title: Regulation of constitutive and alternative mRNA splicing across the human transcriptome by PRPF8 is determined by 5' splice site strength.
abstract: BACKGROUND
  Sequential assembly of the human spliceosome on RNA transcripts regulates splicing across the human transcriptome. The core spliceosome component PRPF8 is essential for spliceosome assembly through its participation in ribonucleoprotein (RNP) complexes for splice-site recognition, branch-point formation and catalysis. PRPF8 deficiency is linked to human diseases like retinitis pigmentosa or myeloid neoplasia, but its genome-wide effects on constitutive and alternative splicing remain unclear.
  
  
  RESULTS
  Here, we show that alterations in RNA splicing patterns across the human transcriptome that occur in conditions of restricted cellular PRPF8 abundance are defined by the altered splicing of introns with weak 5' splice sites. iCLIP of spliceosome components reveals that PRPF8 depletion decreases RNP complex formation at most splice sites in exon-intron junctions throughout the genome. However, impaired splicing affects only a subset of human transcripts, enriched for mitotic cell cycle factors, leading to mitotic arrest. Preferentially retained introns and differentially used exons in the affected genes contain weak 5' splice sites, but are otherwise indistinguishable from adjacent spliced introns. Experimental enhancement of splice-site strength in mini-gene constructs overcomes the effects of PRPF8 depletion on the kinetics and fidelity of splicing during transcription.
  
  
  CONCLUSIONS
  Competition for PRPF8 availability alters the transcription-coupled splicing of RNAs in which weak 5' splice sites predominate, enabling diversification of human gene expression during biological processes like mitosis. Our findings exemplify the regulatory potential of changes in the core spliceosome machinery, which may be relevant to slow-onset human genetic diseases linked to PRPF8 deficiency.
authors.affiliation: The Medical Research Council Cancer Unit, University of Cambridge, Cambridge Biomedical Campus, Box 197, Cambridge, CB2 0XZ, UK.
authors.forename: Vihandha O
authors.initials: VO
authors.lastname: Wickramasinghe
authors.affiliation: European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI), Hinxton, UK.
authors.forename: Mar
authors.initials: M
authors.lastname: Gonzàlez-Porta
authors.affiliation: The Medical Research Council Cancer Unit, University of Cambridge, Cambridge Biomedical Campus, Box 197, Cambridge, CB2 0XZ, UK.
authors.forename: David
authors.initials: D
authors.lastname: Perera
authors.affiliation: The Medical Research Council Cancer Unit, University of Cambridge, Cambridge Biomedical Campus, Box 197, Cambridge, CB2 0XZ, UK.
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Bartolozzi
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Sibley
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.
authors.forename: Martina
authors.initials: M
authors.lastname: Hallegger
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.affiliation: European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI), Hinxton, UK.
authors.forename: John C
authors.initials: JC
authors.lastname: Marioni
authors.affiliation: The Medical Research Council Cancer Unit, University of Cambridge, Cambridge Biomedical Campus, Box 197, Cambridge, CB2 0XZ, UK. arv22@mrc-cu.cam.ac.uk.
authors.forename: Ashok R
authors.initials: AR
authors.lastname: Venkitaraman
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D005091:Exons / Q000235:genetics; D016678:Genome; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000473:pathology; D012261:Ribonucleoproteins / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics; D059467:Transcriptome / Q000235:genetics*
chemical_list: C115374:PRPF8 protein, human; D022821:RNA Splice Sites; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins