PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects
Robert Atkinson, Μαρία Γεωργίου, Chunbo Yang, Katarzyna Szymańska, Albert Lahat, Elton J. R. Vasconcelos, Yanlong Ji, Marina Moya Molina, Joseph Collin, Rachel Queen, Birthe Dorgau, Avril Watson, Marzena Kurzawa‐Akanbi, Ross Laws, Abhijit Saxena, Chia Shyan Beh, Chileleko Siachisumo, Franziska Goertler, Magdalena Karwatka, Tracey Davey, Chris F. Inglehearn, Martin McKibbin, Reinhard Lührmann, David Steel, David J. Elliott, Lyle Armstrong, Henning Urlaub, Robin R. Ali, Sushma‐Nagaraja Grellscheid, Colin A. Johnson, Sina Mozaffari‐Jovin, Majlinda Lako
Abstract
The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.