RP1 Is Required for the Correct Stacking of Outer Segment Discs

Mutations in RP1are a common cause of retinitis pigmentosa (RP).1-3Like many retinal degeneration genes, however, the mechanism by which mutations in RP1lead to photoreceptor cell death is not known. The location of the RP1 protein in the region of the axoneme of rod and cone photo-receptors provides some clues about its function.4The photo-receptor axoneme begins at the basal body in the distal inner segment, passes through the connecting cilium, and continues into the outer segment up to half its length.5Because the outer segment is continually renewed by shedding older discs at the tip and adding new discs at the base, there is a constant demand for polarized transport of proteins from the inner to the outer segment.6,7As the only connection between the inner and outer segments of rods and cones, the connecting cilium is considered to be the primary conduit for the proteins and membrane needed in outer segments.8Protein transport through the cilium is thought to occur by conserved axonemal transport processes, mediated by the motor proteins kinesin and dynein, along with intraflagellar transport (IFT) proteins and myosin VIIa.9-12 The junction between the connecting cilium and the outer segment is also the location of disc morphogenesis. Thus, proteins found in this region may also participate in this process. New outer segment discs are formed by a process of membrane evagination and fusion at the base of the outer segment, adjacent to the axoneme of the outer segment.7,13-15Actin is concentrated at the base of the outer segment and has been implicated in disc formation, based on microscopy studies and use of inhibitors of actin polymerization.16,17RPGRIP is also a component of connecting cilia, and RPGRIP knockout mice demonstrate defects in outer segment disc size and orientation, leading to the hypothesis that RPGRIP is required for disc morphogenesis.18The roles of other proteins shown to be located in or around the cilium, such as α-actinin, cadherin, centrin, and myosin II are not as clearly defined.19-22 Data from mice with targeted removal of exons 2 and 3 of the Rp1gene suggest roles for Rp1 in both protein transport and disc formation.23These mice produce an Rp1mRNA resulting from abnormal splicing of exons 1 and 4 of the Rp1gene. This abnormal mRNA is translated, and an abnormal Rp1 protein that is lacking the N-terminal 269 amino acids is produced in the retinas of heterozygous and homozygous Rp1-exon 2/3 deletion mice.23It is thus not clear whether the defect in outer segment formation observed in these mice is due to the lack of the Rp1 protein or to a dominant effect of the abnormal protein. In addition, the mislocalization of opsin is minimal compared with that observed with other mutations that alter protein trafficking.10,11 All the 20 pathologic mutations in RP1found to date are nonsense or frameshift mutations that are located between codons 500 and 1053; 18 are found between codons 658 and 872.3,24These mutations all cluster at the beginning of exon 4 (codons 263-2156) and are thought to generate a truncated RP1 protein that lacks the C-terminal one half to two thirds of the protein. Because these premature termination mutations are all located after the final intron–exon boundary of the RP1gene, it is predicted that the mutant mRNAs could escape nonsense mediated decay.25To study the function of the RP1 protein and gain insight into disease pathogenesis, we investigated the expression of mutant RP1mRNA in lymphoblasts from patients with RP1 disease. We then used gene-targeting techniques to produce mice with a mutant Rp1allele that mimics the truncation alleles found to cause disease. Our data show that RP1 has a role in controlling the orientation of outer segment discs and in the organization of discs into outer segments.