Structural Organization and Expression Pattern of the Canine RPGRIP1 Isoforms in Retinal Tissue

The RPGRIP1 gene encodes retinitis pigmentosa GTPase interacting protein 1, and mutations in the human gene are associated with Leber congenital amaurosis (LCA),1–3 juvenile retinitis pigmentosa,4 a late-onset cone–rod dystrophy,5 and cone–rod dystrophy type 1 in dogs.6,7 Human RPGRIP1 consists of 25 exons, of which 24 code for a 1259-amino-acid protein.1,3 Exons 6 to 13, 14 to 16, and 18 to 24 encode, respectively, the α-helical coil–coiled protein interaction motif of members of the structural maintenance of chromosomes (SMC) superfamily, two protein kinase C conserved region 2 motifs (C2), and conserved RPGR-interacting domain (RID).8–10 The biological functions of RPGRIP1 are complex. In the eye, it is expressed in amacrine neurons8,11 and photoreceptors,9,12 and in numerous other tissues, albeit at greatly reduced levels.9,12 Moreover, the existence of multiple isoforms, with species-specific subcellular localization patterns (e.g., connecting cilium,11,13 photoreceptor inner14 and outer8,11 segments, and basal bodies of cells with primary cilia15), suggests that different isoforms perform cell-specific functions. RPGRIP1 is required not only for disc morphogenesis of the outer segments (OS),16 but also for the formation of the OS itself, particularly in rods.17 A general role of RPGRIP1 as a scaffold protein has been suggested,13 and it interacts directly or indirectly with RPGR,9,16,18 NPHP4,10 and RanBP2.8 It has been shown that RPGRIP1 can be proteolytically processed, rendering its N-terminal domain competent for nuclear localization,19 suggesting that it may be involved in regulating gene expression. Although a mutation in RPGRIP1 is causally associated with canine cone–rod dystrophy,6,7 a potential large-animal model for gene-based therapies,20 little is known about the canine gene structure, organization, and expression, and the molecular basis of the disease. To assess the structure/function relationship of the RPGRIP1 isoforms, we characterized the full-length transcript of canine RPGRIP1 (cRPGRIP1) and its several alternatively spliced isoforms and evaluated the 5′- and 3′-UTRs of RPGRIP1 transcripts. Our results identified a novel complex 5′ and 3′ splicing pattern and further described the complete structure of six cRPGRIP1 alternatively spliced variants driven by two different promoters.