Metazoan tRNA introns generate stable circular RNAs in vivo

Abstract

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an agedependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science.

Circular RNAs: Unexpected outputs of many protein-coding genes

“…It should be noted that these plasmids do not efficiently circularize small exons (300 -nt), but an alternative strategy that involves processing from tRNA introns can be used to generate small circles. 93,94 Methods, such as splint ligation or the use of selfcleaving ribozymes, can also be used to generate circular RNAs in vitro (For a review see ref. 95 ).…”

Engineering and expressing circular RNAs via tRNA splicing

“…3 It has since become clear that circRNA biogenesis in many eukaryotes proceeds through several diverse and evolutionarily conserved biochemical pathways, [4][5][6][7] resulting in an abundant class of RNA molecules with a host of proposed functions. 8,9 CircRNAs have been shown to be remarkably stable in living cells, [10][11][12] potentially due to their unique structure and lack of free termini on which the exonucleolytic RNA turnover machinery relies. Along with being generally abundant, 2 circRNAs can be differentially expressed in a tissue-dependent manner and can have expression profiles that differ from their parental mRNA, 13 suggesting regulated expression and diversity of function.…”

“…46,47 In RtcB knockdown experiments in Drosophila, tricRNA levels decreased significantly, indicating that tricRNA ligation is indeed dependent upon ligase activity of RtcB. 10 In at least one case, ligation of mRNA splice products by tRNA processing enzymes can also result in intron circularization in vitro. 48 The unfolded protein response in metazoans and yeast causes stress signaling, triggering an unconventional splicing of XBP1 (HAC1 in yeast) mRNA.…”

“…One tricRNA (tric31905) even displays compensatory mutation and preservation of base-pairing motifs, indicating a conserved secondary structure. 10 Additionally, small RNA sequencing analysis indicates that this circular RNA is further processed into microRNA-sized fragments. 10 It is currently unclear whether these small RNAs have a function in the cell or are merely degradation intermediates.…”

“…10 Additionally, small RNA sequencing analysis indicates that this circular RNA is further processed into microRNA-sized fragments. 10 It is currently unclear whether these small RNAs have a function in the cell or are merely degradation intermediates. Inspection of sequence and secondary structure of other Drosophilid introns does not reveal common sequence motifs or elements, suggesting that processing of tric31905 is not necessarily applicable to all tricRNAs.…”