Global identification of mRNA-interacting circular RNAs by CLiPPR-Seq. CLiPPR-Seq

By interacting with proteins and nucleic acids, circular RNAs (circRNAs) are proposed to regulate physiological processes. Although the functional relevance of circRNA interaction with microRNAs and proteins has been studied extensively, circRNA interactions with the protein coding mRNAs in intact cells remain largely unknown. Here, we sought to identify the transcriptome-wide interaction of circRNAs with mRNA transcripts. By employing AMT-mediated proximity ligation of circRNA-RNA duplexes followed by circRNA enrichment and deep sequencing, we report a novel Cross-Linking Poly-A Pulldown RNase R Sequencing (CLiPPR-seq) technology for the global profiling of intermolecular circRNA–mRNA interactions. Using this method, we identified hundreds of mRNA-interacting circRNAs that were enriched in the mRNA samples of mouse βTC6 cells. In addition, CLiPP-seq without RNase R treatment on mouse C2C12 and human HeLa cells also discovered hundreds of mRNA-associated circRNAs, indicating that circRNA-mRNA interactions are evolutionarily conserved cellular mechanisms that need in-depth study. Furthermore, computational analysis of the CLiPPR-Seq identified circRNAs with mRNAs determined their potential complementarity sequences. Pulldown of circRNAs and poly-A RNAs followed by RT-qPCR analysis confirmed the direct interaction of circRNAs with target mRNAs. Silencing of mRNA-interacting circRNAs led to the altered expression of target mRNAs in βTC6 cells suggesting the role of direct interaction of circRNAs with mRNAs in gene expression regulation. This technique not only identified unexpected global interactions of hundreds of circRNAs with mRNAs but also opened up a new regulatory axis for gene regulation by circRNAs. CLiPPR-seq thus represents a novel method for illuminating the myriad of uncharacterized circRNA-mRNAs hybrids that may regulate gene expression by base-pairing interactions.