Biogenesis and Regulatory Roles of Circular RNAs

Abstract

Covalently closed, single-stranded circular RNAs can be produced from viral RNA genomes as well as from the processing of cellular housekeeping noncoding RNAs and precursor messenger RNAs. Recent transcriptomic studies have surprisingly uncovered that many protein-coding genes can be subjected to backsplicing, leading to widespread expression of a specific type of circular RNAs (circRNAs) in eukaryotic cells. Here, we discuss experimental strategies used to discover and characterize diverse circRNAs at both the genome and individual gene scales. We further highlight the current understanding of how circRNAs are generated and how the mature transcripts function. Some circRNAs act as noncoding RNAs to impact gene regulation by serving as decoys or competitors for microRNAs and proteins. Others form extensive networks of ribonucleoprotein complexes or encode functional peptides that are translated in response to certain cellular stresses. Overall, circRNAs have emerged as an important class of RNAmolecules in gene expression regulation that impact many physiological processes, including early development, immune responses, neurogenesis, and tumorigenesis.

Keywords: backsplicing; circRNA; microRNA; noncoding RNA; pre-mRNA splicing; translation.

Figure 1

circRNAs can be produced from the backsplicing of precursor (m)RNAs. (a, top) An RNA precursor containing multiple exons can be subjected to canonical splicing, leading to the formation of a linear RNA with all exons included. (Bottom) In contrast, backsplicing and canonical splicing can lead to the formation of a circRNA with two exons, and an alternatively spliced linear RNA with skipped circle-forming exons. Note that the alternatively spliced linear RNA is only sometimes observed, as indicated by the asterisk. Panel a adapted with permission from X. Li et al. (2018); copyright 2018 Elsevier. (b) Short RNA-sequencing reads are mapped to (top) the splicing junction sites of a linear RNA and (bottom) the BSJ site of a circRNA. The BSJ-mapped, noncolinear reads are critical for identification of circRNAs from transcriptomic studies. Abbreviations: BSJ, backsplicing junction; bss, backsplice site; circRNA, circular RNA from exon backsplicing; mRNA, messenger RNA; ss, splice site.

Figure 2

The life cycle of circRNAs in cells. (a) Backsplicing is coupled to Pol II transcription. Fast Pol II elongation can lead to enhanced efficiency of backsplicing catalyzed by the spliceosome. (b) Regulation of backsplicing. (❶) Long introns usually flank circRNA-forming exons. (❷) ICSs (red arrows) in flanking introns of circRNA-forming exons can facilitate backsplicing by forming transient intronic RNA duplexes that bring the splice sites into close proximity. (❸) trans-factors bind to intronic ICSs or other sequences in the pre-mRNA to directly bridge distal splice sites to promote backsplicing. (❹) cis- and trans-factors synergistically modulate exon backsplicing. (c) Competition of RNA pairing across introns or within an intron modulates splicing and backsplicing reactions. In this example, when repeats flanking exons 2 and 3 base pair to one another, (middle) a two-exon circRNA is produced. In contrast, when repeats flanking exon 2 base pair to one another, (right) a single exon circRNA is produced. (d) circRNA export is modulated by different proteins and occurs in a length-dependent manner. (e) circRNAs are stable in the cytoplasm and sometimes can accumulate to high levels. (f) Pathways of circRNA degradation in the cytoplasm. (g) Secretion of circRNAs from cells in exosomes. Abbreviations: BP, branch point; bss, backsplicing site; circRNA, circular RNA; ICS, intronic complementary sequence; m⁶A, N6-methyladenosine; miRNA, microRNA; Pol II, RNA polymerase II; pre-mRNA, precursor messenger RNA; RNase, ribonuclease.

Figure 3

Altered cellular and physiological expression patterns of circular RNAs (circRNAs). Up- and downregulated circRNA expression has been observed in different biological and physiological contexts, including embryo and neuronal development, spermatogenesis, immune responses, and tumorigenesis. Although some specific circRNAs have been shown to participate in these different processes, the regulatory roles of most of these altered circRNAs await further investigation.

Figure 4

A schematic of different modes of action of circRNAs currently annotated in cells. In addition to the interplay between backsplicing and canonical splicing for the same splice site selection that can ultimately affect pre-mRNA splicing, mature circRNAs themselves can act as decoys or sponges for miRNAs and proteins, and as RNA scaffolds. A small subset of circRNAs can serve as templates for protein translation. Abbreviations: circRNA, circular RNA; miRNA, microRNA.