Cardiac circRNAs arise mainly from constitutive exons rather than alternatively spliced exons

Abstract

Circular RNAs (circRNAs) are a relatively new class of RNA molecules, and knowledge about their biogenesis and function is still in its infancy. It was recently shown that alternative splicing underlies the formation of circular RNAs (circRNA) arising from the Titin (TTN) gene. Since the main mechanism by which circRNAs are formed is still unclear, we hypothesized that alternative splicing, and in particular exon skipping, is a major driver of circRNA production. We performed RNA sequencing on human and mouse hearts, mapped alternative splicing events, and overlaid these with expressed circRNAs at exon-level resolution. In addition, we performed RNA sequencing on hearts of Rbm20 KO mice to address how important Rbm20-mediated alternative splicing is in the production of cardiac circRNAs. In human and mouse hearts, we show that cardiac circRNAs are mostly (∼90%) produced from constitutive exons and less (∼10%) from alternatively spliced exons. In Rbm20 KO hearts, we identified 38 differentially expressed circRNAs of which 12 were produced from the Ttn gene. Even though Ttn appeared the most prominent target of Rbm20 for circularization, we also detected Rbm20-dependent circRNAs arising from other genes including Fan1, Stk39, Xdh, Bcl2l13, and Sorbs1 Interestingly, only Ttn circRNAs seemed to arise from Rbm20-mediated skipped exons. In conclusion, cardiac circRNAs are mostly derived from constitutive exons, suggesting that these circRNAs are generated at the expense of their linear counterpart and that circRNA production impacts the accumulation of the linear mRNA.

Keywords: Rbm20; circRNAs; gene expression; heart; splicing.

FIGURE 1.

Characterization of circRNAs in mouse hearts. (A) Strategy used to elucidate the relation between the circRNAs formation and alternative splicing. (B) Schematic representation of two splice isoforms of the Ttn gene (N2BA-G and N2B) and the location of the 38 identified circRNAs in the mouse hearts and the previously identified TTN circRNAs in human hearts (Khan et al. 2016) (C) Table showing conservation of cardiac circRNAs derived from mouse and human after filtering the human circRNAs for the expression level. Between brackets, the number of circRNAs passing the filter are indicated. (D) Scatter plot showing the relation between the percentage of complementarity and the length of the identified reverse complementary sequences (RCS) passing the P-value cutoff (FDR ≤ 0.05). RCS sharing high sequence similarity with the mouse B1 elements are depicted in black.

FIGURE 2.

A subset of circRNAs are differentially expressed in hearts of Rbm20 knockout mice. (A) Volcano plot showing differentially expressed circRNAs in Rbm20 KO hearts compared to wild-type hearts. Differentially expressed circRNAs (−1 ≥ Log2 fold change ≥ 1 and P-value ≤ 0.05) are marked in gray. (B) RT-PCR for circRNAs on wild-type (WT), heterozygous (HET), and Rbm20 knockout (KO) mouse hearts. GAPDH was used as input control.

FIGURE 3.

Rbm20-dependent alternative splicing and circRNA production. Scatterplots showing the relation between the PSI of the back-spliced exons and the expression of the circRNAs (Log2 bsj reads) in (A) wild-type mice and (B) Rbm20 KO mice. Only the 38 differentially expressed circRNAs are depicted. The names of the circRNA host genes are plotted. (C) Scatterplot showing the relation between the dPSI of the back-spliced exons of the 38 differentially expressed circRNAs (dPSI back-spliced exons) and the expression fold changes (Log2 bsj reads FC) of the corresponding circRNAs between Rbm20 KO hearts and WT hearts.

FIGURE 4.

CircRNAs in the human heart are derived from exons that are typically not alternatively spliced. (A) Scatterplot showing the relation between the PSI of the back-spliced exons with the expression of the corresponding circRNAs (Log2 bsj reads) in the human hearts. Gray dots represent back-spliced exons with PSI > 0.90. Blue dots represent back-spliced exons from TTN gene. Black dots represent back-spliced exons with PSI ≤ 0.90. (B–E) The PSI of the four host genes (PALM2, YAF2, CUX1, and ZEB1), indicated in orange in A are plotted, in which exon skipping is connected with circRNA production. Black dots represent all exon bins of the corresponding genes, and orange dots represent the exon bins that give rise to the circRNAs (e.g., back-spliced exons). (F) Proposed model showing two mechanisms for circRNA production: competition-based and exon skipping-based circRNA production.