RBM20 Regulates Circular RNA Production From the Titin GeneNovelty and Significance
Rationale: RNA-binding motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of noncoding RNA molecules.
Objective: To establish the role of RBM20 in the formation of circRNAs in the heart.
Methods and Results: Here, we performed circRNA profiling on ribosomal depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene that is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy but not in hypertrophic cardiomyopathy. We generated RBM20-null mice and show that they completely lack these titin circRNAs. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript.
Conclusions: We show that RBM20 is crucial for the formation of a subset of circRNAs that originate from the I-band of the titin gene. We propose that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNAs.
Mutations in the RNA-binding motif protein 20 (RBM20) have been shown to cause a clinically aggressive form of dilated cardiomyopathy (DCM).1 A next-generation sequencing study in a large cohort of idiopathic DCM patients revealed that RBM20 belongs to the most frequently affected genes in DCM.2 RBM20 is essential for proper splicing of a large number of genes, and loss of RBM20 induces splicing defects in, for example, titin.3 These splicing defects in titin are thought to be an important reason why these mutations in RBM20 cause DCM.3,4 However, the pathophysiological role of RBM20 mutations may not be limited to abnormal splicing. Given its essential role in splicing, we hypothesized that RBM20 may also regulate other splicing-dependent processes, like the formation of circular RNAs (circRNAs). If so, this would be of importance because it adds a novel potential disease mechanism.
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In This Issue, see p 965
Despite their discovery over 20 years ago, circRNAs have only recently been recognized as a novel class of noncoding RNA molecules. Because of their unusual properties, they were presumed to be by-products of aberrant RNA splicing.5 Decades later, next-generation sequencing has revealed that thousands of endogenous circRNAs are expressed in mammals, including the cardiovascular system,6 and that some of these circRNAs are even more abundant than their linear counterparts.7
CircRNAs are produced by the canonical spliceosome machinery, by back-splicing of exons of pre-mRNA, which results in covalently closed, single-stranded RNA molecules that lack poly(A) tails.7–9 The formation of circRNAs can affect splicing, as it has been shown that the more an exon is circularized, the less it will be represented in the linearly processed mRNA.9,10 How RNA circularization is connected to alternative splicing remains largely unknown, but splicing factors such as muscleblind and quaking have been shown to regulate circRNA formation.8,11 Recent studies revealed that circRNAs may also regulate gene expression by different mechanisms. Specifically, 2 cytoplasmic circRNAs have been shown to serve as microRNA sponges,12,13 and a class of nuclear circRNAs has been shown to promote transcription of their parental genes directly by associating with RNA polymerase II.14
Little is known about circRNA expression and biogenesis in the healthy and diseased human heart. Therefore, we explored circRNAs in cardiac tissue from patients having hypertrophic cardiomyopathy (HCM) or DCM and from nondiseased individuals. We identified thousands of circRNAs expressed in the heart, with some of them regulated in disease. We identified a hotspot of circRNAs produced by the titin gene, precisely within the I-band region, a region known to undergo extensive, RBM20 regulated, alternative splicing.15 We show in a patient with an RBM20 mutation and in Rbm20 knockout mice that RBM20 function is required for the production of circRNAs from the I-band region of titin. This suggests that loss of RBM20 may induce myocardial disease not only by abnormal splicing of linear transcripts but possibly also by loss of a specific class of conserved and regulated circRNAs.
Detailed methods are provided in the Online Data Supplement.
Whole Transcriptome Identification of circRNAs in Human Hearts
To detect circRNAs in diseased and nondiseased human hearts, ribosomal depleted RNA, obtained from left ventricles of 2 control, 2 HCM, and 2 DCM individuals, were used for whole transcriptome sequencing (Figure 1A). We searched for evidence of back-splicing (Figure 1B) by mapping canonical and noncanonical fusion junctions using the software MapSplice16 and uncovered a total number of 7130 putative circRNAs, of which 826 back-spliced junctions were commonly identified in all 6 samples (Figure 1C; Online Table I). Regression analysis confirmed that back-spliced junction counts were positively correlated across replicate samples within each group (Online Figure I). The most striking observation was that a total of 80 different circRNAs were identified within the titin gene (80 titin circRNAs expressed in ≥2 individuals and 22 titin circRNAs in all 6 individuals). Similarly, 59 different back-spliced junctions were identified in the RYR2 gene (59 RYR2 circRNAs expressed in ≥2 individuals and 16 RYR2 circRNAs in all 6 individuals; Online Table II). The expression level of circRNAs did not correlate with the level of expression of the host gene (Figure 1D), neither did we observe a relationship between transcript/gene length and number of circRNAs arising from the corresponding host gene (Online Figure II).
