title: Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene PRPF31.
abstract: OBJECTIVE
  Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function.
  
  
  METHODS
  Splicing activity was assayed using an in vivo assay in transfected mammalian cells with rhodopsin (RHO) and transducin (GNAT1) splicing templates. Pull-down assays were used to study the interaction between PRPF31 and one of its cognate partners in the spliceosome, PRPF6.
  
  
  RESULTS
  Splicing of RHO intron 3 and GNAT1 introns 3-5 mini-gene templates was inefficient with both spliced and unspliced products clearly detected. Assays using the RHO minigene template revealed a direct negative effect on splicing efficiency of the mutant. However, no effect of the mutation on splicing efficiency could be detected using the longer GNAT1 minigene template or using a full-length RHO transcript, splicing of which had an efficiency of 100%. No unspliced RHO transcripts could be detected in RNA from human retina. Pull-down assays between PRPF31 and PRPF6 proteins showed a stronger interaction for the mutant than wild type, suggesting a mechanism for the negative effect.
  
  
  CONCLUSIONS
  Splicing of full-length RHO is more efficient than splicing of the minigene, and assays using a full-length template more accurately mimic splicing in photoreceptors. The RP11 missense mutations exert their pathology mainly via a mechanism based on protein insufficiency due to protein insolubility, but there is also a minor direct negative effect on function.
authors.affiliation: University College London Institute of Ophthalmology, London, United Kingdom.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Wilkie
authors.forename: Veronika
authors.initials: V
authors.lastname: Vaclavik
authors.forename: Huimin
authors.initials: H
authors.lastname: Wu
authors.forename: Kinga
authors.initials: K
authors.lastname: Bujakowska
authors.forename: Christina F
authors.initials: CF
authors.lastname: Chakarova
authors.forename: Shomi S
authors.initials: SS
authors.lastname: Bhattacharya
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Warren
authors.forename: David M
authors.initials: DM
authors.lastname: Hunt
mesh_terms: D002460:Cell Line; D005136:Eye Proteins / Q000235:genetics*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D008957:Models, Genetic; D050505:Mutant Proteins / Q000378:metabolism; D020125:Mutation, Missense / Q000235:genetics*; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000235:genetics; D012160:Retina / Q000473:pathology; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000473:pathology*; D017299:Rod Opsins / Q000235:genetics; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics; D015293:Transducin; D014162:Transfection
chemical_list: D005136:Eye Proteins; C034809:GNAT1 protein, human; D050505:Mutant Proteins; C454222:PRPF31 protein, human; C408605:PRPF6 protein, human; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D017299:Rod Opsins; D014157:Transcription Factors; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin