PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

Nat Commun. 2024 Apr 11;15(1):3138. doi: 10.1038/s41467-024-47253-0.

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.

MeSH terms

  • Alternative Splicing / genetics
  • DNA Helicases / metabolism
  • Humans
  • Mutation
  • Proteomics
  • RNA Splice Sites*
  • RNA Splicing / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Nuclear / genetics
  • RNA, Small Nuclear / metabolism
  • RNA-Binding Proteins / metabolism
  • Retinitis Pigmentosa*
  • Spliceosomes* / genetics
  • Spliceosomes* / metabolism

Substances

  • RNA Splice Sites
  • RNA, Small Nuclear
  • RNA, Messenger
  • DNA Helicases
  • PRPF8 protein, human
  • RNA-Binding Proteins

Supplementary concepts

  • Retinitis Pigmentosa 13