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Ciliary dysfunction is an underlying cause of severe human disorders (collectively called ciliopathies), such as retinitis pigmentosa (RP), Joubert Syndrome (JBTS), and Bardet-Biedl Syndrome. Ciliary proteins form distinct functional networks for localization to cilia as well as regulation of ciliary function. However, not much is known about the mechanism of ciliary localization and function of RPGR (retinitis pigmentosa GTPase regulator), a ciliary protein frequently associated with RP worldwide. Using tandem mass spectrometry analysis, we show that RPGR interacts with two JBTS-associated proteins: PDE6Π (delta subunit of Phosphodiesterase; a prenyl-binding protein) and INPP5E (inositol polyphosphate-5-phosphatase 5E; a ciliary cargo). Whereas PDE6Π binds in a prenylation-dependent manner to the C-terminus of RPGR, INPP5E associates with the N-terminus of RPGR. Prenylation and interaction of RPGR with PDE6Π are critical for its localization to cilia. We further show that loss of RPGR results in reduced amount of INPP5E in cilia of fibroblasts and in photoreceptor outer segment, a modified sensory cilium. Overall, our results suggest that RPGR, in complex with PDE6D, regulates the trafficking of ciliary cargo INPP5E and implicate reduction in ciliary INPP5E in the pathogenesis of RPGR-ciliopathy.

Purpose: Mutations in the CEP290 gene are a frequent cause of childhood blindness disorder, Leber congenital amaurosis (LCA). Not much is known about the mode of action of CEP290 in the retina. We previously showed that CEP290 interacts with RKIP and that RKIP protein is abnormally accumulated in rd16 (retinal degeneration 16) mouse, a model of Cep290- associated retinal degeneration. The purpose of this study is to evaluate the role of RKIP accumulation in mediating photoreceptor degeneration in the rd16 mouse. Methods: rd16/rd16:Rkip ko/Rkip ko double homozygous knock out mice were generated by breeding rd16 and Rkip ko mice (both on C57BL6/J background). Photoreceptor structure and function were evaluated by Histology, Transmission electron microscopy (TEM), Immunofluorescence microscopy and Electroretinography (ERG). Results: We detected improved photoreceptor structure in the double homozygous mutant mice compared to rd16 at postnatal day 18 (P18). In addition, Rhodopsin and M-opsin staining in Rd16 Rkip-/- mouse showed increased outer segment localization in the double mutant mice. Finally, ERG analysis showed 40% scotopic response recovery in Rd16 Rkip-/- from Rd16 mouse. Conclusions: We propose that accumulation of RKIP due to mutation in CEP290, although not the only pathway is a critical step in the pathogenesis of associated severe retinal degeneration. Altering RKIP levels may potentially be used in combination with other modalities as an approach for treating such disorders.