Emerging Functions of Circular RNAs

Abstract

Many thousands of Circular RNAs (circRNAs) have recently been identified in metazoan genomes by transcriptome-wide sequencing. Most circRNAs are generated by back-splicing events from exons of protein-coding genes. A great deal of progress has recently been made in understanding the genome-wide expression patterns, biogenesis, and regulation of circRNAs. To date, however, few functions of circRNAs have been identified. CircRNAs are preferentially expressed in neural tissues and some are found at synapses, suggesting possible functions in the nervous system. Several circRNAs have been shown to function as microRNA "sponges" to counteract microRNA mediated repression of mRNA. New functions for circRNAs are arising, including protein sequestration, transcriptional regulation, and potential functions in cancer. Here, we highlight the recent progress made in understanding the biogenesis and regulation of circRNAs, discuss newly uncovered circRNA functions, and explain the methodological approaches that could reveal more exciting and unexpected roles for these RNAs.

Keywords: EIciRNAs; RNA-seq; aging; alternative splicing; ceRNA; ciRNAs; circRNAs; microRNA; non-coding RNA; transcriptome.

Figure 1

CircRNA biogenesis. In-order splicing occurs to produce linear mRNAs with the introns removed. CircRNAs are most commonly produced from back-splicing events, usually from exons of protein-coding genes. Shown is an example of a single exon circRNA. Note that circRNAs can contain multiple exons and the intervening intronic sequence in multi-exon circRNAs is usually removed.

Figure 2

Quantification of circRNA expression. RNA-seq reads (orange) can align directly to the genome or be split, indicating a spliced junction. Back-spliced split reads that do not align in linear fashion are consistent with detection of a circRNA. PCR primers (red) can be used to detect linear RNAs (inward facing orientation), or circRNAs (outward facing orientation) from cDNA preparations.

Figure 3

Methods to manipulate circRNA expression. A) Expression constructs are available that include intronic elements flanking the laccase2 or ZKSCAN1 circRNAs [51]. These express circularized over linear exons almost exclusively. Overexpression and knockdown approaches to study circRNA function must be designed to specifically alter circRNAs, and not linear RNAs from the same gene. B) siRNA strategies to knockdown circRNAs must have the siRNA designed to specifically target the back-spliced junction. C) Deletion of one pair of a flanking intronic reverse complementary matches can disrupt circularization without impeding linear mRNA [11].

Figure 4

CircRNA localization and molecular functions. A) EIciRNAs retain introns and seem to interact with U1 snRNA and Pol II, acting as regulators of their parental genes’ expression [31]. B) Circular intronic RNAs (ciRNAs) have a characteristic 7nt GU-rich element near the 5’ splice site and an 11nt C-rich element close to the branch point, and interact with phosphorylated Pol II regulating parental gene mRNA expression [8]. C) Not all the circRNAs generated from intronic lariats are in the nucleus. In Xenopus tropicalis oocytes circular lariats are abundant in the cytoplasm [71]. Lariat species can interact with proteins such as TDP-43 [48]. D) f-circRNAs arise from chromosomal translocated regions. They stimulate proliferation and contribute to cellular transformation and tumorigenesis [70]. E) Recent reports refer to the interaction of circ-Foxo3 with several proteins to regulate senescence [45] and cell cycle progression [41]. F) Multiple circRNAs have been shown to have activity as microRNA sponges, most notably CDR1-as [5,12].