There is a general consensus that circRNAs are flanked by significantly longer introns than expected by chance.17 Therefore, we compared the median length of introns flanking the back-splicing junctions of the 826 predicted circ RNAs with a control set comprising an equal number of introns randomly selected from the human genome. The median length of introns flanking our set of circRNAs was 13 071 nt compared with 1727 nt in the control set, corresponding to a 7- to 8-fold difference in length (Online Figure IIIA).
Introns flanking the back-spliced junctions of circRNAs are known to be enriched for paired Alu repeats in inverted orientation (Online Figure IIIB). We performed de novo motif enrichment analysis in introns flanking the back-splicing junctions of all 826 predicted circRNAs and detected the Alu-Ya5 motif as the candidate with the highest information content (Online Figure IIIC). In total, 16% of the circRNAs were flanked by introns containing at least 1 paired inverted Alu repeat, whereas in a control set of introns only 7% contained inverted Alu repeats (Online Figure IIID and Table III).
Recent studies suggest that circRNAs can act as microRNA sponges.17 Using miRbase in conjunction with the RNAhybrid tool, we compared the frequency of putative miRNA-binding sites identified in exons belonging to the set of 826 circRNAs to those identified in 2 control sets of exons, containing either 3′ untranslated region or coding sequences. As shown in Online Figure IV, circRNAs expressed in the heart are not globally enriched for miRNA-binding sites.
Experimental Validation of Next-Generation Sequencing–Derived Human Heart circRNAs
To test whether the identified transcripts are bona fide circRNAs, we selected 22 of the identified circRNAs for reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to amplify the circRNA-specific back-splice junctions. We selected these candidates on the basis of their absolute expression level, location within the host gene, conservation, or function of their host gene in cardiac biology (Online Table V). To avoid amplification of linear transcripts, primers were designed to diverge on linear cDNA, although being convergent on circRNA-derived cDNA. We tested circRNA expression in poly(A)-negative and poly(A)-positive RNA fractions as well as their expected resistance to exoribonuclease RNase R digestion. Although a linear transcript (αMHC [myosin heavy chain]) seemed sensitive to RNase R digestion and was mainly found in the poly(A)-positive RNA fraction, the 22 circRNAs were all resistant to RNase R digestion and were exclusively found in the poly(A)-negative fraction (Figure 2A; Online Figure VA). Interestingly, several host genes produced alternative circRNAs, as can be appreciated from the additional bands for cTTN and cLAMA2 gels in Figure 2. Sanger sequencing confirmed the presence of back-spliced exons, both in the predicted amplicons and in the additional bands observed on gel. The higher bands mostly contained additional exons, and sometimes introns, upstream of the acceptor exon or downstream of the donor exon, which indicates alternative circularization (Online Figure VI). The precise exon and intron composition of the identified titin circRNAs remains unknown because only back-spliced regions of the circRNAs were mapped. Taken together, experimental validation indicates that our RNA-seq and circRNA prediction with MapSplice is a robust approach to identify bona fide circRNAs.
As shown in Figure 2B and Online Figure VB, RT-PCR of these 22 circRNAs on a human panel of adult and fetal tissues revealed that a subset of circRNAs are widely expressed (eg, cPDLIM5, cATP2B4), whereas others are expressed in a tissue-specific manner (eg, cLAMA2 and cTTN). Interestingly, the precise alternative circularization of some of the circRNAs seems tissue specific (eg, cSTRN) or developmental specific (eg, cTMEM38b).
Identification of Disease-Regulated circRNAs
Differential expression analysis revealed 43 out of 826 commonly identified circRNAs to be differentially expressed in DCM compared with control samples and 60 circRNAs in HCM compared with control samples (Figure 3A and 3B; Online Table IVA and IVB). Because of the limited number of samples (n=2 per group), a fold change of >2 and a P value of <0.05 were used as cutoffs, rather than the adjusted P value (note that only 2 circRNAs survived multiple testing; Online Table IVA and IVB). We selected 10 candidates from the set of 22 experimentally validated circRNAs and examined them by semiquantitative RT-PCR in a larger group of patients (7 control, 7 HCM, and 7 DCM). As shown in Figure 3C and quantified in Online Figure VII, we confirmed the loss of circRNA formation from the host genes CAMK2D (in DCM and HCM) and titin (mainly in DCM) in disease. To investigate whether differential expression of circRNAs was caused by altered expression of host genes, we performed qRT-PCR of the linear transcripts (Online Figure VIII). Interestingly, expression of CAMK2D and titin circRNAs did not correlate with expression changes of their linear host transcripts, indicating that the disease-regulated changes in circRNA production are independent of transcriptional regulation.
