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. Overall design: Gene expression profiling by RNA-seq of PMO-Treated ROs after 4 weeks (n=3). ROs were treated at day 180 of differentiation with either PROM1-PMO, PMO1 (targeting exon 4) or Ctrl-PMO (SC) and incubating for 4 weeks, adding a fresh dose of PMO every week.

可变剪接（Alternative splicing, AS）是驱动蛋白质组多样性形成的关键机制，其调控严格遵循组织特异性与发育特异性模式。视网膜组织的可变剪接水平位居全身前列，其中光感受器细胞具有独特的可变剪接谱，包含其他细胞类型中未检测到的微外显子。PROM1基因编码的Prominin-1蛋白定位于光感受器外节（outer segments, OSs），其外显子4保留型剪接从人类发育的第12个受孕后周开始，持续至成年阶段。PROM1的外显子4跳过型剪接与迟发性轻度黄斑病变相关，但其在光感受器成熟与功能调控中的作用尚不明确。
本研究采用极具应用价值的模型系统——视网膜类器官（retinal organoids, ROs），结合磷酰二胺吗啉代寡核苷酸（phosphorodiamidate morpholino oligos, PMOs），探究PROM1外显子4可变剪接在人类视网膜发育中的功能。研究数据显示，PROM1外显子4的跳过比例达55%，导致Prominin-1蛋白表达水平下降40%，可观测到视锥光感受器细胞的纤毛缺陷，且视锥视蛋白等特定外节蛋白的转运功能受损。转录组学与蛋白质印迹（Western blot）分析表明，视锥细胞、内节及连接纤毛基体标志物的表达显著下调，而与应激反应及泛素-蛋白酶体系统相关的基因表达上调，同时自噬过程被抑制。综合上述结果表明，视锥细胞可能对PROM1外显子4跳过更为敏感，这印证了迟发性轻度黄斑病变的发病机制。
值得关注的是，视网膜类器官的应用为在更具生理相关性的环境中研究可变剪接与解析疾病机制提供了宝贵的实验平台，为后续研究与潜在治疗干预开辟了新的方向。
实验整体设计：对经PMO处理的视网膜类器官在培养4周后，通过RNA测序（RNA-seq）开展基因表达谱分析（每组n=3）。视网膜类器官于分化第180天时，分别用靶向外显子4的PROM1-PMO、PMO1或对照PMO（SC）进行处理，孵育周期为4周，且每周更换一次新鲜的PMO制剂。