Circular RNA biogenesis can proceed through an exon-containing lariat precursor

Abstract

Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression programs, yet its function remains largely unknown. The presumed biogenesis of these RNAs involves a non-canonical ‘backsplicing’ event. Recent studies in mammalian cell culture posit that backsplicing is facilitated by inverted repeats flanking the circularized exon(s). Although such sequence elements are common in mammals, they are rare in lower eukaryotes, making current models insufficient to describe circularization. Through systematic splice site mutagenesis and the identification of splicing intermediates, we show that circular RNA in Schizosaccharomyces pombe is generated through an exon-containing lariat precursor. Furthermore, we have performed high-throughput and comprehensive mutagenesis of a circle-forming exon, which enabled us to discover a systematic effect of exon length on RNA circularization. Our results uncover a mechanism for circular RNA biogenesis that may account for circularization in genes that lack noticeable flanking intronic secondary structure.DOI: http://dx.doi.org/10.7554/eLife.07540.001

Circular RNAs: Unexpected outputs of many protein-coding genes

“…The mature circular RNA consisting of mrps16 exon 2 then accumulates in cells, while the double lariat from the re-splicing event and the skipped linear mRNA are both rapidly degraded. 41,88 Interestingly, there are other examples of exon skipping events in S. pombe, 89 yet none of these genes appear to generate circular RNAs. 41 This argues that the production of an exoncontaining lariat is not sufficient for circular RNA biogenesis, and the mechanisms by which cells determine when to re-splice to yield a circular RNA remain unclear.…”

“…41,88 Interestingly, there are other examples of exon skipping events in S. pombe, 89 yet none of these genes appear to generate circular RNAs. 41 This argues that the production of an exoncontaining lariat is not sufficient for circular RNA biogenesis, and the mechanisms by which cells determine when to re-splice to yield a circular RNA remain unclear. The length of the skipped exon appears to be one key determinant, 41 but other factors such as RNA secondary structures and the speed of lariat debranching are also likely to be involved.…”

“…Indeed, the S. pombe mrps16 pre-mRNA lacks repetitive elements, and instead takes advantage of exon skipping to generate a circular RNA. 41 By splicing exon 1 to exon 3, an intron lariat containing exon 2 is released, which is subsequently re-spliced to covalently join the beginning and end of exon 2 together (Fig. 5A).…”

“…37 At certain genes, circular RNAs are produced independently of repetitive elements and the backsplicing reaction is instead coupled to an exon skipping event. 41 In addition, a separate class of circular intronic RNAs are made not by backsplicing but by a failure to debranch intron lariats. 42 It is thus becoming increasingly clear that circular RNAs can be generated via multiple, tightly regulated strategies.…”

“…40 The efficiency of backsplicing is further affected by exon length, with longer exons (>300-nt) able to form circular RNAs better than short exons. 37,41,65 The genomic repeat landscape varies significantly across species, 75 which causes different populations of circular RNAs to be expressed. As pointed out above, the Sry circular RNA is expressed in mouse but not humans.…”