The small ORFeome of Tribolium castaneum reveals deeply conserved small ORFs broadly expressed across tissues

Small open reading frames (small ORFs/smORFs/sORFs) were historically overlooked due to their short length (<100 codons) and detection challenges. It is widely accepted that small ORFs are rarely conserved across distant taxa, particularly in deeply rooted ancestors. Growing evidence supports their biological relevance and potential roles in gene innovation. Here, we integrated genomic, transcriptomic, and proteomic datasets to identify and characterize 454 putative small ORFs in Tribolium castaneum. Conservation analyses revealed 230 small ORFs conserved within Coleoptera, 167 in insects, 85 in arthropods, 39 in eukaryotes, and six in bacteria. Conserved small ORFs displayed higher GC content ([~]44.87%), with bacterial-conserved sequences exceeding 50%. Functional enrichment revealed dozens of small ORFs associated with catalytic, transporter, structural, and regulatory roles. Binding site predictions indicated 254 small ORFs possess protein-protein interaction potential. Expression profiles across 14 RNA-seq libraries spanning multiple developmental stages showed 40.1% of small ORFs expressed (TPM >1) in at least one sample, and 16.5% expressed in five or more libraries. Conserved small ORFs showed broader and more stable expression, while lineage-specific ones displayed restricted expression, mostly in gonads or embryos. Isoformic and small CDSs were the most widely expressed classes. Notably, 29 small ORFs exhibited transcriptional and translational support (Swiss-Prot and/or Ribo-seq), 28 being small CDSs. A total of 390 small ORFs had potential paralogs, supporting gene duplication as a mechanism for small ORF emergence. Altogether, our findings reinforce that small ORFs, contrary to initial assumptions, can be deeply conserved and potentially perform key roles in evolutionary processes.