Regulation of circRNA biogenesis

Abstract

Unlike linear RNAs terminated with 5' caps and 3' tails, circular RNAs are characterized by covalently closed loop structures with neither 5' to 3' polarity nor polyadenylated tail. This intrinsic characteristic has led to the general under-estimation of the existence of circular RNAs in previous polyadenylated transcriptome analyses. With the advent of specific biochemical and computational approaches, a large number of circular RNAs from back-spliced exons (circRNAs) have been identified in various cell lines and across different species. Recent studies have uncovered that back-splicing requires canonical spliceosomal machinery and can be facilitated by both complementary sequences and specific protein factors. In this review, we highlight our current understanding of the regulation of circRNA biogenesis, including both the competition between splicing and back-splicing and the previously under-appreciated alternative circularization.

Keywords: alternative circularization; back-splicing; circular RNA; circularization; complementary sequence; splicing.

Figure 1.

Back-splicing for circRNAs. Pre-mRNA can go through either high efficient canonical splicing to generate a linear RNA with exon inclusion (top) or low efficient back-splicing to produce both a circular RNA and an alternatively spliced linear RNA with exon exclusion (bottom). Canonical splicing signals and spliceosomal machinery are required for back-spliced exon circularization (blue bars). The coupling between splicing and back-splicing ( - ) are discussed in text. ss, splice site.

Figure 2.

Two possible models for circRNA formation. (A) The “exon skipping” or “lariat intermediate” model for circRNA formation. The processing starts with canonical splicing for a linear RNA with skipped exons and a long intron lariat containing these skipped exons (blue bars), which is then further back-spliced to form a circRNA. (B) The “direct back-splicing” model for circRNA formation. The precessing starts with back-splicing for a circRNA together with an exon-intron(s)-exon intermediate, which can be further processed to produce a linear RNA with skipped exons or to be potentially degraded. ss, splice site. BP, branchpoint.

Figure 3.

The competition of RNA pairing for splicing or back-splicing. (A) (B) Both cis-elements (A) and trans-factors (B) can affect back-splicing efficiency by taking the downstream splice donor and upstream acceptor sites close together. (C) The competition model of RNA pairing. Top, RNA pairing formed across flanking introns promotes back-splicing, leading to the formation of a circRNA and a linear RNA with exon exclusion. Bottom, the RNA pairing formed within one individual intron promotes the canonical splicing, resulting in a linear RNA with exon inclusion, but no back-splicing. Red arrows, complementary sequences. ss, splice site. BP, branchpoint.

Figure 4.

Possible mechanisms for alternative circularization. (A) Multiple circRNAs can be processed from a single gene locus with different numbers of exons regulated by the competition of RNA pairing across different introns (red arcs). (B) Multiple circRNAs can be produced from a single gene locus with the internal intron included or excluded with unknown mechanisms. (C) Multiple circular RNAs from either exons or introns can be generated from a single gene locus through distinct circular RNA formation pathways. See text for details. Red arrows, complementary sequences. ss, splice site. BP, branchpoint.