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Medical AI Inquiry: You was designed to assist in medical decision-making based on the provided user questions. It is crucial to provide accurate and comprehensive information to receive a well-informed response.  Labels: OBJECTIVE, METHODS, RESULTS. Meshes: Animals, Blotting, Western, Eye Proteins, Immunoprecipitation, Membrane Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Protein Interaction Mapping, Protein Isoforms, Retina, Retinal Degeneration, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA.
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Does rpgrORF15 connect to the usher protein network through direct interactions with multiple whirlin isoforms? Context: Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are a frequent cause of X-linked retinitis pigmentosa. The RPGR transcript undergoes complex alternative splicing to express both constitutive (Rpgr(ex1-19)) and Rpgr(ORF15) variants. Because functional studies of Rpgr suggest a role in intracellular protein trafficking through the connecting cilia, the goal of this study was to identify potential binding partners for Rpgr(ORF15) and to identify the domains on whirlin necessary for Rpgr binding. The C-terminus of mouse Rpgr(ORF15) was used as bait in a yeast two-hybrid system. Whirlin expression was analyzed using RT-PCR and Western blot analysis. Protein-protein interactions were confirmed using in vitro binding assays and coimmunoprecipitation. Subcellular colocalization was analyzed using immunohistochemistry on retinal cryosections. Yeast two-hybrid analysis identified whirlin, a PDZ-scaffold protein, as a putative binding partner for Rpgr(ORF15). The RPGR(ORF15)-whirlin interaction was confirmed using in vitro binding assays and coimmunoprecipitation from retinal tissue, and both proteins were shown to colocalize in the photoreceptor connecting cilia in vivo. Results from RT-PCR, Western blot analysis, and immunocytochemistry demonstrated that whirlin expressed multiple isoforms in photoreceptors with variable subcellular localization.
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Whirlin expression has been reported in photoreceptors and cochlear hair cells, and mutations in whirlin cause Usher syndrome (USH2D) and nonsyndromic congenital deafness (DFNB31). Because mutations in the 5' end of whirlin are associated with the syndromic phenotype associated with USH2D, the identification of novel N-terminal isoforms in the retina and a novel RPGR(ORF15)-whirlin interaction provide a potential mechanism for the retinal phenotype observed in USH2D. Final Decision: yes