Molecular determinants of metazoan tricRNA biogenesis

ABSTRACTMature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, our laboratory discovered a new class of metazoan circular RNAs formed by ligation of excised tRNA introns; we termed these molecules tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace the native introns of twoDrosophilatRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identifiedcis-acting elements required for tricRNA formation in both human and fly cells. We observed that disrupting the conserved anticodon-intron base pair dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. Furthermore, we found that strengthening weak base pairs in the pre-tRNA also impairs tricRNA production. We also used the reporters to identifytrans-acting tricRNA processing factors. We found that several known tRNA processing factors, such as RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis. Depletion of these factors inhibits tRNA intron circularization. Furthermore, we observed that depletion of Clipper endonuclease results in increased tricRNA levels. In summary, our work characterizes the major players inDrosophilatricRNA biogenesis.