Biogenesis of circular RNAs in vitro and in vivo from the Drosophila Nk2.1/scarecrow gene.

The scarecrow (scro) gene encodes a fly homolog of mammalian Nkx2.1 which is vital for early fly development as well as for optic lobe development. Previously, scro was reported to produce a circular RNA (circRNA) in addition to traditional mRNAs. In this study, we report 12 different scro circRNAs, which are either mono- or multi-exonic forms. The most abundant ones are circScro(2) carrying the second exon (E2) only and bi-exonic circScro(3,4) having both the third (E3) and fourth exon (E4). Levels of circScro(2) show an age-dependent increase in adult heads, supporting a general trend of high accumulation of circRNAs in aged fly brains. In silico analysis of the introns flanking circRNA exons predicts two pairs of intronic complementary sequences (ICSs); one pair residing in introns 1 and 2 and the other in introns 2 and 4. The first pair was demonstrated to be essential for the circScro(2) production in cell-based assays; furthermore, deletion of the region including ICS components in the intron-2 reduces in vivo production of both circScro(2) and circScro(3,4) by 80%, indicating them to be essential for the biogenesis of the two circRNAs. Besides the ICS, the intron regions immediately abutting exons seem to be responsible for a basal level of circRNA formation. Moreover, ectopic ICS derived from the laccase2 locus is comparably effective in circScro production, buttressing the importance of the hairpin-loop structure formed by ICS for the biogenesis of circRNA. Lastly, overexpressed scro alters outcomes of both linear and circular RNAs from the endogenous scro locus, suggesting that Scro plays a direct or indirect role in regulating the expression levels of either or both forms.