Exon-Specific U1s Correct SPINK5Exon 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.

Microprocessor-dependent processing of splice site overlapping microRNA exons does not result in changes in alternative splicing

“…Normal human primary epidermal keratinocytes (NHEK) were cultured as previously described (Zambruno et al 1995). Keratinocytes were seeded at a concentration of 1-1.5 × 10 4 cells/ cm 2 in serum-free Keratinocyte Growth Medium (KGM, Invitrogen) containing 0.15 mM Ca 2+ , cultured till confluence, and then induced to differentiate by culturing in KGM containing 1.2 mM Ca 2+ for up to 5 d (Dal Mas et al 2015). Cells were maintained at 37°C with 5% CO 2 .…”

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

“…However, these U1snRNAs are often tailored on the disease-causing mutation and have the intrinsic risk of off-target effects by recognizing the partially conserved donor splice site 22 in other splicing units. For this reason, we recently developed a second-generation U1snRNAs, named Exon Specific U1snRNAs (ExSpeU1s), that are designed to recognize intronic, often poorly conserved and thus gene-specific, regions downstream of the affected exon, and shown to efficiently rescue exon-skipping in different disease models, 23,24,25,26,27,28 Of great importance, in cellular cultures, a unique ExSpeU1 was able to rescue normal splicing from different type of exon-skipping mutations either at the 5′ss or acceptor splice site (3′ss), or within the exon. 23,29 Although these gene-specificity and activity ExSpeU1 features would significantly extend the applicability of a single therapeutic molecule to panels of mutations and thus cohorts of patients, a key issue when addressing the numerous diseases with heterogeneous mutational patterns (www.hgmd.cf.ac.uk), this effect of ExSpeU1s in vivo has not been investigated yet.…”

Disease-causing variants of the conserved +2T of 5′ splice sites can be rescued by engineered U1snRNAs

“…On the basis of the frequency and relevance of these nucleotide changes and on their mechanism, we and others have devised a correction approach based on variants of the U1snRNA designed to restore complementarity with the defective 5′ss (compensatory U1snRNAs; Pinotti et al., ) or to target downstream intronic regions (exon‐specific U1snRNAs; ExSpeU1; Alanis et al., ). For different human genetic disorders, in both cellular (Balestra et al., ; Dal Mas et al., ; Glaus, Schmid, Da Costa, Berger, & Neidhardt, ; Scalet et al., ; Schmid et al., ; Tajnik et al., ; van der Woerd et al., ) and animal (Balestra et al., ; Balestra et al., ; Dal Mas, Rogalska, Bussani, & Pagani, ; Donadon et al., ; Rogalska et al., ) models, the engineered U1snRNAs were shown to be effective on variants at 5′ss but also within the exon or at the 3′ss. However, these approaches failed to rescue changes at the highly conserved nucleotides +1G and +2T of the 5′ss (Alanis et al., ; Cavallari et al., ), which are the most represented (Buratti et al., ; Krawczak et al., ) and severe ones, and commonly considered to be virtually null.…”