title: HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform.
abstract: Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1.
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
authors.forename: Farhana
authors.initials: F
authors.lastname: Nasrin
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
authors.forename: Mohammad Alinoor
authors.initials: MA
authors.lastname: Rahman
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Masuda
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Ohe
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Takeda
authors.affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
authors.forename: Kinji
authors.initials: K
authors.lastname: Ohno
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D005091:Exons; D015870:Gene Expression; D005786:Gene Expression Regulation; D023061:Gene Order; D056426:Genetic Loci; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000378:metabolism*; D034664:Heterogeneous-Nuclear Ribonucleoprotein L / Q000378:metabolism*; D006801:Humans; D051379:Mice; D024510:Muscle Development / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D011950:Receptors, Cholinergic / Q000235:genetics*; D037941:Silencer Elements, Transcriptional; D051153:Wnt Proteins / Q000378:metabolism; D051840:Y-Box-Binding Protein 1 / Q000378:metabolism*
chemical_list: D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D020033:Protein Isoforms; D012333:RNA, Messenger; D011950:Receptors, Cholinergic; D051153:Wnt Proteins; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases