Molecular roles and function of circular RNAs in eukaryotic cells

Abstract

Protein-coding and noncoding genes in eukaryotes are typically expressed as linear messenger RNAs, with exons arranged colinearly to their genomic order. Recent advances in sequencing and in mapping RNA reads to reference genomes have revealed that thousands of genes express also covalently closed circular RNAs. Many of these circRNAs are stable and contain exons, but are not translated into proteins. Here, we review the emerging understanding that both, circRNAs produced by co- and posttranscriptional head-to-tail "backsplicing" of a downstream splice donor to a more upstream splice acceptor, as well as circRNAs generated from intronic lariats during colinear splicing, may exhibit physiologically relevant regulatory functions in eukaryotes. We describe how circRNAs impact gene expression of their host gene locus by affecting transcriptional initiation and elongation or splicing, and how they partake in controlling the function of other molecules, for example by interacting with microRNAs and proteins. We conclude with an outlook how circRNA dysregulation affects disease, and how the stability of circRNAs might be exploited in biomedical applications.

Keywords: Alu elements; Cancer; Cardiovascular disease; Chromatin; Exosomes; RNA polymerase II; Spliceosome; microRNA.

Fig. 1

Molecular formation of spliceosome-dependent circular RNAs in eukaryotes. a–c Three major spliceosomal mechanisms lead to the formation of circular RNA in eukaryotes. Reaction substrates are on the left, reaction products on the right. a Conventional colinear splicing (top) causes the excision of an intron from a multi-exon gene, resulting in a 2′ → 5′-linked lariat that is usually degraded (bottom). Lariats can be processed to a perfectly circular 2′ → 5′-linked RNA circle that becomes stable (ciRNA). b Formation of 3′–5′-linked circRNAs by cotranscriptional backsplicing. This reaction occurs in nascent pre-mRNA and can be assisted by backfolding of reverse complementary repeats in flanking introns as well as by dimerization of RNA-binding proteins that bind to flanking introns (yellow). When a single exon is involved, the end of this single exon fuses to its other end. As a by-product, a branched linear mRNA is produced that is branched because still containing a 2′ → 5′-linked intron (bottom). c Formation of 3′-5′-linked circRNAs by posttranscriptional backsplicing. In a first step, linear alternative splicing leads to excision of the exon(s)-containing lariat (left), which can become substrate for intralariat backsplicing (middle). As for cotranscriptional backsplicing, a more upstream located branchpoint (A₁) serves as nucleophil to fuse a formerly downstream exon (dark green) to a formerly upstream exon (light green). This results in an intron- and exon(s) containing circular RNA (EIciRNA). Subsequently, from such an EIciRNA, the intron can be spliced out by a second linear splicing reaction (right), resulting in an exon-only 3′–5′-linked circRNA. CircRNA end products produced by co- or posttranscriptional backsplicing are molecularly identical. Introns (grey lines); Position of the linear splice donor junction (orange); backsplice junctions (red triangle); chemical transesterification reactions and their direction are indicated with orange lines. The arrowheads represent the direction of the nucleophilic attacks. Flanking sequences (dashed lines)

Fig. 2

Cellular functions of circRNAs in eukaryotes. a, b Nuclear functions of circRNAs. a EIciRNAs and ciRNAs stimulate RNAP II-dependent transcriptional initiation at the transcriptional start site of a protein-coding gene in the nucleus. Potential roles in elongation are not depicted. b Top: stimulation of parental exon-skipping by DNA-binding circRNAs that form a DNA:RNA hybrid (R-loop) that can impair RNAP II. Bottom: backsplicing in the pre-mRNA antagonizes the production (and/or stability) of the colinearly spliced linear host mRNA. c–e Cytoplasmic functions of circRNAs. c Interaction of circRNAs with proteins and inhibition of their normal functions. Two unrelated cases are shown, binding of circANRIL to PES1 for inhibiting the PeBoW complex during rRNA processing (left) and the sponging of the HuR protein by circPABPN1 (right). d circRNAs can also sponge microRNAs and thereby inhibit the translational blockage in mRNAs targeted by these microRNAs (whether binding is occuring only in the cytoplasm is not known). e Translation of ORFs encoded on circRNAs by 5′Cap-independent initiation using either IRES or, hypothetically, m6A methylation (not shown). See text for details