Extended Data Fig. 5: Hierarchical ORF prediction based on Ribo-seq identifies short, overlapping, tissue-specific nuORFs.

a. nuORFs predictions are more sample and tissue specific than annotated ORFs. Proportion of annotated ORFs (grey) and nuORFs (pink) in the MHC I immunopeptidome (y axis, and pie chart). Hashed: proportion predicted only at the leaf and clade level, but not at the root. b. Two overlapping, MHC I MS-detected 5’ uORFs in LUZP1 as an example of tissue-specific, overlapping nuORFs identified by hierarchical ORF prediction. uORF2 (pink) was predicted in the CLL clade, and not at the root. uORF1 (cyan) was predicted at the root and not in the CLL clade. Detected peptides outlined in red with the HLA alleles where peptides were detected marked below. c. SOCS1 gene as an example of identification of short, overlapping nuORFs. SOCS1 gene encodes three translated proteins: the annotated ORF, an out-of-frame iORF, and a 5’ overlap ouORF. Two MHC I MS-detected peptides from 5’ ouORF outlined in yellow. Detected iORF peptide outlined in red and shown in higher magnification below. Bottom: Histogram of Ribo-seq reads supporting translation of the annotated ORF (blue) and the out-of-frame iORF (green).