RBM20 Is Required for the Production of circRNAs From the Host Gene Titin
We observed a remarkably large number of circRNAs produced from the titin gene, a gene that is known to undergo highly complex alternative splicing. Titin is a protein that spans half of the sarcomere and determines biomechanical properties of the heart. Particularly, the inclusion/exclusion of a large segment within titin, the so-called I-band, which behaves as a molecular spring, importantly determines the passive stiffness of sarcomeres.18 RBM20 is the splicing factor responsible for alternative splicing within the I-band (particularly the elastic PEVK domain and the immunoglobulin (Ig)-rich region), and mutations in RBM20 have been shown to result in the expression of large and highly compliant titin isoforms, suggested to cause DCM.3,4 Strikingly, we noted a hotspot of circRNAs exactly within titin’s I-band (Figure 4A). This prompted us to investigate whether there is an enrichment of RBM20-binding sites in the introns flanking the back-spliced junctions of the 80 predicted titin circRNAs. Interestingly, we found a 5-fold increase in RBM20-binding site frequency in the introns flanking (within 100 bp) the 80 titin circRNAs compared with a control set of introns (see also track in Figure 4A). Examination of RBM20-binding sites in flanking introns of all 826 identified circRNAs revealed a ≈2-fold enrichment compared with the control intron set (Online Figure IXA and IXB and Table VI).
To investigate whether RBM20 is essential for titin-derived circRNAs, we generated Rbm20-null mice and examined their cardiac titin circRNA production. In these mice, loss of Rbm20 resulted in early-onset DCM, which manifested by LV dilatation and impaired cardiac function at an age of 10 weeks and was accompanied by the previously described aberrant titin splicing3 (Online Figure X). From a recent report, which profiled circRNAs in the mouse heart,19 we selected 4 titin circRNAs that arise from the Ig and PEVK regions, regions that critically depend on Rbm20 for alternative splicing (mus_cTtn1-4), and 2 circRNAs that arise from the Rbm20-independent N2A and Z-disk regions (mus_cTtn5-6, Online Figure XI). Interestingly, we identified both Rbm20-dependent and Rbm20-independent circRNAs arising from the titin gene. CircRNAs produced from the Ig and PEVK regions were absent in the Rbm20 knockout hearts, whereas the circRNAs generated from the Rbm20 splicing–independent regions were abundantly expressed in the Rbm20 knockout hearts (Figure 4B). Interestingly, for the Rbm20-dependent circRNAs, the corresponding exons are more included in the linear titin transcript after loss of Rbm20, whereas for Rbm20-independent circRNAs, there is equal exon inclusion between wild-type and Rbm20 knockout mice (Online Figure XII). This implies that when Rbm20-dependent exons are spliced out of the linear titin transcript, they can serve as a substrate for circRNA formation. When, however, Rbm20 is lost and these exons are included in the linear titin transcript, the substrate is lost, and circRNA formation is abolished.
To validate RBM20-dependent circRNA production and its relevance in human disease, we investigated a DCM patient with a heterozygous mutation in RBM20 (E913K). This mutation resulted in a shift in titin from the less compliant N2B toward the highly compliant N2BA isoforms.20 Analysis of circRNA production by RT-PCR in the heart of this patient revealed that titin circRNA production was grossly abnormal, specifically in the Ig and PEVK domain (ie, cTTN1, 2, 4, and 5), when compared with hearts of other idiopathic DCM patients, but not for circRNAs produced partially from the Z-disk region (ie, cTTN3), which is considered RBM20-independent (Figures 3C and 4A).
Our data confirms that alternative splicing and circRNA production are intimately connected. We show that skipping of titin’s I-band region is associated with circRNA formation, likely by providing a substrate for the formation of circRNAs, as illustrated in Figure 4C. This figure depicts the concept based on the established role of RBM20 as a splicing repressor.4 Thus, in the absence of RBM20, these exons are included in the linear titin transcript so that they cannot serve as substrate for circRNA formation.
We identified thousands of circRNAs in the human heart and show that a subset of these circRNAs is differentially expressed in diseased hearts. The identified cardiac circRNAs conform to most of the key properties of circRNAs: (1) circ RNAs were flanked by 7- to 8-fold longer introns compared with a random set of intron, (2) we found an enrichment of inverted Alu repeats in the introns flanking the predicted circ RNAs, (3) cardiac circRNAs were not generally enriched for miRNA-binding sites, (4) circRNAs were resistant to RNase R treatment and lack poly(A) tails.
The main finding of this study is that RBM20 is crucial for the formation of a subset of circRNAs that originate from a specific region within the I-band of the titin gene (ie, PEVK and Ig repeats), a region that is known to undergo extensive alternative splicing to produce titin isoforms with the desired biomechanical properties.15 Given the known role of RBM20 in exon skipping in titin’s I-band, we propose the concept that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNAs. It has been described that idiopathic DCM patients have increased N2BA/N2B isoform ratios and thus more inclusion of Ig repeats and a longer PEVK domain in the I-band.21 In similarity to our observations in the Rbm20 knockout mice, we postulate that increased inclusion of these domains in the linear titin transcript prevents the formation of titin circ RNAs. As shown in Figure 3C, this is indeed the case for the I-band circRNAs. Interestingly, we have also identified RBM20-independent titin circRNAs. These were produced from exons closer to the Z-disk and from exons within the N2A domain, regions that are not spliced by RBM20; these circRNAs were readily expressed in the Rbm20 knockout hearts.
