Table 1 Approaches to interpreting Ribo-seq ORFs

A Ribo-seq ORF encodes a ‘missing’ conserved protein

Ribo-seq ORFs may be recognized as canonical—in accordance with existing protein annotations—on the basis that the sequence of the proteins they encode shows clear evidence of being maintained by evolutionary selection over a significant period of evolutionary time.

A Ribo-seq ORF encodes a taxonomically restricted protein

Ribo-seq ORFs may encode proteins whose sequences and molecular activities are specific to one species or lineage. Evidence for selection or conservation across distant species or lineages is lacking for these ORFs, either because the protein sequence has diverged beyond recognition from its orthologs, or because the protein evolved recently from previously noncoding material and homologs do not exist in other species or lineages.

A Ribo-seq ORF regulates protein or RNA abundance

Ribosome engagement of regulatory ORFs does not result in a protein product under selection but regulates the abundance of a canonical protein or RNA. This paradigm is well established for uORFs and uoORFs, as noted in Table 2, though it is applicable to other transcript scenarios. Regulatory ORFs may compete for ribosomes with their downstream canonical ORFs or produce nascent peptides that stall ribosomes, leading to the controlled ‘dampening’ of protein expression. Alternative modes of action, such as the induction of RNA decay pathways, the processing of small RNA precursors or the adjustment of RNA stability, have also been inferred.

A Ribo-seq ORF is the result of random translation

The translation of some Ribo-seq ORFs may simply be ‘noise’. Because translation has a high bioenergetic cost, a protein that results from random translation is likely to be translated at lower levels than a canonical CDS and evolve neutrally; it may also be comparatively unstable and could be rapidly degraded. Nonetheless, it is theoretically possible that certain proteins do exist as stable ‘junk’ proteins, or that random translation events affect the expression of canonical proteins. The detection of random Ribo-seq ORFs is less likely to be reproducible.

A Ribo-seq ORF encodes a disease-specific protein

This protein would not be produced under normal physiological homeostasis but could be of major interest for diagnostics and therapeutics. Insights of this sort are especially emerging in cancer biology, where transcription and translation are known to be dysregulated. This leads to the production of ‘aberrant’, possibly rapidly degraded proteins that are commonly antigenic and presented on the cell surface by the HLA system, potentially acting as neoantigens. Furthermore, antigens resulting from disease-specific dysregulated ribosome activity—sometimes called defective ribosomal products (DRiPs)—have also been explored.