Circular RNAs: Unexpected outputs of many protein-coding genes

Abstract

Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs, some of which accumulate to higher levels than their associated linear mRNA. Recent work has revealed widespread mechanisms that dictate whether the spliceosome generates a linear or circular RNA. For most genes, circular RNA biogenesis via backsplicing is far less efficient than canonical splicing, but circular RNAs can accumulate due to their long half-lives. Backsplicing is often initiated when complementary sequences from different introns base pair and bring the intervening splice sites close together. This process is further regulated by the combinatorial action of RNA binding proteins, which allow circular RNAs to be expressed in unique patterns. Some genes do not require complementary sequences to generate RNA circles and instead take advantage of exon skipping events. It is still unclear what most mature circular RNAs do, but future investigations into their functions will be facilitated by recently described methods to modulate circular RNA levels.

Keywords: Alternative splicing; RNA stability; backsplicing; biogenesis; ciRNA; circRNA; circularization; exon skipping; noncoding RNA; pre-mRNA splicing.

Figure 1.

A pre-mRNA can be spliced to generate a linear or circular RNA. When the pre-mRNA splice sites (ss) are joined in the canonical order, a linear mRNA is generated that is subsequently polyadenylated and exported to the cytoplasm for translation (top). Alternatively, backsplicing can join a 5′ ss to an upstream 3′ ss to generate a circular RNA whose ends are covalently linked by a 3′-5′ phosphodiester bond (bottom). This competition between canonical splicing and backsplicing helps determine which mature RNAs are generated from a gene.

Figure 2.

Base pairing between intronic complementary sequences facilitates circular RNA biogenesis. (A) Backsplicing is commonly induced when inverted repeats (red arrows) in the flanking introns base pair to one another. This brings the intervening splice sites (ss) into close proximity, facilitating catalysis (left). Extensive mutagenesis of the human ZKSCAN1 locus revealed minimal introns that are sufficient for the generation of a circular RNA from exons 2 and 3. Besides the splice sites, ∼40-nt complementary repeats (red) are needed for backsplicing. (B) Exon/intron structure of the human ZKSCAN1 locus, highlighting the region that contains exons 2 and 3. Complementary AluS elements (red) immediately flank these exons, and facilitate backsplicing from the end of exon 3 to the beginning of exon 2 (purple). (C) Exon/intron structure of the D. melanogaster Laccase2 locus, highlighting a region that contains exon 2. A pair of DNAREP1_DM family transposons are close to the circularizing exon and facilitate backsplicing.

Figure 3.

Alternative circularization is driven by competition for base pairing between intronic repeats. Multiple complementary repeat elements (red arrows) are often present in a pre-mRNA. Depending on which repeats base pair to one another (denoted by blue arcs), different pre-mRNA splicing patterns are triggered. (a) If the repeats flanking exon 2 base pair to one another, backsplicing is induced to generate a circular RNA comprised of exon 2. (b) A larger circular RNA can be generated if the repeats flanking exons 2 and 3 base pair. This allows backsplicing from the end of exon 3 to the beginning of exon 2. Canonical splicing removes the intron between exons 2 and 3 to yield the mature circular RNA. (c) Alternatively, base pairing between repeats in a single intron leads to canonical splicing and the generation of a linear mRNA.

Figure 4.

Chromosomal translocations can lead to aberrant circular RNA expression. Rearrangements between nonhomologous regions can result in the joining of 2 previously separate genes (colored orange and blue). Regulatory sequences, such as intronic Alu repeats (red arrows), that flank the translocation breakpoint become juxtaposed when the fusion gene is transcribed. This can lead to the formation of aberrant circular RNAs when complementary repeat sequences base pair to one another. Recent work has demonstrated that these fusion circular RNAs then act with the fusion mRNA/protein to drive cancer development..

Figure 5.

Generation of circular RNAs via exon skipping or a failure to debranch introns. (A) Circular RNA biogenesis can proceed through an exon-containing lariat. Via an alternative splicing event, exon 2 can first be skipped to generate a linear mRNA consisting of exons 1 and 3 as well as an intron lariat intermediate. The lariat can then be re-spliced to generate a circular RNA comprised of exon 2 along with a double lariat, which is subsequently debranched and degraded. (B) Although most intron lariats are rapidly debranched, some are only trimmed to their branch point. This generates a circular intronic RNA that is covalently joined by a 2′-5′ phosphodiester bond between the 5′ end of the intron and the branch point adenosine.

Figure 6.

Plasmids for ectopic expression of circular RNAs in cells. To facilitate the expression of nearly any circular RNA in mammalian cells, we optimized the ZKSCAN1 and Laccase2 flanking introns and replaced the endogenous exon with a multicloning site exon (blue). These plasmids have been successfully used to express circular RNAs of various sizes, including ones with an IRES that enables translation.