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.
Highlights
Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression; these circular isoforms are ubiquitously expressed in humans and a number of highly diverged eukaryotic organisms and can exceed the level of the cognate mRNA (Salzman et al, 2012; Memczak et al, 2013; Salzman et al, 2013; Wang et al, 2014)
We found that mrps16 lacks any apparent base pairing between the regions flanking the circularized exon (Figure 1—figure supplements 1, 2); we hypothesized that the biogenesis of this circular RNA might proceed through a lariat precursor generated by exon skipping
As canonical lariats are of low abundance and difficult to detect in wild-type fission yeast, we used a strain of yeast lacking the debranching enzyme, which catalyzes the hydrolysis of the branch point 2′–5′ linkage, the first step of lariat decay (Ruskin and Green, 1985)
Summary
Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression; these circular isoforms are ubiquitously expressed in humans and a number of highly diverged eukaryotic organisms and can exceed the level of the cognate mRNA (Salzman et al, 2012; Memczak et al, 2013; Salzman et al, 2013; Wang et al, 2014). One of the only known examples of an abundant circular RNA was discovered in the mouse sex-determining gene, SRY (Capel et al, 1993). This exon is flanked by long (∼15 kb) inverted sequence whose complementarity is required for circularization (Dubin et al, 1995). Modeled after work on SRY, recent studies in mammalian cell culture have found similar results in human genes (Liang and Wilusz, 2014; Zhang et al, 2014b), and additional genome-wide computational sequence analysis suggests this complementary sequence-mediated mechanism may be widespread and associated with Alu elements (Jeck et al, 2013; Zhang et al, 2014b; Ivanov et al, 2015)
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