Retrozymes are a unique family of non-autonomous retrotransposons with hammerhead ribozymes that propagate in plants through circular RNAs

Abstract

BackgroundCatalytic RNAs, or ribozymes, are regarded as fossils of a prebiotic RNA world that have remained in the genomes of modern organisms. The simplest ribozymes are the small self-cleaving RNAs, like the hammerhead ribozyme, which have been historically considered biological oddities restricted to some RNA pathogens. Recent data, however, indicate that small self-cleaving ribozymes are widespread in genomes, although their functions are still unknown.ResultsWe reveal that hammerhead ribozyme sequences in plant genomes form part of a new family of small non-autonomous retrotransposons with hammerhead ribozymes, referred to as retrozymes. These elements contain two long terminal repeats of approximately 350 bp, each harbouring a hammerhead ribozyme that delimitates a variable region of 600–1000 bp with no coding capacity. Retrozymes are actively transcribed, which gives rise to heterogeneous linear and circular RNAs that accumulate differentially depending on the tissue or developmental stage of the plant. Genomic and transcriptomic retrozyme sequences are highly heterogeneous and share almost no sequence homology among species except the hammerhead ribozyme motif and two small conserved domains typical of Ty3-gypsy long terminal repeat retrotransposons. Moreover, we detected the presence of RNAs of both retrozyme polarities, which suggests events of independent RNA-RNA rolling-circle replication and evolution, similarly to that of infectious circular RNAs like viroids and viral satellite RNAs.ConclusionsOur work reveals that circular RNAs with hammerhead ribozymes are frequently occurring molecules in plant and, most likely, metazoan transcriptomes, which explains the ubiquity of these genomic ribozymes and suggests a feasible source for the emergence of circular RNA plant pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1002-4) contains supplementary material, which is available to authorized users.

Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes

“…Moreover, with the exception of the predicted secondary structure of the mussel circRNA retrozyme, RNA circles of most metazoan retrozymes are predicted to allow the HHR to freely adopt the catalytically competent structure of the ribozyme (Figure 2C, 2D, 4E and supplementary Figure S6). This feature, observed for most retrozyme circRNAs of metazoans, contrasts with what is observed in plant LTRretrozymes and viroidal circRNAs with HHRs, which show the ribozyme motif paired with a complementary sequence that should avoid its self-cleavage (26). Consequently, most circRNAs derived from metazoan retrozymes may follow in vivo different mechanisms to control their HHR activity and circRNA self-cleavage.…”

“…Natural type I HHRs keeping tertiary interactions between loops 1 and 2 have been previously reported to achieve a 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop interaction, suggesting that HHR may be almost as efficient at ligation as it is at cleavage. We previously described that RNAs encoded by plant LTR retrozymes with type III HHRs are efficiently circularized in vitro by a chloroplast tRNA ligase (26), similarly to the RNA circularization reported for HHR viroids (42). To get a deeper insight into the circularization mechanism of the RNAs expressed by metazoan retrozymes, linear monomers obtained in vitro from the A. mexicanum DRtzm_350 construct ( Figure 4C) were assayed for either self-or tRNA ligase-mediated circularization.…”

“…Moreover, we also show that repeats with HHRs in the genomes of invertebrates from different phyla are also expressed and efficiently self-processed to result in monomeric linear and circular RNAs. Altogether, these data allow us to conclude that short tandem DNA repeats with HHRs in metazoans, plants (26) and, likely, eukaryotes in general, are a peculiar group of mobile genetic elements that are expressed as linear and circular RNAs, probably in most cell types. Moreover, the differences observed in the ratio of linear to circular molecules among diverse species or RNA extracts (see above), allow us to suggest that the levels of circRNAs in vivo could be much higher than their linear counterparts.…”

“…Prehybridization, hybridization (at 68°C in 50% formamide) and washing was done following the instructions of the manufacturer (GE Healthcare). The riboprobes were obtained by rub-off transcriptions of linearized pBlueScript plasmids containing the full retrozymes in the presence of DIG-UTP (Roche Diagnostic GmbH) (26).…”

“…Rare minimal variants of the type I HHR ( Figure 1A) have been reported in retrotransposons of the Penelope-like (PLEs) (23) and Terminon (24) families, whereas a few copies of highly conserved HHRs in amniotes seem to play a role in mRNA biogenesis (25). More recently, type III HHRs detected in several flowering plants have been found to be involved in the processing of a novel family of non-autonomous LTR retrotransposons, the so-called retrozymes for retroelement with hammerhead ribozymes ( Figure 1B), which spread through circRNA transposition intermediates of 600-800 nt (26).…”