on the left of the heatmap panels. Each pixel line corresponds to the average of 10 transcripts. e Analysis of mean ribosome occupancy at the stop codon relative to mean ribosome occupancy in CDS, performed for NMD targets (identified as in d) and all other transcripts (Others). Violin plots showing the counts distribution of ribosome-derived reads mapping at the stop codon relative to ribosome-derived reads aligning to the CDS as an average of 3 biological replicates. Boxplots (middle) indicate the percentiles (5,25,50,75, and 95) of the ribosome occupancy values distribution with dots representing outliers. Percentage of reads relative to total reads are shown on the y-axis. Anova statistic test, p value = 0.65. Source data are provided as a Source Data File.