Circular RNAs (circRNAs) are an abundant class of non-coding RNAs with largely uncharacterized functions. CircRNAs are formed via backsplicing, whereby a downstream splice donor of an exon covalently bonds to an upstream splice acceptor, linking the 3' and 5' ends in a continuous circle. Hundreds of circRNAs increase in expression during aging, a phenomenon found across phyla, but their function in the aging process is mostly undiscovered. Previously, we identified two highly abundant circRNAs arising from exon 4 of
crh-1, an ortholog of human CREB1. Within
crh-1, two large reverse complementary motifs (RCMs), that can facilitate backsplicing, flank the circularizing exon of
crh-1 (exhibiting 83% base complementarity), giving evidence that these RCMs could drive circularization. Using CRISPR/Cas9, we deleted the downstream RCM sequence of
crh-1 to uncover its effect on age-accumulated circRNA formation, and impact on lifespan. In four independent
crh-1 circRNA (circ-
crh-1) CRISPR-generated alleles, RCM deletion resulted in complete loss of circ-
crh-1 expression in both young and old adults without disruption of linear
crh-1 expression or activated phospho-CRH-1 protein levels. Introduction of exon 4 from
crh-1 with flanking RCM sequences into circ-
crh-1 mutants restored the loss of circ-
crh-1. Interestingly, mutants of
adr-1, a double-stranded RNA-specific adenosine deaminase (ADAR), resulted in increased circ-
crh-1 expression levels in both young and old adults. These results suggest that RCMs likely drive circ-
crh-1 formation, which is depended on ADAR function. Remarkably, two independent RCM deletion alleles of circ-
crh-1 showed a small but significantly increased mean lifespan (~11% change) but decreased thermotolerance, suggesting these mutants may extend the period of frailty. Finally, we performed RNA sequencing on wild-type and circ-
crh-1 mutants and identified 17 significantly downregulated genes, which may contribute to the observed lifespan phenotype. Our findings represent the first circRNA-specific knockout in C. elegans, and this strategy for circRNA specific deletion can be applicable to study the functions of other circRNAs in C. elegans. Furthermore, these results highlight a new role for the
crh-1 circRNA in the regulation of lifespan and suggest that circRNAs may be a target for further understanding healthy aging.