Detecting of chloroplast circular RNAs in <i>Arabidopsis thaliana</i>

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 <i>Arabidopsis thaliana</i> 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: <i>ath_circ_476, ath_circ_477, ath_circ_478</i>, and <i>ath_circ_480</i>. Their expression levels in etiolated and de-etiolated seedlings were quantified using digital PCR. Of the four, the exonic circRNA <i>ath_circ_476</i> was highly expressed in etiolated seedlings. Its parental gene, <i>cytochrome C assembly protein</i> (<i>ycf5</i>), has expression trends similar to <i>ath_circ_476</i>. Given that <i>ycf5</i> is a single-exon gene, we propose that the circularization of <i>ath_circ_476</i> 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.