A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing

Recent improvements in high-throughput sequencing technologies underscore the pervasiveness of circular RNA (circRNA) expression in animal cells. CircRNAs are distinct from their linear counterparts because they lack the 5' caps and 3' tails that typically help determine the cellular fate of a transcript. However, due to the lack of free ends, circRNAs are impervious to exonucleases and thus can evade normal RNA turnover mechanisms. Most circRNAs are derived from protein-coding pre-mRNAs, via a mechanism called "back-splicing." Existing methods of circRNA expression thus typically involve genes that have been engineered to contain sequence elements that promote back-splicing. We recently uncovered an anciently conserved mechanism of RNA circularization in metazoans that involves splicing of tRNA introns. This splicing mechanism is completely independent from that of pre-mRNAs. In this chapter, we detail an orthogonal method that involves splicing of intron-containing tRNAs in order to produce circRNAs in vivo. We utilize fluorescence-based RNA reporters to characterize the expression, localization, and stability of these so-called tRNA intronic circular RNAs. Because tRNA biogenesis is essential for all cellular life, this method provides a means to express ultrastable, high-copy, circRNA effectors in a wide variety of metazoan cell types.