Maatz et al4 previously identified a correlation between RBM20 mRNA levels and the levels of titin splicing, which suggests that titin circRNA expression is also correlated to RBM20 mRNA levels. In the myocardial tissues used for RT-PCR (Figure 3C), we observed a 29%, nonsignificant downregulation of RBM20 mRNA in the DCM samples (data not shown). However, the small group size of this study was not sufficient to find a correlation between RBM20 and titin circRNA expression. This is not surprising considering the observation by Maatz et al that the expression of RBM20 greatly varies between patients with end-stage heart failure. Therefore, the authors used 10 patients with the highest RBM20 expression and 10 patients with the lowest RBM20 expression, selected from a database of 148 heart failure patients to demonstrate a correlation between titin splicing and RBM20 levels. In line with this observation, the RNA-seq experiment in our study was underpowered to test for a correlation between RBM20 mRNA and cTTN expression because none of the RBM20-dependent titin circRNAs were significantly differentially expressed between DCM and controls after correcting for multiple testing (Online Table VII).
It is known that circRNA processing is intimately connected to alternative splicing10; however, our findings suggest that circularization of RNA substrates occurs after alternative splicing has taken place. This order of events is supported by a recent study of Zhang et al,22 who used metabolic tagging of nascent RNAs to show that circRNA processing from pre-mRNA occurs post-transcriptionally. Specifically, they show that the majority of circRNAs were produced after transcription and splicing of their parental gene was completed. However, others have shown that the use of 5′ and 3′ splice sites in circRNAs can compete with pre-mRNA splicing, which would imply that circRNA biogenesis occurs simultaneously and thus can affect or even regulate alternative splicing.8 It therefore is possible that circRNA formation within titin underlies the diversity of titin splicing, and it is tempting to speculate that perturbations in circRNA expression could contribute to the pathophysiology of DCM patients. The general function of circRNAs remains unclear, but an intriguing possibility, which is still under debate, is that circRNAs form a template for protein synthesis. Because most circRNAs are composed of protein-coding exons, it will be interesting to investigate their potential to be translated. If these circRNAs are translatable, it has to be considered that small titin peptides are expressed in the heart.
Taken together, we propose that RBM20 is important in normal cardiac physiology not only by regulating exclusion of specific exons but also by allowing a specific subclass of circRNAs to be generated. This opens the possibility that loss of RBM20 induces disease not only by aberrant splicing of titin and other genes but also by loss of circRNAs.
We thank Aeilko H. Zwinderman for valuable advice on bioinformatics and Anke J. Tijsen for scientific discussions. This work was supported by grants from the Netherlands Organization for Scientific Research (NWO-836.12.002 and NWO-821.02.021) and the Netherlands Cardiovascular Research Initiative (grants CVON-ARENA-2011-11).
In July 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.27 days.
↵* These authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.309568/-/DC1.
- Nonstandard Abbreviations and Acronyms
- circular RNA
- dilated cardiomyopathy
- hypertrophic cardiomyopathy
- RNA-binding motif protein 20
- Received July 14, 2016.
- Revision received August 12, 2016.
- Accepted August 14, 2016.
- © 2016 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Circular RNAs (circRNAs) are a novel class of RNAs that are formed by back-splicing of exons by the spliceosome.
Thousands of different circRNAs are expressed in mammals, but their function remains largely elusive.
RNA-binding protein 20 (RBM20) is an important cardiac splicing factor, and mutations herein cause early-onset dilated cardiomyopathy.
What New Information Does This Article Contribute?
We identified thousands of circRNAs to be expressed in the human heart, with at least 80 originating from the titin gene.
Using Rbm20 knockout mice and myocardial tissue of a patient with a mutated RBM20 allele, we demonstrate a role for RBM20 in the formation of circRNAs of the titin gene.
We propose the model that the splicing factor RBM20, by excluding specific exons from titin’s I-band region, provides the substrate to form circRNAs.
Here, we identified thousands of circRNAs from the human heart, with at least 80 circRNAs originating from the titin gene. Our most important finding is that the splicing factor RBM20 is crucial for the formation of a subset of these titin circRNAs. As such, we provide insights into the biogenesis of titin circRNAs, by showing that exon skipping of titin’s I-band region is required for circRNAs to be formed. We think that these findings open novel avenues to understand the role of RBM20 as a cause of hereditary dilated cardiomyopathy, while contributing to our basic understanding of the biogenesis of circRNAs.