title: An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy.
abstract: SMN1 and SMN2 represent the two nearly identical copies of the survival of motor neuron gene in humans. The most frequent cause of spinal muscular atrophy (SMA) is loss of SMN1 accompanied by the inability of SMN2 to compensate due to an inhibitory mutation at position 6 in exon 7 (C6U) that causes exon 7 exclusion. How this single exonic nucleotide regulates exon 7 recognition has been of major interest. Based on score matrices and in vitro assays, abrogation of an exonic splicing enhancer (ESE) associated with SF2/ASF has been considered as the cause of exon 7 exclusion. However, a recent report supports the creation of an exonic splicing silencer (ESS) associated with hnRNP A1 as the determining factor for exon 7 exclusion. Here we show that C6U strengthens an inhibitory context that covers a larger sequence than the hnRNP A1 binding site. The inhibitory context can also be strengthened by the addition of a G residue at the first position of exon 7 in SMN1, promoting exon 7 skipping despite the presence of SF2/ASF binding site. Through in vivo selection and a series of mutations we demonstrate that the strengthening of the extended inhibitory context at the 5' end of exon 7 is exercised through overlapping sequence motifs that collaborate to regulate exon usage.
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605-2324, USA.
authors.forename: Natalia N
authors.initials: NN
authors.lastname: Singh
authors.forename: Elliot J
authors.initials: EJ
authors.lastname: Androphy
authors.forename: Ravindra N
authors.initials: RN
authors.lastname: Singh
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D018388:Codon, Terminator; D017362:Cyclic AMP Response Element-Binding Protein; D004742:Enhancer Elements, Genetic; D005091:Exons; D020868:Gene Silencing; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009134:Muscular Atrophy, Spinal / Q000235:genetics*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055532:SMN Complex Proteins; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D014162:Transfection
chemical_list: D018388:Codon, Terminator; D017362:Cyclic AMP Response Element-Binding Protein; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D012313:RNA