Translational landscape of XUTs. (A) Experimental scheme. Ribo-seq libraries were prepared from biological duplicates of WT and upf1Δ cells grown in native conditions (no CHX) or treated for 15 min with CHX (100 μg/mL final concentration). SmORFs (≥5 codons, starting with an AUG) were detected using the Ribotricer software (Choudhary et al. 2020), pooling all conditions together (list 1) or analyzing them separately (list 2). A third list was produced from list 2 upon application of a signal threshold (≥10 reads/smORF). See lists in Supplemental Table S2. (B) Venn diagram showing the number of XUTs detected as translated by Ribotricer (list 2) in each of the indicated conditions. See also Supplemental Table S2. (C) Metagene of Ribo-seq signals along the 633 translated XUTs (list 2). For each condition, the densities (tag/nt, log2) along the XUTs ±200 nt were piled up, then the average signal was plotted. The shading surrounding each line denotes the 95% confidence interval. (D) Heatmap view of the Ribo-seq signals (densities, tag/nt) from positions −50 to +150 relative to the AUG codon of the smORF showing the highest signal for the 510 NMD-sensitive and 123 NMD-insensitive XUTs detected as translated. A separate heatmap is shown for each condition.
