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The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy
Author(s) -
Pedrotti Simona,
Bielli Pamela,
Paronetto Maria Paola,
Ciccosanti Fabiola,
Fimia Gian Maria,
Stamm Stefan,
Manley James L,
Sette Claudio
Publication year - 2010
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/emboj.2010.19
Subject(s) - smn1 , biology , exon , rna splicing , spinal muscular atrophy , rna binding protein , exonic splicing enhancer , alternative splicing , microbiology and biotechnology , minigene , repressor , exon skipping , rna , splicing factor , gene expression , gene , genetics
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.