Circular RNAs (circRNAs) are covalently closed circular RNA molecules that are now recognised to be widely expressed in most if not all tissues in higher eukaryotes and to be long-lived in cells due to their circular nature (lacking 5’ and 3’ ends). It is widely assumed they can carry out regulatory functions within cells and can be involved in pathologies such as cancer, although to date only a few circRNAs have had definitve functions described. We report the regulation, biogenesis and function of circDOCK1(2-27), a large, unusually abundant circular RNA whose formation depends on the epithelial-specific splicing regulator ESRP1, making it strongly regulated during Epithelial to Mesenchymal Transition (EMT). Its synthesis in epithelial cells represses cell motility, both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA.
Formation of the circRNA involves a back splice to ligate exons that bridge 24 intervening exons and introns and are over 150 kb apart. Given this means intron 1 which precedes the back splice must remain unspliced for nearly an hour to allow transcription to proceed to the back-splicing exon 27, there has clearly been strong evolutionary pressure to engender formation of this circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 modifies the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.