A comparative analysis of Mitochondrial ORFans: new clues on their origin and role in species with Doubly Uniparental Inheritance of Mitochondria

Despite numerous comparative genomics studies revealed that animal mitochondrial genomes (mtDNAs) are highly conserved in terms of gene content, supplementary genes are sometimes found. Many mechanisms are responsible for the origin of such new genes. For example, novel mitochondrial Open Reading Frames (ORFs) can arise from gene duplication. In bivalve molluscs, a cox2 duplication is found in the clam Ruditapes philippinarum and in the mussel Musculista senhousia. Moreover, nad2 duplication are at the origin of two novel ORFs in the oyster genus Crassostrea. Extra-elements are also found in cnidarian mtDNA, some attributed to the duplication of extant genes others to an ancient invasion by a linear plasmid. Interestingly, mitochondrial ORFans (ORFs having no detectable homology and unknown function) were found in bivalve molluscs with Doubly Uniparental Inheritance (DUI) of mitochondria. In DUI animals two mitochondrial lineages are present: one transmitted through females (F-type), the other through males (M-type), each showing a specific and conserved ORF. The analysis of 34 mitochondrial major Unassigned Regions of Musculista senhousia F- and M- mtDNA allowed us to verify the presence of novel mitochondrial ORFs in this species and to compare them with ORFs from other species with ascertained DUI, with other bivalves and with animals showing new mitochondrial elements. Overall, 17 ORFans from 9 species were analyzed for structure and function. Our results generally excluded an origin from gene duplication for such sequences, instead many clues suggest that the analyzed ORFans arose from endogenization of viral genes. The co-option of such novel genes by viral hosts may have determined some evolutionary aspects of host life cycle, possibly involving mitochondria. These putative proteins may have a role in the maintenance of sperm mitochondria during embryo development, possibly masking them from the degradation processes that normally affects sperm mitochondria in species with strictly maternal inheritance. It is well established that the nucleus regulates mitochondrial gene expression through anterograde regulation. On the other hand, several studies have recently demonstrated that signals from organelles regulate nuclear gene expression by retrograde signalling. It appears likely that, given the complex cross talk between the nucleus and mitochondria, not only chemical messengers but also exported proteins may participate in transducing signals from mitochondrion to nucleus, as, for example, the retrograde signalling that characterize plants with Cytoplasmic Male Sterility. Moreover, the presence of mitochondrial proteins in diverse cellular extra-mitochondrial sites supports the existence of specific export mechanisms by which certain proteins exit mitochondria. In our case, given the presence of a signal peptide in many of the analyzed ORFs, this N-terminal sequence may be used to target the proteins to sites outside mitochondria. The structure similarity of DUI ORFans within evolutionary lineages may indicate that they originated from independent events. If these novel ORFs are in some way linked to DUI establishment, a multiple origin of DUI has to be considered. We can hypothesize that viral selfish elements may have colonized the mitochondrial genome in male bivalves promoting its segregation into primordial germ cells thus allowing the transmission to next generations, and leading to DUI achievement. If this is true, the insertion event and the appearance of DUI might be causally linked, and some implications on the origin and evolution of DUI become evident. Finally, the general mechanism proposed above for the transmission of selfish elements would imply that bivalves are in some way prone to viral integration in the mitochondrial genome and therefore in DUI establishment, and maybe that other animals can have experienced such kind of mitochondrial transmission modification but no evidence has been found so far.