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Tanner A. Slayden, James Haley, Xun Sun, Tiansen Li; Identification of Novel Mouse RPGR Splice Variants that End in Alternative Exons. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5401.
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Mutations in the gene for Retinitis pigmentosa GTPase regulator (RPGR) are the major cause of X-linked retinitis pigmentosa (XLRP), a severe form of retinal degenerative disease. The expression pattern of RPGR is complex, with known default and ORF15 variants that arise from alternative splicing. Our initial data in mice suggest that additional complexity may exist in the default and ORF15 family of transcripts. Elucidation of RPGR variants and their functional significance is a prerequisite for gene therapeutic interventions.
Cytoplasmic-enriched RNA was extracted from wild-type C57/B1 mouse retinas, and cDNA templates were prepared from total RNA. Screening of mouse retinal cDNA libraries was conducted with probes in the RPGR default and ORF15 specific regions. Analytical PCR and DNA sequencing was performed to see if Sushi-repeat-containing protein (SRPX) sites appeared at the end of RPGR. Sense primers specific to the default RPGR form and ORF15 form, which is the functionally significant transcript in photoreceptors, were used as well as sense primers specific to both. Antisense primers were specific to exon 2 and intron 2 of SPRX.
Library screening identified cDNA clones that terminate with an exon from the downstream SRPX gene. By RT-PCR, the RPGR sense primers and SRPX antisense primers produced bands of relative high abundance, especially for the default variant. Primers spanning the ORF15 variant and SRPX produced products of lower abundance. DNA sequencing of PCR products showed that default RPGR variably splices together with SRPX’s exon 2, resulting in loss of exon 19 and in some instances both exons 18 and 19. Consistent with findings on the RPGR transcripts, immunoblotting studies confirmed that a sizable fraction of RPGR does not carry the C terminal sequence (exon 19 in default and the unique sequence downstream from the repetitive region in ORF15).
The results suggest that default and ORF15 RPGR variants undergo complex processing at their C terminal ends. This additional level of heterogeneity is likely to have important functional implications.
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