Deeply conserved and expressed small ORFs in the model beetle Tribolium castaneum

Abstract 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. However, growing evidence supports their biological relevance and potential roles in gene innovation. Integrating genomic, transcriptomic, and proteomic datasets, we identified and characterized 454 putative small ORFs in Tribolium castaneum . Conservation analyses revealed 230 potential orthologs 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, stable expression, while lineage-specific genes displayed restricted expression, mostly in gonads or embryos. Isoformic and small CDSs (coding DNA sequences) were the most widely expressed classes. Notably, 29 small ORFs exhibited transcriptional and translational support (Swiss-Prot/Ribo-seq), 28 being small CDSs. Furthermore, 390 small ORFs had potential paralogs, some homologous to described proteins. Our findings highlight the small ORF diversity and provide a framework for distinguishing conserved small proteins from lineage-specific candidates, emphasizing the need for experimental validation to determine their functional relevance and evolutionary significance.