Characterization and validation of predicted uORFs. a The number of genes with at least one predicted ORF (bar plot) in the 5′ leader of evaluated protein-coding genes. The number of genes with a predicted ORF terminated upstream in the 5′ leader only (orange), terminated in the CDS only (blue), or with both a predicted upstream- and CDS-terminated ORF (overlap). b Distribution of predicted ORF translation initiation position relative to the annotated protein-coding CDS start site. c Distribution of predicted ORF lengths. d Distribution of uORF translation start sites (TIS). AUG represents all AUGs predicted by SPECtre, or upstream/downstream 30-nt from the SPECtre predicted start site if no intervening stop codon is present. Near-cognate start codons are utilized in the majority of uORFs, while AUG is the single most common start site. e Plots show mRNA reads (grey) and RPF counts (cyan/gold) for ARF4. The annotated uORF is characterized by the presence of consistent RPF coverage in the 5′ leader. f Schematic of the uORF nanoluciferase (nLuc) reporters used in this study. GGG-nLuc serves as a negative control, as its AUG initiation start codon is mutated to a GGG codon. This reporter supports very little translation. A table of the predicted start sites for each uORF reporter. g nLuc assays performed in SH-SY5Y cells confirmed expression of these uORFs (teal). 5′ leaders not included in the uORF dataset (black) are below the GGG-nLuc reporter activity and considered not translated. All values are normalized to the GGG-nLuc control performed in parallel, data for individual reporters was collected in triplicate in multiple experiments. Student’s t test, all teal uORFs in panel F) have a p value ≤0.0001. Graph represents mean ± S.E.M. h Frameshifting the uORF relative to nLuc decreases translation of the reporter. The reporter was cloned so that the nLuc tag was frameshifted (f.s.) out of frame with the predicted uORF and the CDS start site. n = 3, Student’s t test, ****p ≤ 0.0001. Graph represents mean ± S.E.M.