The Major Ciliary Isoforms of RPGR Build Different Interaction Complexes with INPP5E and RPGRIP1L

X-linked retinitis pigmentosa (XLRP) is frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. A complex splicing process acts on the RPGR gene resulting in three major isoforms: RPGR(ex1-19), RPGR(ORF15) and RPGR(skip14/15). We characterized the widely expressed, alternatively spliced transcript RPGR(skip14/15) lacking exons 14 and 15. Using the CRISPR/eSpCas9 system, we generated HEK293T cell lines exclusively expressing the RPGR(skip14/15) transcript from the endogenous RPGR gene. RPGR(ex1-19) and RPGR(ORF15) were knocked out. Immunocytochemistry demonstrated that the RPGR(skip14/15) protein localizes along primary cilia, resembling the expression pattern of RPGR(ex1-19). The number of cilia-carrying cells was not affected by the absence of the RPGR(ex1-19) and RPGR(ORF15) isoforms. Co-immunoprecipitation assays demonstrated that both RPGR(ex1-19) and RPGR(skip14/15) interact with PDE6D, further supporting that RPGR(skip14/15) is associated with the protein networks along the primary cilium. Interestingly, interaction complexes with INPP5E or RPGRIP1L were only detectable with isoform RPGR(ex1-19), but not with RPGR(skip14/15), demonstrating distinct functional properties of the major RPGR isoforms in spite of their similar subcellular localization. Our findings lead to the conclusion that protein binding sites within RPGR are mediated through alternative splicing. A tissue-specific expression ratio between RPGR(skip14/15) and RPGR(ex1-19) seems required to regulate the ciliary concentration of RPGR interaction partners.