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Daniele Ottaviani, Amelia Lane, David A Parfitt, Katarina Jovanovic, Paul Edward Sladen, Jessica Clare Gardner, Alison J Hardcastle, Michael E Cheetham; Temporal resolution of alternative splicing in the developing human retina using 3D retinal organoids. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3333.
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© ARVO (1962-2015); The Authors (2016-present)
Human photoreceptors maximise alternative gene splicing, leading to a highly specialised transcriptome during development and differentiation. This makes the retina particularly vulnerable to intronic mutations, compared to other tissues, as previously observed for the c.2991+1655A>G variant in CEP290 associated with Leber Congenital Amaurosis; however, the mRNA processing mechanisms causing this retinal sensitivity to aberrant splicing is unknown. In this study, we aim to correlate the inclusion of photoreceptor specific exons with retina differentiation in 3D retinal organoids.
We developed 3D retinal organoids in vitro exploiting the method published by Zhong et al. (Nature communications, 2014). The retinal organoid transcriptome was analysed by whole-tissue RNA-Sequencing (RNA-Seq) after 150 days of differentiation. We then used StringTie software to build the retinal transcriptome de novo. Novel exons and alternative splicing events were identified and quantified by rMATS tools. Expression of retinal isoforms was assessed by quantitative-PCR (qPCR) at different time-points of differentiation; day 20, 40, 70, 90, 120, 150, 180 and 200. Total mRNA was extracted from organoids (RNeasy Micro Kit, Qiagen) and cDNA was synthesised using the Tetro cDNA Synthesis Kit (Bioline) followed by qPCR on a QuantStudio 6 Flex machine (Life Technologies).
We identified putative novel retinal gene isoforms by RNA-Seq from retinal organoids (n=6). Of particular interest, we found a previously unreported micro-exon of 17bp in length in the IMPDH1 gene. We used this novel isoform as a marker of retinal organoid alternative splicing together with known retinal specific exons; BBS8-exon2A, RPGRorf15 and REEP6.1. Transcript quantification by qPCR (n>3) revealed this unique splicing mechanism is switched on at early stages during retinal development. Moreover, we correlated expression of retinal specific exons with expression of the master regulators PAX6, VSX2, NRL CRX, NR2E3.
We have identified novel retinal gene isoforms in human 3D retinal organoids, and defined their temporal expression in comparison with known markers of retinal development. Photoreceptor-specific splicing starts early during development of retinal organoids. Future work will focus on identifying the regulators(s) of this process and on targeting intronic mutations we are currently investigating.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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