Systematic identification and characterization of eukaryotic ...

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Highlights

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  HMMER search reveals thousands of previously unknown 2A peptide sequences

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  Analysis of 2A peptide sequences uncovers a unique class, class B

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  Class B activity is dependent on identity and register of key amino acids

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  2A peptide activity is dependent on both length and inclusion of the GSG linker

Summary

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.

Keywords

2A peptide

translation

peptide bond skipping

ribosome

exit tunnel

Research topic(s)

CP: Molecular biology

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These authors contributed equally

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Lead contact

© 2025 The Author(s). Published by Elsevier Inc.