Abstract
BackgroundMany pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known.ResultsHere, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5’ splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5′ splice site can be blocked by SSOs to activate the exon.ConclusionsThe hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0279-9) contains supplementary material, which is available to authorized users.
Highlights
Many pathogenic genetic variants have been shown to disrupt mRNA splicing
We tested our dataset with the iCLIPro analysis tool [51], which confirmed that our individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) reads do cluster around the 5′ end of the reads (Additional file 3: Figure S2)
We searched for enriched motifs in the hnRNP A1 binding peaks and, confirming the validity of our iCLIP approach, the most highly enriched motif has high similarity to the SELEX-based motif UAGGGA/ U [25] and to the previous HITS-Cross-linking immunoprecipitation (CLIP) generated motifs [17] (Fig. 1a)
Summary
Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. Stringent regulation of splicing is crucial, since missplicing may lead to the production of nonfunctional or malfunctional mRNA isoforms. Up to one third of all disease-causing mutations may disrupt. A well-studied example of a disease caused by disruption of an SRE is spinal muscular atrophy (SMA). SMN2 is highly homologous to SMN1, but a single nucleotide difference in SMN2 exon 7 (c.840C > T) simultaneously disrupts a splicing enhancer and introduces a splicing silencer, which binds heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) [3,4,5,6,7]. In patients with MCAD deficiency a mutation in exon 5 (c.362C > T) disrupts a splicing enhancer and
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