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.

环状RNA（circRNA）是具有封闭环状结构的稳定的非编码RNA。研究最深入的circRNA之一是ciRS-7（CDR1as），它可作为microRNA miR-7的调节子。然而，其生物合成途径仍然是一个谜。在这里，我们描述了ciRS-7的生物合成途径。灵长类特异性Alu的反向重复序列侧翼通常促进形成circRNA的反向剪接事件元素。ciRS-7基因缺少这些元件，但是，我们发现了一组侧翼倒置元件，它们属于哺乳动物范围的散布重复序列（MIR）家族。在HEK293细胞中剪接报告基因分析表明，这些反向MIR是通过反向剪接生成ciRS-7所必需的，而CRISPR / Cas9介导的缺失证实了SH-SY5Y细胞中内源MIR元素的需求。使用生物信息学搜索，我们鉴定了其他几种依赖MIR的circRNA，并通过实验进行了确认。我们建议使用MIR介导的RNA环化来生成哺乳动物circRNA的子集。