Exon-specific U1s correct SPINK5 exon 11 skipping caused by a synonymous substitution that affects a bifunctional splicing regulatory element

Andrea Dal Mas, Paola Fortugno, Irving Donadon, Lauretta Levati, Daniele Castiglia, Franco Pagani

Research output: Contribution to journalArticle


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. The most frequent mutation in European patients with Netherthon syndrome, a severe genodermatosis, is a synonymous mutation in Spink 5 that affects a bifucntional splicing regulatory element and induces exon 11 skipping. An RNA based therapy with U1 snRNA specific for the defective exon recovers splicing and protein in primary keratinocytes. Exon-specific U1s can be used to correct splicing defects in skin-disease genes.

Original languageEnglish
Pages (from-to)504-512
Number of pages9
JournalHuman Mutation
Issue number5
Publication statusPublished - May 1 2015



  • ESE
  • ESS
  • SPINK5
  • Splicing defect
  • U1 snRNA

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)
  • Medicine(all)

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