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.