A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation

Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life 1 , yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis , including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 ( SEP3 ) gene increase abundance of the cognate exon-skipped AS variant ( SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA:DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS 2 – 4 . This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation. While circular RNAs (circRNAs) have been identified in all eukaryotic kingdoms of life, their functions have remained elusive. Now, a study shows that circRNAs can promote alternative splicing of their cognate mRNA, thus driving homeotic phenotypes.