Insights into circular RNA biology

Abstract

Circular RNAs (circRNAs) are a novel class of non-coding RNA characterized by a covalently closed-loop structure generated through a special type of alternative splicing termed backsplicing. CircRNAs are emerging as a heterogeneous class of molecules involved in modulating gene expression by regulation of transcription, protein and miRNA functions. CircRNA expression is cell type and tissue specific and can be largely independent of the expression level of the linear host gene, indicating that regulation of expression might be an important aspect with regard to control of circRNA function. In this review, a brief introduction to the characteristics that define a circRNA will be given followed by a discussion of putative biogenesis pathways and modulators of circRNA expression as well as of the stage at which circRNA formation takes place. A brief summary of circRNA functions will also be provided and lastly, an outlook with a focus on unanswered questions regarding circRNA biology will be included.

Keywords: Alternative splicing; backsplicing; biogenesis; circRNA; circular RNA; inverted repeats.

Figure 1.

Putative pathways for circRNA biogenesis. A constitutive linear RNA splicing pattern is shown for comparison purposes (A). CircRNA formation may proceed through a direct backsplicing pathway in which circularization can be driven by intron pairing (B, left upper panel) or RNA-binding protein pairing (B, right upper panel) that brings the appropriate splice signals within proximity of each other. A linear RNA containing the skipped exons may be formed as shown in the gray box or may be degraded depending on the kinetics of splicing vs. debranching and exonuclease mediated degradation. Alternatively, circRNAs may be produced through a lariat driven pathway (C) where exon skipping removes the exons to be backspliced from the primary transcript and promotes circularization because the splice signals of the circRNA exon to be are juxtaposed in the lariat structure. The circRNA may be formed concurrently with the linear RNA as shown in the gray box or may not be formed depending on the kinetics of intra-lariat splicing vs. debranching and exonuclease mediated degradation. In all instances, the intronic lariat products are likely rapidly degraded (gray, hatched boxes).

Figure 2.

Modulators of circRNA biogenesis. (A) Competitive base-pairing between multiple pairs of inverted repeats can modulate circRNA formation as only base-pairing between IRs across an exon (inter-intronic pairing, up) promotes circRNA formation whereas intra-intronic base-pairing does not (down). The 2 mutually exclusive base-pairing patterns are indicated with arrows in the middle panel. (B) Additionally, when dependent on intron-pairing, the efficiency with which circRNAs are formed may be modulated by the activity of the RNA editing enzyme ADAR 1 that acts as an inhibitor of circRNA formation by mediating the conversion of adenosines (A) to inosines (I) thereby destabilizing the base-pairing that promotes backsplicing. (C) Splicing factors, both tissue specific and general, can affect circRNA expression and may either inhibit or promote the expression of a circRNA. (D) CircRNA formation is affected by the rate of transcription, with a high transcriptional elongation rate being favorable for circRNA expression.

Figure 3.

Functions of circular RNAs. In the cytoplasm, circRNAs can interact with miRNA-Ago2 complexes to inhibit miRNA action on linear targets (A). Additionally, circRNAs can interact with RNA-binding proteins (RBPs) as can linear RNAs and may either facilitate interaction between RBPs to inactivate/activate them (B) or sequester these to prevent them from functioning (C). In the nucleus, (D) circRNAs with retained introns can promote transcription of their parental gene by interacting with RNA polymerase II (Pol II) and U1 snRNP at the promoter of the gene. (E) During splicing, the elements facilitating backsplicing and linear splicing compete with each other. As a result of this competition either a linear RNA or an alternatively spliced linear RNA and a circRNA is generated. Hence, the backsplicing pattern may alter the expression of the linear gene product.