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

Abstract

The scarecrow (scro) gene encodes a fly homolog of mammalian Nkx2.1, which is vital for early fly development and for optic lobe development. Previously, scro was reported to produce a circular RNA in addition to traditional mRNAs. In this study, we report 12 different scro circular RNAs, which are either mono or multiexonic 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 circular RNAs in aged fly brains. In silico analysis of the introns flanking circular RNA exons predicts 2 pairs of intronic complementary sequences; 1 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 intronic complementary sequence 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 2 circular RNAs. Besides the intronic complementary sequence, the intron regions immediately abutting exons seem to be responsible for a basal level of circular RNA formation. Moreover, ectopic intronic complementary sequence derived from the laccase2 locus is comparably effective in circScro production, buttressing the importance of the hairpin loop structure formed by intronic complementary sequence for the biogenesis of circular RNA. Last, 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.