Identification of novel murine- and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization.

PURPOSE: Mutations in RPGRIP1 cause Leber congenital amaurosis. The human and bovine RPGRIP1 undergo alternative splicing. A single murine rpgrip1 transcript has been reported, but distinct expression profiles of RPGRIP1 isoforms exist between species in the retina. To elucidate the heterogeneity of RPGRIP1 isoforms and the degree of functional redundancy among these, we extended the analysis of RPGRIP1 to the region between exons 12 and 14, which undergoes significant alternative splicing. METHODS: Identification of alternative splice transcripts of murine and human RPGRIP1 was performed by reverse transcription-polymerase chain reaction (RT-PCR). The murine rpgrip1 isoforms were analyzed by immunoblot and immunocytochemistry analysis of murine retinas and transient transfected cultured cells. RESULTS: A novel murine-specific transcript, rpgrip1b was identified. It arises from the extension of exon 13, leading to the premature truncation of rpgrip1 and deletion of its C2 and RID domains. It is predominantly expressed in the retina, where it is more abundant than the transcript(s) encompassing the constitutive exons 12 to 14. Conversely, the human retina lacks rpgrip1b, and the constitutive transcript is the major isoform. The subcellular distribution of rpgrip1b is distinct from its larger isoform, rpgrip1. In the photoreceptor inner segments and cells expressing enhanced green fluorescent protein (EGFP)-rpgrip1b, rpgrip1b is dispersed as punctate foci throughout the perikarya, where it colocalizes with a subpopulation of lysosomes. CONCLUSIONS: These data support the RPGR-independent function of the isotype- and species-specific rpgrip1b in lysosome-related processes. The results further strengthen the model of the selective participation of distinct RPGRIP1 isoforms in different subcellular processes and molecular pathogenesis of RPGRIP1-allied diseases.

Duke Scholars

Author Paulo Alexandre Ferreira Ophthalmology, Vitreoretinal Diseases & Surgery