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One of most important factors for messenger RNA (mRNA) transcription is the spliceosomal component U1 small nuclear RNA (snRNA), which recognizes 5′ splicing donor sites at specific regions in pre‐mRNA. Mutations in these sites disrupt U1 snRNA binding and cause abnormal splicing. In this study, we investigated mutations at splice sites in SLC26A4 (HGNC 8818), one of the major causative genes of hearing loss, which may result in the synthesis of abnormal pendrin, the channel protein encoded by the gene. Seventeen SLC26A4 variants with mutations in the U1 snRNA binding sites were assessed by minigene splicing assays, and 11 were found to result in abnormal splicing. Interestingly, eight of the 11 pathogenic mutations were intronic, suggesting the importance of conserved sequences at the intronic splice site. The application of modified U1 snRNA effectively rescued the abnormal splicing for most of these mutations. Although three were cryptic mutations, they were rescued by cotransfection of modified U1 snRNA and modified antisense oligonucleotides. Our results demonstrate the important role of snRNA in SLC26A4 mutations, suggesting the therapeutic potential of modified U1 snRNA and antisense oligonucleotides for neutralizing the pathogenic effect of the splice‐site mutations that may result in hearing loss. [ABSTRACT FROM AUTHOR]

ABSTRACT The c.891 C> 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 hn RNPA1 and Tra2β splicing factors, respectively. The C-to- T substitution concomitantly increases hn RNPA1 and weakens Tra2β-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs ( Ex Spe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891 C> T mutation. Ex Spe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 m RNA 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, Ex Spe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of Ex Spe U1 may represent a general approach for correction of splicing defects affecting skin disease genes. [ABSTRACT FROM AUTHOR]

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., (© 2015 WILEY PERIODICALS, INC.)