Cytoplasmic DIS3 degrades circular RNAs independently of the exosome

Circular RNAs (circRNAs) are frequently generated during splicing. Their physiological roles, however, remain largely elusive. Some specific circRNAs function as microRNA (miRNA) or RNA binding protein (RBP) sponges or encode for proteins and are actively translated. Due to the lack of accessible ends, circRNAs are resistant to exonucleolytic cleavage and thus are more stable than linear RNAs. Nevertheless, circRNAs are most likely turned over by cellular degradation pathways. Here, we used a biochemical purification strategy to find nucleases that could degrade circRNAs. We identified the exosome-associated nuclease DIS3, which contains an endonucleolytic PIN domain and an exonuclease domain. We show that DIS3 can degrade circRNAs independently of the exosome in vitro. Although moderately, RNA-seq reveals that selected circRNAs are stabilized when DIS3 is reduced in vivo. Based on our data, we propose a model, in which cytoplasmic DIS3 contributes to the turnover of circRNAs in human cells. Overall design: To study the physiological relevance of DIS3 in circRNAs degradation in vivo, we performed knockout experiments using the CRISPR/Cas9 system. First, we stably integrated an inducible tagged DIS3 variant (Flag-HA DIS3) into Flp-In T-REx 293 cell line using the Flp-In system. We then knocked out the endogenous gene DIS3 using CRISPR/Cas9. We refer to these cells as knock down cells since some DIS3 expression is still present in the uninduced state. To identify circRNAs candidates that could be regulated by DIS3, we performed gene expression analysis from two different cell lines (DIS3 KD clone 8 and control, two replicates each).