Circular RNAs in cardiovascular diseases

Abstract

In eukaryotes, precursor mRNAs (pre-mRNAs) produce a unique class of biologically active molecules namely circular RNAs (circRNAs) with a covalently closed-loop structure via back-splicing. Because of this unconventional circular form, circRNAs exhibit much higher stability than linear RNAs due to the resistance to exonuclease degradation and thereby play exclusive cellular regulatory roles. Recent studies have shown that circRNAs are widely expressed in eukaryotes and display tissue- and disease-specific expression patterns, including in the cardiovascular system. Although numerous circRNAs are discovered by in silico methods, a limited number of circRNAs have been studied. This review intends to summarize the current understanding of the characteristics, biogenesis, and functions of circRNAs and delineate the practical approaches for circRNAs investigation. Moreover, we discuss the emerging roles of circRNAs in cardiovascular diseases.

Introduction

Transcriptomic analyses have revealed that human genome can transcribe ~90% of the genomic DNA, but only 1-2% of transcripts encode proteins, whereas a large amount of the transcripts are categorized as noncoding RNA (ncRNAs) (Consortium, 2004). Emerging evidence has shown that aberrant ncRNA expression is associated with a number of diseases, including developmental, oncological, neurological, cardiovascular, autoimmune, and cutaneous disorders (Amaral & Mattick, 2008; Esteller, 2011; Li, Baptista, & Kissinger, 2020; Mattick & Rinn, 2015; Mehler & Mattick, 2007; Tang et al., 2019; Tang et al., 2020; Tang, Zhang, Wang, Mei, & Chen, 2017; Uchida & Dimmeler, 2015; Wu et al., 2015). Recently, a novel class of ncRNA, circRNA, has drawn much attention from researchers in different fields, ascribed to its unusual structure and diverse biological features compared to other long noncoding RNAs. The major difference between circRNAs and long noncoding RNAs is their structure, in which circRNAs show a covalently closed continuous loop without 5′–3′ polarity, but long noncoding RNAs show linear form with or without the polyadenylated tail. With this unique form, circRNAs have longer half-life than long noncoding RNAs. Another difference is the cellular location. Compared with long noncoding RNAs, a large portion of circRNAs is in the cytoplasm.

The first circular form of RNA molecule was reported in 1976, due to the discovery of plant viroids that are covalently closed circular RNA molecules. Two ends of viroids were ligated by host cellular enzymes (Sanger, Klotz, Riesner, Gross, & Kleinschmidt, 1976). Since then, sporadic studies have identified a number of circRNAs in different species such as humans, mice, and rats, although most of them were considered as byproducts of RNA splicing or so-called “scrambled exons” (Capel et al., 1993; Cocquerelle, Daubersies, Majerus, Kerckaert, & Bailleul, 1992; Hsu & Coca-Prados, 1979; Nigro et al., 1991; Patop, Wust, & Kadener, 2019; Zaphiropoulos, 1996). In the 2000s, advances in next-generation sequencing (NGS) and computational pipelines led to an explosion of RNA-related researches and the scale of NGS datasets (Mardis, 2008). However, due to the lack of polyadenylation tail (poly-A tail), circRNAs have not caught the attention of scientists in the first wave of NGS, which has mainly focused on the polyadenylated transcriptomes. Starting in the early 2010s, new pre-sequencing approaches were developed to preserve non-polyadenylated RNAs, which revealed that circRNAs are expressed in many species, including mice, rats, pigs, monkeys, humans, zebrafish, and fruitflies, as well as plants, fungi, and protists, etc (Barrett, Wang, & Salzman, 2015; Broadbent et al., 2015; Dong, Ma, Chen, & Yang, 2017; Fan et al., 2015; Guo, Agarwal, Guo, & Bartel, 2014; Jeck et al., 2013; Lu et al., 2015; Luo et al., 2018; Memczak et al., 2013; Salzman, Gawad, Wang, Lacayo, & Brown, 2012; Shen, Guo, & Wang, 2017; Westholm et al., 2014; Yang, Duff, Graveley, Carmichael, & Chen, 2011; Zhang et al., 2014). So far, more than 183,000 circRNAs have been identified in human tissues/cells (Dong, Ma, Li, & Yang, 2018). Unlike linear mRNAs that are generated from precursor mRNAs (pre-mRNAs) and undergo normal splicing following RNA polymerase II (Pol II)-mediated transcription, circRNAs are formed via back-splicing, in which a downstream 5′ splice site is ligated to an upstream 3′ splice site across exon or exons (Black, 2003; Li, Yang, & Chen, 2018). Besides, intron lariats derived from the normal splicing process erratically escape from debranching at times and cause a circular formation and are referred as circular intronic RNAs (ciRNAs) (Zhang et al., 2013). Additionally, another type of circRNAs called exon-intronic circRNAs or exon-intron-containing-circular-RNAs (EIciRNAs) is predominantly localized in the nucleus and retrain intronic information. This type of circRNAs is largely caused by escaping of EIciRNAs from decompression of debranching enzymes following splicing based on the nucleotide information in these introns (Geng, Jiang and Wu, 2018, Li et al., 2015).

Given the unique characteristics, circRNAs exhibit excellent stability compared to the linear mRNA transcripts because they are resistant to exonucleolytic decay by the exosome ribonucleases (Szabo & Salzman, 2016). The average half-life of circRNAs ranges from 19 to 48 hours, whereas linear RNA transcripts only have a lifetime of 4-9 hours (Enuka et al., 2016; Jeck et al., 2013; Schwanhausser et al., 2011). Therefore, despite a relatively low expression level compared to the linear RNAs, circRNAs have shown disease- and tissue-specific expression patterns and extraordinary potential in modulating the pathogenesis of diseases, such as oncological, cardiovascular, neurodegenerative, and immune disorders (Aufiero, Reckman, Pinto, & Creemers, 2019; Chen et al., 2020; Kumar, Shamsuzzama Haque, Baghel, & Nazir, 2017; Li et al., 2019; Saaoud et al., 2021; Shang, Yang, Jia, & Ge, 2019). In this review, we provide an overview of the recent progress in circRNA biology, including characteristics, biogenesis, and functions of circRNAs, as well as strategies for investigating circRNAs and up-to-date available online resources. Moreover, the emerging roles of circRNAs in cardiovascular diseases are discussed.