Mapping subcellular localizations of unannotated microproteins with MicroID

Identification of translated small open reading frames using proteogenomic approaches and ribosomal profiling has revealed thousands of previously unannotated cellular microproteins, or polypeptides of less than 150 amino acids, and alternative proteins (alt-proteins) that are co-encoded with canonical proteins and can be longer than 150 amino acids. The majority of microproteins and alt-proteins remain uncharacterized, in part because their short lengths preclude analysis of homology to known protein domains. The subcellular localizations of microproteins and alt-proteins remain largely unknown but can have significant implications for their functions. Proximity-dependent biotinylation has provided an attractive approach to define the canonical protein composition of subcellular compartments in living cells and animals. Here, we developed a high-throughput technology for global mapping of unannotated microproteins and alt-proteins to subcellular localizations by proximity-dependent biotinylation with TurboID (MicroID). We showed that more than 150 microproteins and alt-proteins are associated with subnuclear organelles and with complexes that carry out critical cellular functions. One of these novel microproteins, alt-LAMA3, localizes to the nucleolus and functions in pre-rRNA transcription. As a demonstration of its in vivo utility, we applied MicroID in a mouse model, identifying a conserved nuclear microprotein translated from a pseudogene, and establishing the foundation for use of this technology in discovery and characterization of microproteins in animals.