Short intronic repeat sequences facilitate circular RNA production

Abstract

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from proteincoding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery ''backsplices'' and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (~30-to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 39 end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.

Diverse alternative back-splicing and alternative splicing landscape of circular RNAs

“…Since the complementary sequences in flanking introns can promote exon circularization (Liang and Wilusz 2014;Zhang et al 2014), we evaluated whether the existence of multiple pairs of intronic complementary sequences would have an effect on alternative back-splice site selection. Theoretically, an across-intron RNA pair flanking the proximal back-splice sites would lead to proximal back-splice site selection (left panels of Fig.…”

Circular RNA participates in the carcinogenesis and the malignant behavior of cancer

“…Generally, as a result of canonical linear mRNA splicing, circRNAs are regarded as co-transcriptional products, and this process shows a strong dependence on short intronic repeat sequences. 13 In eukaryotic cells, precursor mRNAs (premRNAs), comprising exons as well as introns, are first synthesized in the nucleus in the pattern of a radial arrangement, mostly by RNA polymerase II from DNA. 14 Pre-mRNAs then move to the cytoplasm, where they are precisely dissected into pieces containing one complete isolated intron or exon, which is catalyzed by spliceosomal machinery or by groups I and II ribozymes (only responsible for the production of intronic circRNAs).…”

Circular RNAs remain peculiarly unclear in biogenesis and function

“…Genome-wide bioinformatic analysis actually confirmed the association of these features to circRNA biogenesis [5][6][7]. The exonic sequences within the circRNAs might also be important in some cases [6,7]. At current stage, these associations are linked to the biogenesis of most but not all circRNAs, and are not connected with any actual molecular mechanism.…”

“…Biogenesis of certain circRNAs had long been linked to the relatively long introns and the complementary repeat sequences flanking the region of circularization [5][6][7]. Genome-wide bioinformatic analysis actually confirmed the association of these features to circRNA biogenesis [5][6][7]. The exonic sequences within the circRNAs might also be important in some cases [6,7].…”

“…At current stage, these associations are linked to the biogenesis of most but not all circRNAs, and are not connected with any actual molecular mechanism. There are also disputes about whether circRNAs are generated co-transcriptionally or post-transcriptionally [5,6]. A recent publication showed that the RNA-binding protein quaking I 5 (QKI5) might participate in the circularization [8], although we have to keep in mind with several points before considering QKI5 as an essential player in circRNA biogenesis.…”