Pervasive functional translation of noncanonical open reading frames

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). Here, we exploit a systematic CRISPR-based screening strategy to identify hundreds of non-canonical CDSs that are essential for cellular growth and whose disruption elicit specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds that are presented by the HLA system. Interestingly, we find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same mRNA, thus revealing the diverse use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles. Paired RNA-seq and ribosome profiling sequencing data for human induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes, either with or without the translational inhibitor harringtonine. Three replicates are availble for the no drug treatment samples.