Biosynthesis of Circular RNA ciRS-7/CDR1as Is Mediated by Mammalian-Wide Interspersed Repeats, MIRs

Summary Circular RNAs (circRNAs) are stable non-coding RNAs with a closed circular structure. One of the best studied circRNAs is ciRS-7 (CDR1as) that acts as a regulator of the microRNA miR-7; however, its biosynthesis pathway has remained an enigma. Here we delineate the biosynthesis pathway of ciRS-7. The back-splicing events that form circRNAs are often facilitated by flanking inverted repeats of the primate-specific Alu elements. The ciRS-7 gene lacks these elements but, instead, we identified a set of flanking inverted elements belonging to the mammalian-wide interspersed repeat (MIR) family. Splicing reporter assays in HEK293 cells demonstrated that these inverted MIRs are required to generate ciRS-7 through back-splicing, and CRISPR/Cas9-mediated deletions confirmed the requirement of the endogenous MIR elements in SH-SY5Y cells. Using bioinformatic searches, we identified several other MIR-dependent circRNAs and we confirmed them experimentally. We propose that MIR-mediated RNA circularization is used to generate a subset of mammalian circRNAs.