Biogenesis, identification, and function of exonic circular RNAs

Abstract

Circular RNAs (circRNAs) arise during post-transcriptional processes, in which a single-stranded RNA molecule forms a circle through covalent binding. Previously, circRNA products were often regarded to be splicing intermediates, by-products, or products of aberrant splicing. But recently, rapid advances in high-throughput RNA sequencing (RNA-seq) for global investigation of nonco-linear (NCL) RNAs, which comprised sequence segments that are topologically inconsistent with the reference genome, leads to renewed interest in this type of NCL RNA (i.e., circRNA), especially exonic circRNAs (ecircRNAs). Although the biogenesis and function of ecircRNAs are mostly unknown, some ecircRNAs are abundant, highly expressed, or evolutionarily conserved. Some ecircRNAs have been shown to affect microRNA regulation, and probably play roles in regulating parental gene transcription, cell proliferation, and RNA-binding proteins, indicating their functional potential for development as diagnostic tools. To date, thousands of ecircRNAs have been identified in multiple tissues/cell types from diverse species, through analyses of RNA-seq data. However, the detection of ecircRNA candidates involves several major challenges, including discrimination between ecircRNAs and other types of NCL RNAs (e.g., trans-spliced RNAs and genetic rearrangements); removal of sequencing errors, alignment errors, and in vitro artifacts; and the reconciliation of heterogeneous results arising from the use of different bioinformatics methods or sequencing data generated under different treatments. Such challenges may severely hamper the understanding of ecircRNAs. Herein, we review the biogenesis, identification, properties, and function of ecircRNAs, and discuss some unanswered questions regarding ecircRNAs. We also evaluate the accuracy (in terms of sensitivity and precision) of some well-known circRNA-detecting methods.

Figure 1

Possible models of ecircRNA biogenesis. (a) Lariat‐driven circularization, (b) Intron‐pairing‐driven circularization, and (c) Resplicing‐driven circularization. B, branch point.

Figure 2

Regulation of exon circularization. (a) The presence of flanking intronic RCSs (e.g., Alu elements) can lead to exon circularization. (b) Some splicing factors (e.g., QKI and MBL) can promote ecircRNA generation. (c) ADAR proteins can antagonize circRNA production. RCS, reverse complementary sequence.

Figure 3

Two RNA‐seq‐based strategies for detecting NCL junctions of ecircRNA candidates: (A) pseudo‐reference‐based and (B) fragment‐based strategies. The former identifies NCL junction sites at annotated exon junctions; whereas the latter does not. NCL, nonco‐linear.

Figure 4

Usage of paired‐end RNA sequencing reads for identifying circRNAs. (a) Removal of noncircular RNA events. Noncircular RNA events (e.g., trans‐splicing events in the figure) can be distinguished if the paired‐end of a read spanning a NCL junction maps outside the predicted circle. (b) Possible scenarios for circles based on the mapping of paired‐end reads (from left to right): circles containing a single exon, partial fragment(s) of an annotated exon, or an intron‐containing fragment.

Figure 5

Comparison of identified ecircRNAs based on different bioinformatics methods or the use of sequencing data from different treatments. (a) Venn diagram of identified circRNAs based on HeLa cell transcriptome data with different RNA‐library treatments for each individual algorithm. The percentage of ecircRNA events identified from each RNA‐library treatment is showed in parentheses. (b) Evaluation of sensitivity (S _n) and precision (S _p) of five ecircRNA‐detecting algorithms, based on simulated datasets of different expression levels of NCL transcripts. S _n and S _p, both of which range from 0 to 1, are defined as TP/(TP+FN) and TP/(TP+FP), respectively. TP (true positive), FP (false positive), and FN (false negative) represent the number of correctly identified events, the number of incorrectly identified events, and the number of missing events, respectively.

Figure 6

Summary of selected unanswered questions regarding ecircRNA biogenesis, function, and identification.