An evolutionary preserved intergenic spacer in gadiform mitogenomes generates a long noncoding RNA

Abstract

Background: Vertebrate mitogenomes are economically organized and usually lack intergenic sequences other than the control region. Intergenic spacers located between the tRNA(Thr) and tRNA(Pro) genes ("T-P spacers") have been observed in several taxa, including gadiform species, but information about their biological roles and putative functions is still lacking.

Results: Sequence characterization of the complete European hake Merluccius merluccius mitogenome identified a complex T-P spacer ranging in size from 223-532 bp. Further analyses of 32 gadiform species, representing 8 families and 28 genera, revealed the evolutionary preserved presence of T-P spacers across all taxa. Molecular complexity of the T-P spacers was found to be coherent with the phylogenetic relationships, supporting a common ancestral origin and gain of function during codfish evolution. Intraspecific variation of T-P spacer sequences was assessed in 225 Atlantic cod specimens and revealed 26 haplotypes. Pyrosequencing data representing the mito-transcriptome poly (A) fraction in Atlantic cod identified an abundant H-strand specific long noncoding RNA of about 375 nt. The T-P spacer corresponded to the 5' part of this transcript, which terminated within the control region in a tail-to-tail configuration with the L-strand specific transcript (the 7S RNA).

Conclusions: The T-P spacer is inferred to be evolutionary preserved in gadiform mitogenomes due to gain of function through a long noncoding RNA. We suggest that the T-P spacer adds stability to the H-strand specific long noncoding RNA by forming stable hairpin structures and additional protein binding sites.

Figure 1

Organization of T-P spacer. A. Gene content and organization of European hake mitochondrial genome (Mm1 specimen) presented as a linear map of the circular mtDNA. All protein genes, except ND6, are encoded by the H-strand. Abbreviations: SSU and LSU, mitochondrial small- and large-subunit ribosomal RNA genes; ND1-6, NADH dehydrogenase subunit 1 to 6; COI-III, cytochrome c oxidase subunit I to III; A6 and A8, ATPase subunit 6 and 8; CytB, cytochrome B; oriH and oriL, origin of H-strand and L-strand replication; CR, control region containing the D-loop; tRNA genes are indicated by the standard one-letter symbols for amino acids. H-strand and L-strand encoded tRNA genes are indicated above and below the diagram, respectively. T-P spacer is indicated below the diagram, and position of the 42-bp insertion in the ND6 gene corresponding to 14 amino acids is indicated by arrow above. B. Organization of the European hake T-P spacer in specimens Mm1 and Mm2. Different direct repeat motifs are indicated by green (conserved repeats), yellow (optional repeats), and blue (heteroplasmic tandem repeats, HTR) boxes. Two copies of the Box motif are present. C. Organization of the Silvery pout (G. argenteus) T-P spacer containing three Box-motifs. The third copy of the direct repeat (red boxes) is truncated. Heteroplasmic sites (H) are indicated. D. Organization of the Atlantic cod (G. morhua) T-P spacer. About 1% (3/225) of analyzed specimens harbor a direct repeat duplication (blue box).

Figure 2

Intraspecific variation of Atlantic cod T-P spacer. Alignment of 225 specimens revealed 26 distinct haplotypes. Dots indicate identical positions to the reference Gm-I haplotype and dashes indicate deletions. Two haplotypes (Gm-XXV and Gm-XXVI) harbor a 29 bp insertion. The conserved 17-bp motif is boxed, and the 16 variable sites are indicated by stars above the Gm-I haplotype sequence. Right; numbers of specimens and frequency (%) belonging to each haplotype.

Figure 3

Long noncoding RNAs generated from CR and T-P spacer. A. 454 read coverage of the Atlantic cod liver mitochondrial transcriptome at the T-P spacer and control region. H-strand specific reads (green) start exactly 3 nt downstream the first position of the T-P spacer, and represent lncCR-H. L-strand specific reads (red) represent lncCR-L. Grey line in lncCR-H indicates missing HTR motif due to HTR copy number variation. B. Secondary structure diagram of lncCR-H in Atlantic cod. The T-P spacer is located at the 5’ end of the RNA, and Box-motifs are indicated (yellow boxes). The mirror tRNA^Pro structure and TAS hairpin structure (red box) are located upstream and downstream, respectively, of the heteroplasmic tandem repeat (HTR) motifs.

Figure 4

Evolutionary history of gadiform T-P spacer. Key events in the evolutionary history of the T-P spacer are indicated by numbers 1–4 and explained in the main text. In short: 1, first occurrence of spacer; 2, first occurrence of Box-motif; 3abc, duplication of Box-motif; 4, second duplication of Box motif. Genus containing T-P spacer with heteroplasmic feature is indicated by star (*). The Bregmaceros spacer is involved in gene order rearrangements (#). The 32 gadiform species represent 20 genera and 8 families (color-coded). Pattern of relationship between gadiform families is extrapolated from [38]–[40].