Biogenesis and Functions of Circular RNAs Come into Focus

Abstract

Many eukaryotic protein-coding genes are able to generate exonic circular RNAs. Most of these covalently linked transcripts are expressed at low levels, but some accumulate to higher levels than their associated linear mRNAs. We highlight several methodologies that have been developed in recent years to identify and characterize these transcripts, and which have revealed an increasingly detailed view of how circular RNAs can be generated and function. It is now clear that modulation of circular RNA levels can result in a variety of molecular and physiological phenotypes, including effects on the nervous system, innate immunity, microRNAs, and many disease-relevant pathways.

Keywords: backsplicing; circRNA; innate immunity; microRNA; pre-mRNA splicing; translation.

Figure 1.. Eukaryotic pre-mRNAs can be spliced to generate a linear or circular RNA.

(Top) When splice sites (ss) are joined in a linear order by the pre-mRNA splicing machinery, a canonical linear mRNA is generated that is also capped and polyadenylated. (Bottom) Alternatively, backsplicing can join a 5′ ss to an upstream 3′ ss, resulting in production of a circular RNA whose ends are covalently linked by a 3′-5′ phosphodiester bond and can function via a number of distinct molecular mechanisms.

Figure 2.. Enrichment of circular RNAs using RNase R.

(A) To simultaneously deplete linear RNAs and enrich for circular RNAs, the 3′-5′ exonuclease RNase R is commonly used. In standard protocols (left), total RNA is treated with RNase R in a K⁺-containing buffer, but a number of linear RNAs fail to be digested by this protocol. We thus recently developed an improved protocol (right) in which total RNA is first treated with E. coli poly(A) polymerase (E-PAP) followed by incubation with RNase R in a Li⁺-containing buffer. (B-F) HeLa total RNA was subjected to the indicated treatments and RNA-seq libraries were then prepared [data from 24]. Sequencing read coverage is shown for (B) LGALS3, (C) HIPK3, (D) HIST2H2AB, a member of the histone H2A family, (E) RPPH1, the RNA component of RNase P, and (F) PPP1R8. The HIPK3 circular RNA is designated in purple and the G-quadruplex in the 3′ UTR of the PPP1R8 mRNA is indicated in green. The standard RNase R protocol using KCl fails to digest HIST2H2AB mRNA and the RPPH1 noncoding RNA, and only digests the PPP1R8 mRNA up until the G-quadruplex structure. In contrast, the improved protocol allows efficient digestion of these RNAs coupled to increased enrichment of the HIPK3 circular RNA.

Figure 3.. Circular RNA biogenesis can be controlled by intronic repeat sequences, exon skipping, and the levels of core spliceosome components.

(A) Backsplicing can be induced when inverted repeat sequences (orange arrows) in the flanking introns base pair to one another, thereby bringing the intervening splice sites into close proximity. (B) There are often multiple intronic repeat sequences (labeled as i, ii, iii, and iv) present in a pre-mRNA, which allows distinct mature RNAs to be produced depending on which repeats base pair to one another. When repeats in different introns base pair, backsplicing can be induced to yield the indicated circular RNAs. In contrast, a linear mRNA is produced if repeats in a single intron (ii and iii) base pair to one another. (C) Exon skipping events result in a mature linear mRNA and an intron lariat, which can be re-spliced to generate a stable circular RNA and a double lariat structure that is then debranched and degraded. (D) In pre-mRNAs with long introns, spliceosome components, including the U1 and U2 snRNPs, first assemble on exons to form exon junction complexes. These cross-exon interactions must then be replaced with cross-intron interactions to allow generation of a mature linear mRNA (left). When spliceosome activity is limiting and the exon is sufficiently long, the full spliceosome tends to instead assemble across an exon, resulting in backsplicing and generation of a circular RNA (right).

Figure 4.. Circular RNAs are exported to the cytoplasm in a length-dependent manner.

The export of many linear mRNAs from the nucleus requires binding of a set of proteins, including UAP56 (Hel25E in Drosophila), which enable recruitment of the NXT1/NXF1 heterodimeric export receptor and trafficking through the nuclear pore complex. RNAi screening has now revealed roles for Drosophila Hel25E and its human homologs UAP56/URH49 in nuclear export of circular RNAs. The length of the mature circular RNA dictates which of these factors is required.

Figure 5.. Circular RNAs can modulate microRNA activity or be translated.

(A) The CDR1as/ciRS-7 circular RNA contains many sites complementary to the seed region of the miR-7 microRNA along with a single, near perfect target site for miR-671. Binding of miR-671 triggers endonucleolytic cleavage of the circular RNA by Argonaute-2 (Ago2), which likely leads to recruitment of exonucleases and full degradation of CDR1as/ciRS-7. This allows release of the miR-7 transcripts that can then bind and down-regulate the expression of specific mRNAs. (B) Linear and circular RNAs generated from a protein-coding gene locus may be translated to yield distinct protein products. Here, the linear mRNA can be translated in a cap-dependent manner to yield a full-length protein, while the circular RNA is translated in a cap-independent manner to generate a truncated protein that terminates at a stop codon encountered after the backsplicing junction.

Figure 6.. Circular RNAs and the immune system.

(Top) The biogenesis of circular RNAs can be modulated by factors that stabilize (NF90/NF110) or de-stabilize (DHX9 or ADAR) base pairing between intronic repeat sequences. After backsplicing, circular RNAs are exported to the cytoplasm where they are recognized as self and can bind a number of protein factors, including immune regulators such as PKR and NF90/NF110. (Bottom Left) It has been proposed that foreign circular RNAs may trigger an immune response in a RIG-I dependent manner, although this may be due to contaminants (e.g. short triphosphorylated RNAs) in the circular RNA preparation. (Bottom Right) Furthermore, viral infection can result in activation of RNase L, resulting in cleavage of many endogenous transcripts, including circular RNAs. Recent work suggests that digestion of the circular RNAs releases key immune regulators, including PKR, which can then bind the pathogenic dsRNAs and become activated to inhibit viral infection.