Non-canonical open reading frames encode functional proteins essential for cancer cell survival

Although genomic analyses predict many non-canonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here, we experimentally interrogated 553 candidates selected from non-canonical ORF datasets. Of these, 57 induced viability defects when knocked out of human cancer cell lines. Upon ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. CRISPR tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. Our experiments suggest that non-canonical ORFs can express biologically active proteins that are potential therapeutic targets.