Circular RNAs (circRNAs) are stable non-coding RNAs with a closed circular structure. One of the best studied circRNAs is ciRS-7 (CDR1as), which acts as a regulator of the microRNA miR-7; however, its biosynthetic pathway has remained an enigma. Here we delineate the biosynthetic 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 confirmed them experimentally. We propose that MIR-mediated RNA circularization is used to generate a subset of mammalian circRNAs.
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