Identification of circular RNAs hosted by the <i>RPGR</i> ORF15 genomic locus

Mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGR^orf15) cause X-linked retinitis pigmentosa, a severe and early onset inherited retinal degeneration. The underlying pathogenic mechanisms and variability in disease severity remain to be fully elucidated. The present study examines structural features of the ORF15 exonic region to provide new insights into the disease pathogenesis. Using canine and human RNA samples, we identified several novel <i>RPGR</i> ORF15-like linear RNA transcripts containing cryptic introns (exitrons) within the annotated exon ORF15. Furthermore, using outward-facing primers designed inside exitrons in the ORF15 exonic region, we found many of previously unidentified circular RNAs (circRNAs) that formed via back fusion of linear parts of the <i>RPGR^orf15</i> pre-mRNAs. These circRNAs (resistant to RNAse R treatment) were found in all studied cells and tissues. Notably, some circRNAs were present in cytoplasmic and polysomal RNA fractions. Although certain <i>RPGR</i> circRNAs may be cell type specific, we found some of the same circRNAs expressed in different cell types, suggesting similarities in their biogenesis and functions. Sequence analysis of <i>RPGR</i> circRNAs revealed several remarkable features, including identification of N6-methyladenosine (m6A) consensus sequence motifs and high prevalence of predictive microRNA binding sites pointing to the functional roles of these circRNAs. Our findings also illustrate the presence of non-canonical <i>RPGR</i> circRNA biogenesis pathways independent of the known back splicing mechanism. The obtained data on novel <i>RPGR</i> circRNAs further underline structural complexity of the <i>RPGR</i> ORF15 region and provide a potential molecular basis for the disease phenotypic heterogeneity.