Systematic identification and characterization of eukaryotic and viral 2A peptide-bond-skipping sequences

2A peptides are 18- to 22-amino-acid sequences that cause an unusual co-translational peptide-bond-skipping event. Initially discovered in viruses, they allow multiple proteins to be produced from a single open reading frame. Despite their utility, their evolutionary prevalence and sequence diversity remain unclear. Our computational analyses predict ∼2,200 2A peptides, significantly expanding the known class of 2A peptides (class A) and identifying a previously unrecognized class (class B). Predicted 2A peptides are widespread in RNA viruses and eukaryotes. Functional tests in human cells confirm skipping activity in most cases, suggesting that thousands of active 2A peptides exist. Mutational analysis reveals key residues near the skipped bond and within the upstream region; for instance, class B 2A peptides contain a conserved N-terminal tryptophan, whose register and identity are critical for activity. Together, our findings reveal that 2A peptides are more diverse and widespread than previously appreciated.