title: Global identification of hnRNP A1 binding sites for SSO-based splicing modulation.
abstract: Many pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known.
  
  
  
  Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5' splice site can be blocked by SSOs to activate the exon.
  
  
  
  The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.
authors.forename: Gitte H
authors.initials: GH
authors.lastname: Bruun
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Doktor
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.
authors.forename: Jonas
authors.initials: J
authors.lastname: Borch-Jensen
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Masuda
authors.affiliation: Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY, 11724, USA.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Krainer
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Kinji
authors.initials: K
authors.lastname: Ohno
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark. bragea@bmb.sdu.dk.
authors.forename: Brage S
authors.identifier: 0000-0001-7488-3035
authors.initials: BS
authors.lastname: Andresen
mesh_terms: D000072283:A549 Cells; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D005091:Exons / Q000235:genetics; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D009711:Nucleotides / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D059467:Transcriptome / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009711:Nucleotides; D009841:Oligonucleotides; D022821:RNA Splice Sites
keywords: Alternative splicing; Cross-linking immunoprecipitation (CLIP); Pseudoexons; RNA-seq; Splicing silencer; Splicing splice-switching oligonucleotides (SSOs); Surface plasmon resonance imaging (SPRi); hnRNP A1; iCLIP