July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Regulation of RPGR (retinitis pigmentosa GTPase regulator) isoform expression by miRNAs: insights into retinitis pigmentosa pathogenesis.
Author Affiliations & Notes
  • Laura Moreno Leon
    Department of Ophthalmology, UMASS Medical School, Worcester, Massachusetts, United States
  • Ruhi Sikka
    Department of Ophthalmology, UMASS Medical School, Worcester, Massachusetts, United States
  • Wei Zhang
    Department of Ophthalmology, UMASS Medical School, Worcester, Massachusetts, United States
  • Linjing Li
    Department of Ophthalmology, UMASS Medical School, Worcester, Massachusetts, United States
  • Hemant Khanna
    Department of Ophthalmology, UMASS Medical School, Worcester, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Laura Moreno Leon, None; Ruhi Sikka, None; Wei Zhang, None; Linjing Li, None; Hemant Khanna, None
  • Footnotes
    Support  NIH funding number ROIEY022372
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4925. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Laura Moreno Leon, Ruhi Sikka, Wei Zhang, Linjing Li, Hemant Khanna; Regulation of RPGR (retinitis pigmentosa GTPase regulator) isoform expression by miRNAs: insights into retinitis pigmentosa pathogenesis.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4925. doi: https://doi.org/.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : Mutations in the RPGR (retinitis pigmentosa GTPase regulator) gene account for >80% of X-linked RP. The RPGR gene encodes two major isoforms: RPGRconst (19 exons) and RPGRORF15 (exons 1-intron 15). Both isoforms localize to the cilia and exhibit a temporally regulated expression; however, the mechanism of regulation of their expression is unclear. Our aim is to assess the involvement of microRNAs (miRNAs), which are small non-coding RNAs that bind to the 3’UTR (untranslated region) and silence target gene expression, in regulating RPGR isoform expression.

Methods : In silico analysis using the miRDB, miRbase and TargetScan algorithms was used to identify miRNAs predicted to bind to the 3’-UTR sequence of RPGR isoforms. To validate these results, luciferase assay was performed using wild-type and mutated forms of the miRNA target sites of RPGR 3'-UTR sequence, cloned downstream of a firefly luciferase reporter. Furthermore, temporal miRNAs and RPGR isoform expression in mouse retina was assessed at different ages using RT-qPCR assay. RPE1 cells overexpressing the identified miRNAs were used to determine RPGR isoform expression and cilia formation after serum starvation by staining with anti-acetylated a-tubulin antibody. The cilia length was quantified using the ImageJ software. For in vivo studies, AAV-mediated delivery of the miRNAs was performed by subretinal injection into 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 : Using in silico analysis and luciferase assay, we identified two miRNAs that specifically regulate the expression of the RPGR isoforms. We also found that RPGR isoforms and miRNA expression levels are temporally correlated during retinal development. Interestingly, overexpression of the miRNAs recapitulated RPGR-knockdown associated phenotype of reduced cilia length in cultured cells. In addition, AAV-mediated overexpression of the miRNAs into mouse retina led to the downregulation of specific RPGR isoforms and exhibited ciliary trafficking defects.

Conclusions : Our studies provide a platform to understand how each RPGR isoform functions in the photoreceptors and assist in developing new strategies to modulate the levels of the isoforms as novel treatment paradigms.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×