Characterization of RPGR Variants and Their Role in Inherited Retinal Degeneration
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Retinitis Pigmentosa (RP) refers to a group of inherited retinal dystrophies resulting from progressive photoreceptor degeneration and accumulation of intra-retinal pigment-like deposits. X-linked forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The RPGR transcript undergoes complex alternative splicing to express both constitutive (RPGR^ex1-19) and RPGR^ORF15 variants. Although RPGR is thought to play a role in ciliary function, little is known about the physiological significance of expressing two distinct groups of variants. This study compares Rpgr^ex1-19 and Rpgr^ORF15 expression in developing photoreceptors using immunoblot analysis and immunohistochemistry, assesses ciliary affinity in adult photoreceptors by protein fractionation, examines Rpgr function in transgenic mouse models and identifies a novel Rpgr^ORF15 binding partner using a yeast two-hybrid screen. Our data reveal that Rpgr expression undergoes dynamic temporal regulation during retinal development and indicates variability in ciliary localization of Rpgr variants in adult photoreceptors. Utilization of distinct Rpgr variants during stages of photoreceptor development suggests independent roles. Further examination of Rpgr function using transgenic mouse models over-expressing either the Rpgr^ex1-19 or Rpgr^ORF15 variant reveals that despite normal ciliary localization, an excess of RPGR^ex1-19 results in atypical accumulation of Rpgr in photoreceptor outer segments, abnormal photoreceptor morphology and severe retinal degeneration. The data indicate that the constitutive variant cannot substitute for Rpgr function in photoreceptors and suggest that proper maintenance of the Rpgr isoform ratio is critical to photoreceptor viability. Using mouse retinal cDNA in a yeast two-hybrid screen with the C-terminus of the Rpgr^ORF15 variant, we identified a novel variant of whirlin as an interacting partner. Mutations in whirlin result in Usher syndrome, a disorder characterized by hearing loss and RP. RT-PCR and immunoblot analysis were used to confirm the presence of selected candidate partners in the retina and interaction was confirmed by pull-down assays and co-immunoprecipitation from retinal homogenate. Immunohistochemistry showed co-localization of RPGR and whirlin within photoreceptors and identified isoform specific localization of whirlin. These findings indicate that whirlin binds Rpgr^ORF15 and that this novel isoform may be required for photoreceptor function, thus providing a potential mechanism for the RP phenotype observed in Usher syndrome.
Wright, Rachel (2011). Characterization of RPGR Variants and Their Role in Inherited Retinal Degeneration. Doctoral dissertation, Texas A&M University. Available electronically from