June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Impact of alternative splicing of ciliary genes on the development and maturation of human photoreceptors.
Author Affiliations & Notes
  • Marina Moya Molina
    Stem Cell, Newcastle University Biosciences Institute, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom
    Newcells Biotech, Newcastle Upon Tyne, Tyne and Wear, United Kingdom
  • Emily Flood
    Stem Cell, Newcastle University Biosciences Institute, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom
  • Birthe Hilgen
    Stem Cell, Newcastle University Biosciences Institute, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom
  • Sushma Nagaraja-Grellscheid
    Universitetet i Bergen, Bergen, Hordaland, Norway
  • Lyle Armstrong
    Stem Cell, Newcastle University Biosciences Institute, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom
    Newcells Biotech, Newcastle Upon Tyne, Tyne and Wear, United Kingdom
  • Majlinda Lako
    Stem Cell, Newcastle University Biosciences Institute, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom
  • Footnotes
    Commercial Relationships   Marina Moya Molina None; Emily Flood None; Birthe Hilgen None; Sushma Nagaraja-Grellscheid None; Lyle Armstrong None; Majlinda Lako None
  • Footnotes
    Support  Grant no 861329 SCilS
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3169. doi:
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      Marina Moya Molina, Emily Flood, Birthe Hilgen, Sushma Nagaraja-Grellscheid, Lyle Armstrong, Majlinda Lako; Impact of alternative splicing of ciliary genes on the development and maturation of human photoreceptors.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3169.

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

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Abstract

Purpose : Retinal tissue has one of the highest levels of alternative splicing. As an example of this, PROM1, which is found on the photoreceptor outer segments, undergoes developmental stage-specific alternative splicing. In particular, PROM1 displayed increased exon 4 inclusion during the 12th-18th week of human development. Several studies have shown that exon 4 skipping is associated with late-onset mild maculopathy. The aim of this study is to investigate the dynamics of PROM1 alternatively spliced isoforms generation and their impact on photoreceptor differentiation and maturation using an in vitro model.

Methods : We used morpholino oligos to block PROM1 alternative splicing, resulting in exon 4 exclusion, on pluripotent stem cell-derived retinal organoids. A morpholino Standard Control oligo that targets a human beta-globin intron mutation that causes beta-thalassemia was used as a negative control. RT-PCR and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors.

Results : Retinal organoids (ROs) recapitulated the PROM1 splicing profile observed in vivo, where exon 4 was included from 12-18 post-conception week, reaching its maximum inclusion at adult stage. In vitro, exon 4 was present as early as day 60 of ROs differentiation and its inclusion reached its maximum at day 150 of differentiation (98.5%). ROs treated with morpholino oligos weekly for one month showed a decrease in exon 4 inclusion demonstrating that the treatment is still effective after 4 weeks of incubation. Immunofluorescence analysis showed that PROM1 mis-splicing resulted in the internalization of photoreceptors in the basal side of ROs. In addition, cone cells were affected earlier than rods, after 2 weeks of treatment.

Conclusions : PROM1 exon 4 and its codified internal N-terminal segment may play an important role in photoreceptor development, maturation and maintenance. The link between PROM1 exon 4 exclusion and photoreceptor internalisation needs to be further studied to gain precise insights on PROM1 function in photoreceptor development and maturation.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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