The functional circular RNA, ciRS-7 (CDR1as), is biosynthesized using back-splicing promoted by inverted mammalian-wide MIRs but not primate-specific Alus

CircRNAs are abundant stable noncoding RNAs with covalently closed circular structure. The first characterized circRNA, ciRS-7 (CDR1as), acts as a 9sponge9, or decoy, that regulates the stability and activity of miR-7 micro RNA. Here we demonstrate the definitive biosynthesis pathway of this important ciRS-7. By anti-sense oligoribonucleotide targeting of the relevant splice sites, we found that ciRS-7 is directly generated by back-splicing between the flanking splice sites rather than by subsequence splicing of the excised lariat RNA containing skipped exon and introns. We further validated this observation by CRISPR/Cas9-mediated genomic deletion of the relevant splice sites. Back-splicing circularization events are promoted by the flanking intronic complementary sequences, and only inverted repeats of Alu, primate-specific SINE, was previously identified. ciRS-7 lacks flanking available Alu elements, however, we identified conserved flanking inverted elements of MIR (mammalian-wide interspersed repeat). Splicing reporter assays in stably transfected HEK293 cells with mini-genes, with and without the inverted MIR elements, demonstrated they are required to generate ciRS-7. Using bioinformatics searches followed by mini-gene reporter tests, we identified several other MIR-dependent circRNAs. The MIR-dependent circRNAs are thus a new category of widely conserved mammalian circRNAs.