Abstract
Purpose :
RPGR, a retinal ciliopathy gene is one of the most frequently mutated genes in Retinitis Pigmentosa (RP) patients. The RPGR gene encodes two major isoforms: RPGRCONST(19 exons) and RPGRORF15(exons 1-intron 15), however, post transcriptional regulation of these isoforms is unclear. MicroRNAs (miRNAs), which are small noncoding RNAs that bind to the 3’ UTR (untranslated region), play a critical role in regulating expression of genes involved in retinal diseases. Our aim is to understand the involvement of miRNAs in modulating RPGR expression in mammalian retina.
Methods :
The miRDB, miRbase and TargetScan algorithms were used to identify miRNAs predicted to bind to the 3’-UTR of the RPGR isoforms. Further validation was performed using luciferase assays with wild-type and mutated forms of the miRNA target sites of the RPGR 3'-UTR sequence cloned downstream of a firefly luciferase reporter. Temporal miRNA and RPGR isoform expression in mouse retina was assessed at different ages using RT-qPCR. RPE1 cells exhibiting overexpression or knockdown of the identified miRNAs were used to determine their effect on RPGR isoform expression and cilia formation using ciliary markers. The cilia length was quantified using the ImageJ software. AAV-mediated delivery of the miRNAs was performed by subretinal injection into neonatal C57BL6/J retina. Effect on RPGR isoform expression and photoreceptor morphology was determined using RT-qPCR, immunoblotting, and immunostaining using ciliary and outer segment markers.
Results :
We identified two miRNAs that specifically regulate the expression of the distinct RPGR isoforms. Interestingly, overexpressing the miRNA targeting RPGRORF15 increased cilia length while the RPGRCONST-targeted miRNA reduced cilia length. In addition, AAV-mediated overexpression of the miRNAs in mouse retina led to the downregulation of specific RPGR isoforms, exhibited ciliary trafficking defects, and showed opposing roles of the two miRNAs in protein trafficking in photoreceptors.
Conclusions :
Our results show that miRNAs that regulate RPGR isoform expression are also involved in modulating cilia length. We suggest that cilia length regulation by miRNAs in RPGR-associated disease can be an excellent tool to develop novel treatment paradigms.
This is a 2020 ARVO Annual Meeting abstract.