Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing.
Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.
Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
Elisabete
E
Hernández-Imaz
Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
Yolanda
Y
Martín
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
Laura
L
de Conti
Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
German
G
Melean
Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
Ana
A
Valero
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
Marco
M
Baralle
Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
Concepción
C
Hernández-Chico
D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D016514:Genes, Neurofibromatosis 1; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D022821:RNA Splice Sites; D012326:RNA Splicing; D012045:Regulatory Sequences, Nucleic Acid; D012261:Ribonucleoproteins / Q000378:metabolism
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D022821:RNA Splice Sites; D012261:Ribonucleoproteins; C000619656:hnRNPA1 protein, human