Evidence for a fourteenth mtDNA-encoded protein in the female-transmitted mtDNA of marine Mussels (Bivalvia: Mytilidae)

Abstract

Background: A novel feature for animal mitochondrial genomes has been recently established: i.e., the presence of additional, lineage-specific, mtDNA-encoded proteins with functional significance. This feature has been observed in freshwater mussels with doubly uniparental inheritance of mtDNA (DUI). The latter unique system of mtDNA transmission, which also exists in some marine mussels and marine clams, is characterized by one mt genome inherited from the female parent (F mtDNA) and one mt genome inherited from the male parent (M mtDNA). In freshwater mussels, the novel mtDNA-encoded proteins have been shown to be mt genome-specific (i.e., one novel protein for F genomes and one novel protein for M genomes). It has been hypothesized that these novel, F- and M-specific, mtDNA-encoded proteins (and/or other F- and/or M-specific mtDNA sequences) could be responsible for the different modes of mtDNA transmission in bivalves but this remains to be demonstrated.

Methodology/principal findings: We investigated all complete (or nearly complete) female- and male-transmitted marine mussel mtDNAs previously sequenced for the presence of ORFs that could have functional importance in these bivalves. Our results confirm the presence of a novel F genome-specific mt ORF, of significant length (>100aa) and located in the control region, that most likely has functional significance in marine mussels. The identification of this ORF in five Mytilus species suggests that it has been maintained in the mytilid lineage (subfamily Mytilinae) for ∼13 million years. Furthermore, this ORF likely has a homologue in the F mt genome of Musculista senhousia, a DUI-containing mytilid species in the subfamily Crenellinae. We present evidence supporting the functionality of this F-specific ORF at the transcriptional, amino acid and nucleotide levels.

Conclusions/significance: Our results offer support for the hypothesis that "novel F genome-specific mitochondrial genes" are involved in key biological functions in bivalve species with DUI.

Figure 1. A typical Mytilus mitochondrial genome.

All genes are encoded on the same strand. Gene identities: nd1-6 and nd4l, NADH dehydrogenase subunits 1–6 and 4L; cytb, cytochrome b; cox1-3, cytochrome c oxidase subunits I–III; atp6-8, ATP synthase subunit 6 and 8 (protein-coding genes in white); 12SrRNA and 16SrRNA, small and large subunits of ribosomal RNA (in light gray). Transfer RNA genes are depicted by one-letter amino acid codes (in gray). The red and blue lines at the inner periphery of the ring represent EST sequences for the F and M mt genomes of M. edulis/M. galloprovincialis, respectively. Schematics of the structure of a typical F-type (left) and M-type (right) control regions, which are located between the 16SrRNA and trnY genes, are shown. The F-ORF-VD1 is identified in the F-type control region. CR, control region; CD, conserved domain; VD1, variable domain 1; VD2, variable domain 2 , . The mean size of each domain of the CR is shown. *The “standard” F-type CR of M. trossulus, which is a F/M recombinant CR, is not presented.

Figure 2. T-COFFEE alignment of the translated F-specific ORF sequences of mytilid mussels.

Three of more identical amino acids within a column are highlighted in blue. Conservation (Cons.) score values and quality (Qual.) of the alignment are indicated. Dashes (–) denote a missing residue at this position in comparison with other sequence(s). Mca, M. californianus; Mco, M. coruscus; Med, M. edulis; Mga, M. galloprovincialis; Mse, M. senhousia; Mtr, M. trossulus.

Figure 3. Amino acid composition of mytilid F-specific ORFs, protein-coding gene ATP8 and putative M-ORF-VD1.

(A) Overall amino acid composition of Mytilus spp. F-ORF-VD1 and M. senhousia F-ORF protein sequences. (B) Composition of chemically equivalent amino acids of Mytilus spp. F-ORF-VD1 and M. senhousia F-ORF protein sequences. (C) Overall amino acid composition of Mytilus spp. F-ATP8 and M. senhousia F-ATP8 protein sequences. (D) Composition of chemically equivalent amino acids of Mytilus spp. F-ATP8 and M. senhousia F-ATP8 protein sequences (M. coruscus atp8 sequence is not available in GenBank). (E) Overall amino acid composition of the putative Mytilus spp. M-ORF-VD1 (*mean values for several M-ORF-VD1 GenBank sequences for each species). (F) Composition of chemically equivalent amino acids of the putative Mytilus spp. M-ORF-VD1 (such ORF has not been found in M. senhousia). Amino acid composition is reported as percentage. Mca, M. californianus; Mco, M. coruscus; Med, M. edulis; Mga, M. galloprovincialis; Mse, M. senhousia; Mtr, M. trossulus.

Figure 4. Comparisons of Mytilus spp. F-ORF-VD1 and M. senhousia F-ORF hydropathy profiles.

(A) Profiles for each species were calculated by the method of Kyte and Doolittle . Numbers below profiles designate amino acid positions in each protein. Predicted transmembrane domains according to TMpred (all with significant scores >500) are shown in light gray (It has to be noted that a single TMH in the N terminal portion of each F-ORF proteins has also been identified using HMMTOP [101]). (B) T-COFFEE alignment of the translated N terminal portions of mytilid F-specific ORF sequences. Predicted transmembrane domains according to TMpred are shown in light gray. (C) Kyte and Doolittle profiles for the F-ORF protein alignment with the homologous amino acid sites in the same position on the x axis. The predicted transmembrane domain according to TMpred is shown in light gray. Mca, M. californianus; Mco, M. coruscus; Med, M. edulis; Mga, M. galloprovincialis; Mse, M. senhousia; Mtr, M. trossulus.

Figure 5. Schematics of the structure of a typical F-type (above), recently-masculinized (center, according to Venetis et al. [47]) and typical M-type (below) control regions.

The F-ORF-VD1 is identified in the F-type control region. The amino acids that constitute the putative transmembrane helix are indicated in boldface type and bigger characters. The stretch of ∼60 residues showing the greatest similarity among the species is underlined. Dashes (–) denote the missing amino acid residues in the truncated F-ORF-VD1. CD, conserved domain; VD1, variable domain 1; VD2, variable domain 2 , . *The “standard” F-type CR of M. trossulus, which is a F/M recombinant CR, is not presented.