Conserved novel ORFs in the mitochondrial genome of the ctenophore Beroe forskalii

To date, five ctenophore species’ mitochondrial genomes have been sequenced, and each contains open reading frames (ORFs) that if translated have no identifiable orthologs. ORFs with no identifiable orthologs are called unidentified reading frames (URFs). If truly protein-coding, ctenophore mitochondrial URFs represent a little understood path in early-diverging metazoan mitochondrial evolution and metabolism. We sequenced and annotated the mitochondrial genomes of three individuals of the beroid ctenophore Beroe forskalii and found that in addition to sharing the same canonical mitochondrial genes as other ctenophores, the B. forskalii mitochondrial genome contains two URFs. These URFs are conserved among the three individuals but not found in other sequenced species. We developed computational tools called pauvre and cuttlery to determine the likelihood that URFs are protein coding. There is evidence that the two URFs are under negative selection, and a novel Bayesian hypothesis test of trinucleotide frequency shows that the URFs are more similar to known coding genes than noncoding intergenic sequence. Protein structure and function prediction of all ctenophore URFs suggests that they all code for transmembrane transport proteins. These findings, along with the presence of URFs in other sequenced ctenophore mitochondrial genomes, suggest that ctenophores may have uncharacterized transmembrane proteins present in their mitochondria.