Abstract
Purpose :
Mutations in RPGR exon ORF15 cause X-linked retinitis pigmentosa characterized by severe visual impairment early in life. The underlying disease mechanism and variability in the disease severity remains to be fully elucidated. The present study examines structural features of the ORF15 exonic region to provide new insights into the disease pathogenesis.
Methods :
RNA samples from canine and human retinas and cultured cells (canine fibroblasts and human cell lines Y79 and ARPE19) were used to examine RPGR transcript heterogeneity, the presence of internal introns (exitrons) in ORF15 exonic region and RPGR circular RNAs (circRNAs). Treatment with RNase R was performed for circRNAs enrichment and validation of circularization. Polysomes from a crude cell homogenate were obtained by ultracentrifugation, using a sucrose cushion.
Results :
We found a subset of novel RPGR ORF15-like transcripts in canine and human retina and in cultured cells. Annotation of these transcripts showed that portions of the ORF15 sequence were removed in a variable manner through utilization of noncanonical splicing sites. Notably, the presence of the RPGRorf15 transcript was detected only in the retina. Furthermore, using outward-facing primers designed inside exitrons in ORF15 exonic region we discovered a previously unidentified RPGR circRNAs formed via back-splicing events. The RPGR circRNAs were found in all studied cells and tissues and were present in total RNA pool, in cytoplasmic and polysomal RNA fractions.
Conclusions :
The obtained data on novel RPGR circRNAs further underline the complexity of RPGR gene organization and provide potential molecular basis of disease phenotypic heterogeneity.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.