Abstract
Purpose :
Prph2 is an integral membrane protein expressed in rod and cone photoreceptors that is involved in disk morphogenesis. It is localized to the highly curved rim regions of the outer segment disks. In X. laevis, rods also express another homolog xrds35/36 or rom1. A search of the X. laevis genome also revealed the presence of a third prph2-like sequence, gp2l. We knocked out rom1 in X. laevis in order to study its functions, and characterized gp2l localization and expression.
Methods :
We conducted database searches for sequences related to prph2 using tblastn, and generated a phylogenetic tree. Cas9 and sgRNAs targeting X. laevis rom1 or gp2l were injected into single-cell embryos to yield chimeric F0 tadpoles. Antibodies against prph2, rom1 and gp2l were generated or obtained from other investigators. F0 edited and wild type eyes were cryosectioned and examined by immunofluorescence confocal microscopy.
Results :
We determined that xrds35/36 is the ortholog of human rom1, as many of the surrounding genes are identical in both species. In X. laevis and other non-mammalian vertebrates, prph2 and rom1 share much greater sequence identity than in mammals.
We found that a third paralog, gp2l is present in many non-mammalian vertebrates but absent from most mammals. X. laevis rom1 localizes to rod disk rims and incisures and is not expressed in cones. In contrast, gp2l is absent from red rods, but co-localizes with prph2 in cones along the cone outer segment axoneme, and is also expressed in green rods. Elimination of rom1 expression via genome editing did not result in any obvious phenotype. Overall rod outer segment morphology was normal, as was prph2 localization.
Conclusions :
The genomes of many non-mammalian vertebrates encode three prph2 family members, including prph2 and rom1 homologues, and a third related protein, gp2l. In X. laevis, gp2l is expressed only in cones while rom-1 is expressed only in rods. Despite significant sequence differences between non-mammalian and mammalian vertebrates, non-mammalian rom-1 plays a similarly minor role in outer segment morphogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.