Understanding the impact of PROM1 alternative splicing on the development and maturation of human photoreceptors

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue-specific and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, detectable photoreceptor cilium defects and impaired transport of specific OS proteins in cone photoreceptors, such as cone opsin. Transcriptomic and Western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, and increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Together these data indicate that cones may be more sensitive to PROM1 exon 4 skipping, corroborating the pathogenesis of late-onset mild maculopathy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions.