Vv-circSIZ1 mediated by pre-mRNA processing machinery contributes to salt tolerance.

CircRNAs exist widely in plants, but the regulatory mechanisms for the biogenesis and function of plant circRNAs remain largely unknown. Using extensive mutagenesis of expression plasmids and genetic transformation methods, we analyzed the biogenesis and anti-salt functions of a new grape circRNA Vv-circSIZ1. We identified Vv-circSIZ1 that is mainly expressed in the cytoplasm of xylem. CircSIZ1 is species-specific, and genomic circSIZ1-forming region of seven tested species could be backspliced in Nicotiana benthamiana, but not in Arabidopsis. The retention length of Vv-circSIZ1 flanking introns was significantly positively correlated with its generation efficiency. The precise splicing of Vv-circSIZ1 does not depend on its mature exon sequence or internal intron sequences, but on the AG/GT splicing signal sites and branch site of the flanking introns. The spliceosome activity was inversely proportional to the expression level of Vv-circSIZ1. Furthermore, RNA-binding proteins can regulate the expression of Vv-circSIZ1. The overexpression of Vv-circSIZ1 improved salt tolerance of grape and N. benthamiana. Additionally, Vv-circSIZ1 could relieve the repressive effect of VvmiR3631 on its target VvVHAc1. Vv-circSIZ1 also promoted transcription of its parental gene. Overall, these results broaden our understanding of circRNAs in plants.