The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

• Published in: The EMBO journal 2010-04, Vol.29 (7), p.1235-1247
• Main Authors: Pedrotti, Simona, Bielli, Pamela, Paronetto, Maria Paola, Ciccosanti, Fabiola, Fimia, Gian Maria, Stamm, Stefan, Manley, James L, Sette, Claudio
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Alternative Splicing; Cell Line; Cellular biology; Consensus Sequence; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; EMBO24; EMBO36; Exons; Fibroblasts - metabolism; Gene expression; Genetic disorders; Heterogeneous Nuclear Ribonucleoprotein A1; Heterogeneous-Nuclear Ribonucleoprotein Group A-B - metabolism; hnRNP A1; Humans; Molecular biology; Muscular Atrophy, Spinal - genetics; Muscular Atrophy, Spinal - metabolism; Mutation; Protein Binding; RNA - metabolism; RNA, Messenger - genetics; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; RNA-protein interactions; Sam68; SMA; SMN2; Survival of Motor Neuron 2 Protein - genetics; Survival of Motor Neuron 2 Protein - metabolism
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2010.19

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.