Thousands of eukaryotic protein-coding genes are noncanonically spliced to generate circular RNAs. Because they have covalently linked ends, circular RNAs are resistant to degradation by exonucleases and some accumulate to higher levels than their associated linear mRNAs. The functions of most circular RNAs are still unknown, but recent work has revealed key insights into how the pre-mRNA splicing machinery catalyzes backsplicing. Exons that circularize are often flanked by intronic repeat sequences that are complementary to one another, and backsplicing is triggered when these repeats base pair and bring the intervening splice sites into close proximity. Here, we describe how this knowledge has been translated into a simple plasmid-based method for ectopically expressing circular RNAs in eukaryotic cells. The sequence of interest is cloned into an artificial exon that is flanked by complementary intronic repeats. The plasmid is then transfected into cells, transcription is induced, and the cellular splicing machinery generates the desired circular RNA. Total RNA is isolated and the efficiency/specificity of circular RNA biogenesis is validated by Northern blot analysis. Beyond allowing overexpression of natural circular RNAs to define their functions, this approach can be used to produce designer RNA circles that are translated or bind specific cellular factors, such as microRNAs or proteins.
Keywords: Alternative splicing; Backsplicing; Biogenesis; Circularization; Drosophila; Laccase2; Noncoding RNA; Northern blot; Transfection; circRNA.