title: A T to G mutation in the polypyrimidine tract of the second intron of the human beta-globin gene reduces in vitro splicing efficiency: evidence for an increased hnRNP C interaction.
abstract: In a patient with a beta-thalassemia intermedia, a mutation was identified in the second intron of the human beta-globin gene. The U-->G mutation is located within the polypyrimidine tract at position -8 upstream of the 3' splice site. In vivo, this mutation leads to decreased levels of the hemoglobin protein. Because of the location of the mutation and the role of the polypyrimidine tract in the splicing process, we performed in vitro splicing assays on the pre-messenger RNA (pre-mRNA). We found that the splicing efficiency of the mutant pre-mRNA is reduced compared to the wild type and that no cryptic splice sites are activated. Analysis of splicing complex formation shows that the U-->G mutation affects predominantly the progression of the H complex towards the pre-spliceosome complex. By cross-linking and immunoprecipitation assays, we show that the hnRNP C protein interacts more efficiently with the mutant precursor than with the wild-type. This stronger interaction could play a role, directly or indirectly, in the decreased splicing efficiency.
authors.affiliation: Centre de Génétique Moléculaire, CNRS, Laboratoire Propre Associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette, France.
authors.forename: P
authors.initials: P
authors.lastname: Sébillon
authors.forename: C
authors.initials: C
authors.lastname: Beldjord
authors.forename: J C
authors.initials: JC
authors.lastname: Kaplan
authors.forename: E
authors.initials: E
authors.lastname: Brody
authors.forename: J
authors.initials: J
authors.lastname: Marie
mesh_terms: D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D066298:In Vitro Techniques; D007438:Introns; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D017354:Point Mutation; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D004268:DNA-Binding Proteins; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D046911:Macromolecular Substances; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D005914:Globins