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bioRxiv · 10.1101/2024.02.26.582126

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

Son, S.·Jeong, H.·Lee, G. G.·Park, J. H.·Yoo, S.
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Abstract

scarecrow (scro) encodes a fly homolog of mammalian Nkx2.1 that is vital for early fly development as well as for optic lobe development. Interestingly, scro was reported to produce a circular RNA (circRNA). In this study, we identified 12 different scro circRNAs, which are either mono- or multi-exonic forms. The most abundant forms are circE2 carrying the second exon only and bi-exonic circE3-E4. Levels of circE2 show an age-dependent increase in adult heads, supporting a general trend of high accumulation of circRNAs in aged fly brains. Aligning sequences of introns flanking exons uncovered 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 circE2 production in cell-based assays; furthermore, deletion of the region including potential ICS components in the intron-2 reduced in vivo production of circE2 and circE3-E4 by 80%, indicating them to be essential for the biogenesis of these isoforms. Besides the ICS, the intron regions immediately abutting exons seemed to be responsible for a basal level of circRNA formation. Moreover, the replacement of scro-ICS with those derived from laccase2 was comparably effective in scro-circRNA production, buttressing the importance of the hairpin-loop structure formed by ICS for the biogenesis of circRNA. Lastly, overexpressed scro affected outcomes of both linear and circular RNAs from the endogenous scro locus, suggesting that Scro plays a direct or indirect role in regulating expression levels of either or both forms.

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Son, S., Jeong, H., Lee, G. G., Park, J. H., Yoo, S.. 2024-02-29. Biogenesis of circular RNAs in vitro and in vivo from the Drosophila Nk2.1/scarecrow gene. https://doi.org/10.1101/2024.02.26.582126

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