Regulation, biogenesis and function of circDOCK1(2,27), an abundant epithelial-specific circRNA

We describe the regulation, biogenesis and function of circDOCK1(2,27), a large, abundant EMT-regulated circular RNA whose formation is dependent on the epithelial-specific splicing regulator ESRP1. CircDOCK1(2,27) 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. HITS-CLIP analysis along with 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. Overall design: HITS-CLIP was performed on endogenous ESRP1 from 3 biological replicates of wt HMLE cells using an ESRP1 specific antibody and on 2 biological replicates each of N- and C-terminally 3XFLAG-tagged ESRP1 expressed in HMLE-i-ESRP1_FLAG cells after induction with 1 µg/ml doxycycline for 72 hours, using anti-FLAG antibody.