A 360° view of circular RNAs: From biogenesis to functions

Abstract

The first circular RNA (circRNA) was identified more than 40 years ago, but it was only recently appreciated that circRNAs are common outputs of many eukaryotic protein-coding genes. Some circRNAs accumulate to higher levels than their associated linear mRNAs, especially in the nervous system, and have clear regulatory functions that result in organismal phenotypes. The pre-mRNA splicing machinery generates circRNAs via backsplicing reactions, which are often facilitated by intronic repeat sequences that base pair to one another and bring the intervening splice sites into close proximity. When spliceosomal components are limiting, circRNAs can become the preferred gene output, and backsplicing reactions are further controlled by exon skipping events and the combinatorial action of RNA binding proteins. This allows circRNAs to be expressed in a tissue- and stage-specific manner. Once generated, circRNAs are highly stable transcripts that often accumulate in the cytoplasm. The functions of most circRNAs remain unknown, but some can regulate the activities of microRNAs or be translated to produce proteins. Circular RNAs can further interface with the immune system as well as control gene expression events in the nucleus, including alternative splicing decisions. Circular RNAs thus represent a large class of RNA molecules that are tightly regulated, and it is becoming increasingly clear that they likely impact many biological processes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Evolution and Genomics > Computational Analyses of RNA.

Keywords: CDR1as; Laccase2; R-loop; alternative splicing; backsplicing; circRNA; exon skipping; immune surveillance; microRNA; noncoding RNA; pre-mRNA splicing; scrambled exons; spliceosome; translation; transposable elements.

Figure 1. Pre-mRNAs can be alternatively spliced to generate a linear mRNA or a circular RNA

If the pre-mRNA splice sites (ss) are joined in a linear order, a mature linear mRNA is generated that is also capped and polyadenylated (top). Alternatively, the pre-mRNA splicing machinery can backsplice and join a 5′ ss to an upstream splice acceptor (3′ ss), resulting in production of a circular RNA whose ends are covalently linked by a 3′–5′ phosphodiester bond (bottom).

Figure 2. Base pairing between intronic repeats facilitates many backsplicing events

(A) Exon/intron structure of the D. melanogaster laccase2 gene, highlighting exon 2 that can generate a 490-nt circular RNA. A pair of DNAREP1_DM transposons (red arrows) flanks exon 2. Base pairing between these intronic sequences brings the intervening splice sites into close proximity, facilitating backsplicing. (B) When multiple intronic repeat elements (red arrows) are present in a pre-mRNA, distinct mature RNAs can be generated depending on which repeats base pair to another (denoted by gray arcs). (a–c) Base pairing between repeats in different introns results in backsplicing and production of a circular RNA that contains a single exon (a and c) or multiple exons (b). (d) In contrast, a linear mRNA is produced when repeats in a single intron base pair to one another.

Figure 3. Exon skipping can be directly coupled to circular RNA production

At the S. pombe mrps16 gene, exon skipping results in production of a mature linear mRNA as well as an intron lariat containing exon 2. This lariat is re-spliced to generate a mature circular RNA as well as a double lariat structure that is subsequently debranched and degraded.

Figure 4. Model for how early steps in spliceosome assembly determine whether canonical splicing or backsplicing occurs

In pre-mRNAs with long introns, spliceosomal components first assemble across each exon. U1 snRNP (red) and U2 snRNP (green) recognize the 5′ and 3′ splice sites, respectively, with additional factors, such as SR proteins, serving to stabilize the exon definition complex. These cross-exon interactions must then be replaced with cross-intron interactions in order generate a mature linear mRNA (left). However, when spliceosome activity is limiting (e.g., due to depletion of core spliceosomal components), recent work suggests that cross-exon interactions may not be easily disrupted and the full spliceosome instead assembles across an exon, resulting in backsplicing.

Figure 5. Circular RNAs can have a variety of molecular functions

(A) Some circular RNAs, including CDR1as and Sry, are able to bind many copies of particular microRNAs (in complex with an Argonaute (AGO) protein). In response to a specific stimulus (e.g. cleavage of CDR1as by miR-671), the circular RNA can potentially release these microRNA transcripts, which then bind to mRNAs to down-regulate their expression. (B) Recent work suggests that linear mRNAs and circular RNAs from the same gene locus can be translated to generate distinct protein products. In the example shown, the mature linear mRNA (top) and the circular RNA (bottom) use the same AUG start codon (green), but the circular RNA generates a truncated protein that terminates at a stop codon encountered after the backsplicing junction. (C) Some copies of the Arabidopsis SEP3 circular RNA are retained in the nucleus, where they form R-loops (an RNA:DNA hybrid) at the endogenous SEP3 gene loci to impact alternative splicing of its host gene.