title: The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy.
abstract: Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C-to-T transition at position +6 in exon-7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C-to-T transition in SMN2 creates a putative binding site for the RNA-binding protein Sam68. RNA pull-down assays and UV-crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon-7 skipping. Moreover, mutations in the Sam68-binding site of SMN2 or in the RNA-binding domain of Sam68 completely abrogated its effect on exon-7 skipping. Retroviral infection of dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon-7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.
authors.affiliation: Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Pedrotti
authors.forename: Pamela
authors.initials: P
authors.lastname: Bielli
authors.forename: Maria Paola
authors.initials: MP
authors.lastname: Paronetto
authors.forename: Fabiola
authors.initials: F
authors.lastname: Ciccosanti
authors.forename: Gian Maria
authors.initials: GM
authors.lastname: Fimia
authors.forename: Stefan
authors.initials: S
authors.lastname: Stamm
authors.forename: James L
authors.initials: JL
authors.lastname: Manley
authors.forename: Claudio
authors.initials: C
authors.lastname: Sette
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C489559:KHDRBS1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C526477:SMN2 protein, human; D055540:Survival of Motor Neuron 2 Protein; D012313:RNA