Detecting of chloroplast circular RNAs in Arabidopsis thaliana

Circular RNAs (circRNAs) are covalently closed single-strand RNA molecules identified in eukaryotes. Here we report 10 sequences of chloroplast genome (cpDNA) encoded circRNAs (cp-circRNA) from 3-day-old Arabidopsis thaliana seedlings via next-generation sequencing. Their parental sequences were referred to as exonic, intronic, and intergenic gene sequences. Due to the very low level of cp-circRNA expression, we attempted to use a digital polymerase chain reaction technique to validate their existence. Four of these circRNAs were confirmed: ath_circ_476, ath_circ_477, ath_circ_478, and ath_circ_480. Their expression levels in etiolated and de-etiolated seedlings were quantified using digital PCR. Of the four, the exonic circRNA ath_circ_476 was highly expressed in etiolated seedlings. Its parental gene, cytochrome C assembly protein (ycf5), has expression trends similar to ath_circ_476. Given that ycf5 is a single-exon gene, we propose that the circularization of ath_circ_476 occurs via an unknown process rather than via RNA-splicing-based mechanisms. Therefore, we provided a reliable method for detecting cp-circRNAs in low copies and implied an unknown circularisation mechanism of RNA in chloroplasts.