Global Identification of Hnrnp A1 Binding Sites for SSO-Based Splicing Modulation Gitte H
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Bruun et al. BMC Biology (2016) 14:54 DOI 10.1186/s12915-016-0279-9 RESEARCH ARTICLE Open Access Global identification of hnRNP A1 binding sites for SSO-based splicing modulation Gitte H. Bruun1, Thomas K. Doktor1, Jonas Borch-Jensen1, Akio Masuda2, Adrian R. Krainer3, Kinji Ohno2 and Brage S. Andresen1* Abstract Background: Many 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. Results: Here, 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. Conclusions: The 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. Keywords: hnRNP A1, iCLIP, Splicing splice-switching oligonucleotides (SSOs), Pseudoexons, Alternative splicing, Splicing silencer, Cross-linking immunoprecipitation (CLIP), RNA-seq, Surface plasmon resonance imaging (SPRi) Background pre-mRNA splicing [1, 2]. Mutations that affect pre- Splicing of pre-mRNA is a key step in gene expression, mRNA splicing may directly or indirectly affect the rec- and alternative splicing, the production of multiple ognition of the splice sites or the SREs. Alternatively, mRNA isoforms from one gene, is a key mechanism to mutations may create or disrupt SREs and in this way expand the diversity of the human proteome. Stringent disturb the complex regulatory network of splicing. regulation of splicing is crucial, since missplicing may A well-studied example of a disease caused by dis- lead to the production of nonfunctional or malfunctional ruption of an SRE is spinal muscular atrophy (SMA). mRNA isoforms. Splicing regulatory proteins regulate Patients with SMA lack a functional SMN1 gene and de- splicing by binding to cis-acting splicing regulatory pend on the SMN2 gene to survive. SMN2 is highly elements (SREs). A fine balance between negative and homologous to SMN1, but a single nucleotide difference positive SREs determines the splicing fate of exons. Up in SMN2 exon 7 (c.840C > T) simultaneously disrupts a to one third of all disease-causing mutations may disrupt splicing enhancer and introduces a splicing silencer, which binds heterogeneous nuclear ribonucleoprotein * Correspondence: [email protected] A1 (hnRNP A1) [3–7]. Hence, SMN2 has a high degree 1Department of Biochemistry and Molecular Biology and The Villum Center of exon 7 skipping, which results in a nonfunctional pro- for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark tein. In patients with MCAD deficiency a mutation in Full list of author information is available at the end of the article exon 5 (c.362C > T) disrupts a splicing enhancer and © 2016 Bruun et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bruun et al. BMC Biology (2016) 14:54 Page 2 of 19 induces exon skipping. Interestingly, a silent polymorph- creates an hnRNP A1-binding splicing silencer, thereby ism in a flanking hnRNP A1-binding splicing silencer causing aberrant splicing and phenylketonuria (PKU) [41]. can protect against exon 5 skipping [8]. Also, a SNP affecting an hnRNP A1-binding SRE plays a A promising approach to treat diseases caused by ab- potential role in cardiovascular disease risk by altering the errant splicing is the use of antisense oligonucleotide- alternative splicing of HMGCR [42]. Furthermore, a syn- mediated splicing modulation [9, 10]. One way to do this onymous SNP in exon 11 of ACADM disrupts an hnRNP is to block SREs by complementary antisense oligonucle- A1-binding SRE, which improves exon inclusion and may otides (also called splice-switching oligonucleotides, influence fatty acid oxidation capacity [43]. SSOs) to make them inaccessible to splicing regulatory Despite the fact that hnRNP A1 is a well-studied splicing proteins and thereby redirect splicing. One of the most regulatory factor, transcriptome-wide in vivo binding of promising SSO-based therapies is targeting of the hnRNP A1 has previously only been carried out as part of hnRNP A1-binding splicing silencer N1 in SMN2 intron a study comparing binding of several hnRNP proteins. In 7 to correct SMN2 splicing [11–14]. However, identifica- that study, crosslinking and immunoprecipitation followed tion of SREs which are suitable for this type of therapy is by high-throughput sequencing (HITS-CLIP) analysis was not easy. Mutations that disrupt or create new SREs are performed in HEK293T cells [17]. In the present study, we difficult to identify, since the binding sites for most spli- have examined the transcriptome-wide binding of hnRNP cing regulatory proteins have mainly been characterized A1 by a newer method, iCLIP coupled with high- by in vitro studies. Thus, there is limited knowledge of throughput sequencing [44] in a different cell type, namely the in vivo binding sites of splicing regulatory proteins; HeLa cells. Our main goal is to create a more comprehen- moreover, splicing regulatory proteins may compete and sive catalogue of hnRNP A1 binding sites to enable identi- cooperate for binding in an unpredictable manner [15]. fication of disease-associated binding sites. In this study, To identify in vivo binding sites, a number of RNA bind- we increase the number of CLIP-identified hnRNP A1 ing proteins have been subjected to crosslinking and im- binding sites in the human transcriptome approximately munoprecipitation (CLIP) analysis [16–24]. 20 times. This dramatically increases the number of iden- Here, we performed individual-nucleotide resolution tified hnRNP A1-binding SREs, which can serve as targets crosslinking immunoprecipitation (iCLIP) analysis of the for SSO-based therapy. While some binding sites overlap multifunctional RNA binding protein hnRNP A1. hnRNP between the two studies, many seem to be cell type- or A1 is involved in many RNA processing events including method-specific. Furthermore, we hypothesize that, by both constitutive and alternative splicing [25, 26]. Trad- combining the hnRNP A1 binding map with the validated itionally, hnRNP A1 has been considered a splicing repres- hnRNP A1 binding motif, one will be able to better predict sor, though it may in some cases also stimulate splicing. the effect of sequence variations on splicing. We find that hnRNP A1 may repress splicing by antagonizing the func- hnRNP A1 binding in the proximal intron is important for tion of positive splicing factors such as the SR proteins hnRNP A1-mediated splicing repression, and that target- [27–29]; alternatively, hnRNP A1 may sterically block ing these binding sites with SSOs can improve exon inclu- binding of SR proteins or members of the spliceosome to sion. This will be useful for correction of splicing of prevent splice site recognition [30]. Furthermore, hnRNP constitutive exons which have been inactivated by muta- A1 proteins are able to multimerize after initial binding to tions in SREs or to shift between alternatively spliced a high-affinity site and spread across an exon to inhibit its exons. We selected pseudoexons as a model of constitutive inclusion [31]. Protein-protein interactions between exons, which have been inactivated by mutations that either different hnRNP A1 molecules may also promote either abolish a splicing enhancer or create a splicing silencer, and exon inclusion by looping out introns to reduce intron show that blocking of downstream hnRNP A1 binding sites size and stimulate splice site pairing, or cause exon improves exon inclusion. Furthermore, we show that we skipping by looping out exons to inhibit splice site recog- can shift the alternative splicing of SKA2 exon 3 towards nition [32–34]. more inclusion when blocking an identified hnRNP A1- Diseases associated with deregulated expression of binding splicing silencer downstream of the 5′ splice site. hnRNP A1 include