Pervasive functional micropeptides in humans encoded by non-canonical open reading frames

Protein-coding regions of the genome continue to expand beyond canonical annotations and remain incompletely understood. Despite recent reports demonstrating important cellular functions played by micropeptides from unannotated open reading frames (ORFs), there exists no systematic method to fully uncover the prevalence of functional ORFs and their encoded peptides. Here, using ribosome profiling and mass spectrometry based proteomics, we reveal pervasive translation of ORFs previously annotated as non-coding, including those encoded on long non-coding RNAs (lncRNA ORFs) and upstream regions of protein-coding messages (uORFs). Cross-validation of novel ORFs in six different cell lines via HLA peptidomics confirm their allotype-specific MHC receptor presentation and their engagement with the endogenous HLA processing machinery. We further developed systematic CRISPR-based screens to show that hundreds of these ORFs are essential for cellular growth and encode stable, functional peptides that play diverse roles in cells. By tagging with a split-GFP system, we show that many of the peptides have specific cellular localization patterns and form functional protein complexes. We uncovered uORFs encoding stable peptides that interact with the downstream protein encoded on the same mRNA, suggesting possible regulatory or feedback roles from the bicistronic message in addition to the previously assumed role of uORFs in translational repression. Our results uncover functional micropeptides encoded in the human genome with essential roles in diverse biological processes.