title: Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.
abstract: The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2β splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2β-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes.
authors: International Centre for Genetic Engineering and Biotechnology (ICGEB), Human Molecular Genetics, Trieste, Italy.
authors: Andrea
authors: A
authors: Dal Mas
authors: Paola
authors: P
authors: Fortugno
authors: Irving
authors: I
authors: Donadon
authors: Lauretta
authors: L
authors: Levati
authors: Daniele
authors: D
authors: Castiglia
authors: Franco
authors: F
authors: Pagani
mesh_terms: D017398:Alternative Splicing; D002460:Cell Line; D005091:Exons; D015870:Gene Expression; D020868:Gene Silencing; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D056770:Netherton Syndrome / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D053491:Proteinase Inhibitory Proteins, Secretory / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D000068103:Serine-Arginine Splicing Factors
chemical_list: D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D053491:Proteinase Inhibitory Proteins, Secretory; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; C097083:TRA2B protein, human; C405483:U1 small nuclear RNA; C439768:hnRNP A2; C000619656:hnRNPA1 protein, human; D000068103:Serine-Arginine Splicing Factors
keywords: ESE; ESS; LEKTI; SPINK5; U1 snRNA; splicing defect