Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation.

Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.

Phylogenetic analysis of the metazoan carotenoid oxygenase superfamily: a new ancestral gene assemblage of BCO-like (BCOL) proteins.

Here we describe a new family of carotenoid cleavage oxygenases (CCOs) in metazoans, the BCO2-like (BCOL) clade, which contains lancelet, nematode, and molluscan carotenoid oxygenase sequences. Phylogenetic analysis of CCOs in all kingdoms of life confirmed that the BCOL enzymes are an independent clade of ancient origin. One of the predicted lancelet BCOL proteins, cloned and analyzed for carotenoid cleavage activity in a bacterial carotenoid expression system, had activity similar to lancelet BCO2 proteins, although with a preference for cis isomers. Our docking predictions correlated well with the cis-favored activity. The extensive expansions of the new animal BCOL family in some species (e.g., lancelet) suggests that the carotenoid cleavage oxygenase superfamily has evolved in the "extremely high turnover" fashion: numerous losses and duplications of this family are likely to reflect complex regulation processes during development, and interactions with the environment. These findings also serve to provide a rationale for the evolution of the BCO-related outlier RPE65 retinol isomerase, an enzyme that does not utilize carotenoids as substrate or perform double-bond cleavage.