Evolutionary origins and interactomes of human, young microproteins and small peptides translated from short open reading frames

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids—all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome. [Display omitted] •Most sORF-encoded human microproteins emerged in primates and often evolved de novo•We identify new human sORF-encoded peptides that are smaller than 16 amino acids•We detect interacting proteins for 266 sORF-encoded proteins in MS-based screens•This study builds a resource to investigate small and evolutionarily young proteins Sandmann et al. explore the evolution and interactomes of the smallest and evolutionarily youngest members of the human proteome. They identify previously unknown human short ORF-encoded microproteins and show that conserved and young microproteins that emerged de novo in primates can engage with vital biological processes.