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G. D. Aguirre, Z. Li, D. Gu; Novel Splicing Isoforms of the Canine Retinitis Pigmentosa GTPase Regulator (RPGR) Gene. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4649.
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Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are associated with naturally occurring retinal degeneration in man and dog, and experimentally in mice. The very complex alternative splicing of this gene has been reported in mice (Hong et al.,2002). We are investigating the alternative splicing pattern of RPGR in dogs to gain a better understanding of the expression and function of this gene in the normal retina, and in retinas of dogs with RPGR stop and frameshift mutations.
Total RNA was extracted from normal dog retina, brain and other tissues, and reverse transcription-polymerase chain reaction (RT-PCR) was performed. RT was carried out with oligo-dT primer, and PCR with primers from different characterized exons of RPGR. The RT-PCR products were cloned, and the sequence of the insert fragments analyzed.
Novel splicing isoforms of RPGR gene were identified in canine retina. One isoform with ORF15 as a terminal exon has an in-frame deletion of exons 5-12 with no interruption of the reading frame. Two splicing isoforms are in exon ORF15. One has a deletion of 1347 bp (449 amino acids), and the second a deletion of 1341 bp (447 amino acids); in both the reading frame is maintained and the C-terminal end of ORF15 is unchanged. Another splicing isoform has a small in-frame insert of 48 bp after exon 15 with a stop codon after 12 bp. All splicing isoforms are now being examined in retinal and non-retinal tissues from normal and RPGR mutant dogs with stop and frameshift mutations to identify tissue- and mutation-specific splicing patterns.
Alternative splicing is an important mechanism to maximize amplification of the protein coding capacity of a gene. These novel splicing isoforms of RPGR gene reflect the extraordinary complexity of the transcription and expression pattern of RPGR. Further analysis of the relationship between the structure and function will explore what the role RPGR is playing in the normal retina, how mutations alter splicing, and the association of the splicing isoforms with the disease.
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