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Exon‐Specific U 1s Correct SPINK 5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element
Author(s) -
Dal Mas Andrea,
Fortugno Paola,
Donadon Irving,
Levati Lauretta,
Castiglia Daniele,
Pagani Franco
Publication year - 2015
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22762
Subject(s) - exon , rna splicing , exon skipping , exonic splicing enhancer , biology , intron , genetics , alternative splicing , enhancer , splice site mutation , point mutation , minigene , mutation , microbiology and biotechnology , rna , gene , transcription factor
The c.891 C > T synonymous transition in SPINK5 induces exon 11 ( E 11) skipping and causes N etherton 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 hn RNPA 1 and T ra2β splicing factors, respectively. The C ‐to‐ T substitution concomitantly increases hn RNPA 1 and weakens T ra2β‐binding sites, leading to pathological E 11 skipping. In hybrid minigenes, exon‐specific U 1 small nuclear RNA s ( E x S pe U 1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E 11 skipping defect caused by the c.891 C > T mutation. E x S pe U 1 lentiviral‐mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full‐length SPINK 5 m RNA and the corresponding functional lympho‐epithelial K azal‐type related inhibitor protein in a dose‐dependent manner. This study documents the reliability of a mutation‐specific, E x S pe U 1‐based, splicing therapy for a relatively large subset of E uropean NS patients. Usage of E x S pe U 1 may represent a general approach for correction of splicing defects affecting skin disease genes.