Abstract
Overlapping genes were thought to be essentially absent from the human genome until the discovery of abundant, frameshifted internal open reading frames (iORFs) nested within annotated protein coding sequences. However, it is currently unclear how many functional human iORFs exist and how they are expressed. We demonstrate that, in hundreds of cases, alternative transcript variants that bypass the start codon of annotated coding sequences (CDSs) can recode a human gene to express the iORF-encoded microprotein. While many human genes generate such non-coding alternative transcripts, they are poorly annotated. Here we develope a new analysis pipeline enabling the assignment of translated human iORFs to alternative transcripts, and provide long-read sequencing and molecular validation of their expression in dozens of cases. Finally, we demonstrate that a conserved DEDD2 iORF switches the function of this gene from pro- to anti-apoptotic. This work thus demonstrates that alternative transcript variants can broadly reprogram human genes to express frameshifted iORFs, revealing new levels of complexity in the human transcriptome and proteome.