Prediction and evaluation of Split-ORFs using Ribo-seq data

Abstract Split Open Reading frames (Split-ORFs) occur in transcripts containing at least two open reading frames, each encoding a part of the same full-length protein. These multiple open reading frames arise from alternatively spliced transcript isoforms. Split-ORFs have been described in the SR protein family of splicing factors, where the resulting protein halves play important autoregulatory roles. Here, we present the Split-ORF pipeline, a computational tool that predicts Split-ORFs from transcripts’ sequences and identifies regions unique to the predicted Split-ORF products. Using this pipeline, we predicted more than 14,000 Split-ORF transcripts from alternatively spliced human transcripts containing premature termination codons or retained introns. Hundreds of the Split-ORF unique regions show significant Ribo-seq coverage across diverse cell types and diseases. The candidate Split-ORF genes with significant Ribo-seq coverage are enriched for RNA-binding and RNA-processing functions and the majority of them encodes RNA-binding proteins. Together, these results suggest that Split-ORFs are more widespread than previously assumed and are expressed across diverse cellular contexts. This work paves the road for future studies of the Split-ORF candidates, the mechanisms of their biogenesis and their functions within the RNA-binding protein class.