title: Differential 3' splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP.
abstract: Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF(65) with the 3' splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF(65) crosslinking. Furthermore, blocking recognition of a Tra2-beta1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF(65) crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation.
authors.affiliation: Centre de Regulació Genòmica, Barcelona, Spain.
authors.forename: Mafalda
authors.initials: M
authors.lastname: Martins de Araújo
authors.forename: Sophie
authors.initials: S
authors.lastname: Bonnal
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Hastings
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Krainer
authors.forename: Juan
authors.initials: J
authors.lastname: Valcárcel
mesh_terms: D002474:Cell-Free System; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D055532:SMN Complex Proteins / Q000235:genetics*; D000072668:Splicing Factor U2AF; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics*; D055540:Survival of Motor Neuron 2 Protein
chemical_list: D009687:Nuclear Proteins; D022821:RNA Splice Sites; D017413:Ribonucleoprotein, U2 Small Nuclear; D012261:Ribonucleoproteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D000072668:Splicing Factor U2AF; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; C000607494:U2AF2 protein, human