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T. N. Kuznetsova, B. Zangerl, O. Goldstein, G. M. Acland, G. D. Aguirre; Characterization of Canine RPGRIP1 cDNA and Identification of Two Alternatively Spliced Isoforms in Dog Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):489.
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© ARVO (1962-2015); The Authors (2016-present)
RPGRIP1 encodes the RPGR interacting protein 1, and mutations have been shown to cause a LCA in man, and cone-rod dystrophy in dogs. The structure and expression of the gene is not characterized in dog. In this study we characterize the full length transcript, and two alternatively spliced retinal isoforms of canine RPGRIP1 (cRPGRIP1).
Total RNA was isolated from archived dog retinal tissues using TRIzol. Human and bovine RPGRIP1 cDNA sequences were aligned with canine genomic sequence to deduce the most likely positions of the exon/intron boundaries, and primers were designed based on the alignments. The full RPGRIP1 was cloned from 4 overlapping fragments using TOPO TA cloning kit.
cRPGRIP1 encompasses 24 coding exons, and forms a 3,630 bp ORF encoding a predicted protein product of 1,210 aa. cRPGRIP1 cDNA shares 80.6% homology with human cDNA. Analysis of the N-terminal canine sequence indicated the existence of a canonical NLS (KK/RXR) motif for nuclear import of the Nuclear Domain of RPGRIP1. This sequence, K50RAR, is also conserved in the human (K72RLR) and bovine (K50RMR) proteins. Two alternatively 5’ spliced gene products were found in retinal tissue that create downstream reading frames shorter than the full length cDNA. One transcript (A) arises from skipping of exons 3, 8-9, and 14-16, with the first Met in exon 7. The second transcript (B) is generated by use of an alternative donor splice site in exon 13, and lacks exons 3, and 14-16, predicting the first Met in exon 2.
The complete exon-intron structure of cRPGRIP1 has been established in dog retina. In addition to full length transcript, 2 alternatively spliced isoforms were identified. These data are important for identification of mutations underlying inherited retinal diseases in the dog, and necessary for understanding the multitude of clinical manifestations associated with mutations in cRPGRIP1.
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