Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.
Faà
Valeria
V
From the Istituto di Neurogenetica e Neurofarmacologia, Consiglio Nazionale delle Ricerche, 09042 Cagliari, Italy.
Incani
Federica
F
Meloni
Alessandra
A
Corda
Denise
D
Masala
Maddalena
M
Baffico
A Maria
AM
Seia
Manuela
M
Cao
Antonio
A
Rosatelli
M Cristina
MC
D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D006801:Humans; D007438:Introns / Q000235:genetics*; D009154:Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D000068103:Serine-Arginine Splicing Factors
D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; C081934:SRSF4 protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000068103:Serine-Arginine Splicing Factors