Diversification processes of teleost intron-less opsin genes.

Opsins are universal photosensitive proteins in animals. Vertebrates have a variety of opsin genes for visual and non-visual photoreceptions. Analysis of the gene structures shows that most opsin genes have introns in their coding regions. However, teleosts exceptionally have several intron-less opsin genes that are presumed to have been duplicated by an RNA-based gene duplication mechanism, retroduplication. Among these retrogenes, we focused on the Opn4 (melanopsin) gene responsible for non-image-forming photoreception. Many teleosts have five Opn4 genes including one intron-less gene, which is speculated to have been formed from a parental intron-containing gene in the Actinopterygii. In this study, to reveal the evolutionary history of Opn4 genes, we analyzed them in teleost (zebrafish and medaka) and non-teleost (bichir, sturgeon, and gar) fishes. Our synteny analysis suggests that the intron-less Opn4 gene emerged by retroduplication after the branching of the bichir lineage. In addition, our biochemical and histochemical analyses showed that, in the teleost lineage, the newly acquired intron-less Opn4 gene became abundantly used without substantial changes in the molecular properties of the Opn4 protein. This stepwise evolutionary model of Opn4 genes is quite similar to that of rhodopsin genes in the Actinopterygii. The unique acquisition of rhodopsin and Opn4 retrogenes would have contributed to the diversification of the opsin gene repertoires in the Actinopterygii and the adaptation of teleosts to various aquatic environments.

Convergent evolutionary counterion displacement of bilaterian opsins in ciliary cells.

Opsins are universal photoreceptive proteins in animals. Vertebrate rhodopsin in ciliary photoreceptor cells photo-converts to a metastable active state to regulate cyclic nucleotide signaling. This active state cannot photo-convert back to the dark state, and thus vertebrate rhodopsin is categorized as a mono-stable opsin. By contrast, mollusk and arthropod rhodopsins in rhabdomeric photoreceptor cells photo-convert to a stable active state to stimulate IP3/calcium signaling. This active state can photo-convert back to the dark state, and thus these rhodopsins are categorized as bistable opsins. Moreover, the negatively charged counterion position crucial for the visible light sensitivity is different between vertebrate rhodopsin (Glu113) and mollusk and arthropod rhodopsins (Glu181). This can be explained by an evolutionary scenario where vertebrate rhodopsin newly acquired Glu113 as a counterion, which is thought to have led to higher signaling efficiency of vertebrate rhodopsin. However, the detailed evolutionary steps which led to the higher efficiency in vertebrate rhodopsin still remain unknown. Here, we analyzed the xenopsin group, which is phylogenetically distinct from vertebrate rhodopsin and functions in protostome ciliary cells. Xenopsins are blue-sensitive bistable opsins that regulate cAMP signaling. We found that a bistable xenopsin of Leptochiton asellus had Glu113 as a counterion but did not exhibit elevated signaling efficiency. Therefore, our results show that vertebrate rhodopsin and L. asellus xenopsin regulate cyclic nucleotide signaling in ciliary cells and displaced the counterion position from Glu181 to Glu113 via convergent evolution, whereas subsequently only vertebrate rhodopsin elevated its signaling efficiency by acquiring the mono-stable property.

Amino acid residue at position 188 determines the UV-sensitive bistable property of vertebrate non-visual opsin Opn5.

Opsins are G protein-coupled receptors specialized for photoreception in animals. Opn5 is categorized in an independent opsin group and functions for various non-visual photoreceptions. Among vertebrate Opn5 subgroups (Opn5m, Opn5L1 and Opn5L2), Opn5m and Opn5L2 bind 11-cis retinal to form a UV-sensitive resting state, which is inter-convertible with the all-trans retinal bound active state by photoreception. Thus, these opsins are characterized as bistable opsins. To assess the molecular basis of the UV-sensitive bistable property, we introduced comprehensive mutations at Thr188, which is well conserved among these opsins. The mutations in Opn5m drastically hampered 11-cis retinal incorporation and the bistable photoreaction. Moreover, T188C mutant Opn5m exclusively bound all-trans retinal and thermally self-regenerated to the original form after photoreception, which is similar to the photocyclic property of Opn5L1 bearing Cys188. Therefore, the residue at position 188 underlies the UV-sensitive bistable property of Opn5m and contributes to the diversification of vertebrate Opn5 subgroups.

Evolutionary history of teleost intron-containing and intron-less rhodopsin genes.

Recent progress in whole genome sequencing has revealed that animals have various kinds of opsin genes for photoreception. Among them, most opsin genes have introns in their coding regions. However, it has been known for a long time that teleost retinas express intron-less rhodopsin genes, which are presumed to have been formed by retroduplication from an ancestral intron-containing rhodopsin gene. In addition, teleosts have an intron-containing rhodopsin gene (exo-rhodopsin) exclusively for pineal photoreception. In this study, to unravel the evolutionary origin of the two teleost rhodopsin genes, we analyzed the rhodopsin genes of non-teleost fishes in the Actinopterygii. The phylogenetic analysis of full-length sequences of bichir, sturgeon and gar rhodopsins revealed that retroduplication of the rhodopsin gene occurred after branching of the bichir lineage. In addition, analysis of the tissue distribution and the molecular properties of bichir, sturgeon and gar rhodopsins showed that the abundant and exclusive expression of intron-containing rhodopsin in the pineal gland and the short lifetime of its meta II intermediate, which leads to optimization for pineal photoreception, were achieved after branching of the gar lineage. Based on these results, we propose a stepwise evolutionary model of teleost intron-containing and intron-less rhodopsin genes.