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

Abstract

Ribozymes are catalytic RNAs present in modern genomes but regarded as remnants of a prebiotic RNA world. The paradigmatic hammerhead ribozyme (HHR) is a small self-cleaving motif widespread from bacterial to human genomes. Here, we report that most of the classical type I HHRs frequently found in the genomes of animals are contained within a novel family of non-autonomous non-LTR retrotransposons of the retrozyme class. These retroelements are expressed as abundant linear and circular RNAs of ∼170-400 nt in different animal tissues. Bioinformatic and in vitro analyses indicate an efficient self-cleavage of the HHRs harboured in most invertebrate retrozymes, whereas HHRs in retrozymes of vertebrates, such as the axolotl and other amphibians, require to act as dimeric motifs to reach higher self-cleavage rates. Ligation assays of retrozyme RNAs with a protein ligase versus HHR self-ligation indicate that, most likely, tRNA ligases and not the ribozymes are involved in the step of RNA circularization. Altogether, these results confirm the existence of a new and conserved pathway in animals and, likely, eukaryotes in general, for the efficient biosynthesis of RNA circles through small ribozymes, which opens the door for the development of new tools in the emerging field of study of circRNAs.

Figure 1.

(A) Schematic representation of the three possible hammerhead ribozyme (HHR) topologies (Types I, II and III). The most frequent nucleotides in the catalytic core (black boxes) and in the loop–loop interactions are indicated. Dotted and continuous lines refer to non-canonical and Watson-Crick base pairs. Black arrows indicate the self-cleavage site. The three HHR types have been reported in prokaryotic/phages genomes, whereas metazoan and plant genomes mostly show type I and III motifs, respectively. (B) Schematic representation of a typical LTR retrozyme from plant genomes containing type III HHRs. The average size encompassed by the HHRs (∼700 bp) and GC content (>55%) are indicated. (C) Canonical type I HHRs in metazoan genomes can be found within short tandem repeats (∼300 bp monomer size, from dimers to large multimers) of low GC content (<45%), which can be regarded as a new family of non-LTR retrozymes.

Figure 2.

(A) A genomic retrozyme copy from the coral Acropora millepora was cloned 3′ to a T7 promoter. Schematic representation of the clone and the expected sizes of the fragments after HHR self-cleavage (SC) are indicated. Below, autoradiography of a run-off transcription of this construct at different times run in a denaturing gel. The volumes of the samples at each time were adjusted to show an equivalent signal. RNA self-cleavage of the retrozyme reaches almost completion after 5 minutes. (B) Northern blot analysis of RNA extracts from two different samples of the coral A. millepora (Am_RNAs_1 and Am_RNAs_2) and a quantified RNA from the in vitro transcribed A. millepora retrozyme (Am Rtzm) under native (left), partially denaturing (center, 89 mM TBE buffer) and strongly denaturing conditions (right, 23 mM TBE buffer). The approximate amounts of RNAs for every sample are indicated in each gel line. The approximate position of the bands corresponding to the DNA 100–1000 bp (native blot) and RNA LR RiboRuler (denaturing blots) markers, and of the linear and circular RNAs are indicated. Ethidium bromide-staining of the 5S rRNAs are shown at the bottom as loading controls. (C) A minimum free energy secondary structure prediction of a circRNA derived from a representative A. millepora retrozyme. A typical 20 nt insertion found in many genomic retrozymes is indicated. The sequence of the HHR motif is shown in purple letters. The expected 3D structure of the HHR is shown in an inset. Non-canonical tertiary interactions between loops 1 and 2 are shown in red (61). (D) Minimum free energy secondary structure prediction for some of the retrozyme sequences detected in the genomes of the jellyfish Morbakka virulenta (left, 233 nt) and Nemopilema nomurai (right, 227 nt).

Figure 3.

(A) Schematic representation of a genomic copy of the mussel Mytilus galloprovincialis retrozyme cloned 3′ to a T7 promoter. The expected sizes of the fragments after HHR self-cleavage (SC) are indicated. Below, autoradiography of a run-off transcription of this construct at different times run in a denaturing gel. The volumes of the samples were adjusted to show an equivalent signal. (B) Northern blot analysis under denaturing conditions of RNA extracts from different tissues of the mussel M. galloprovincialis (gonads, hepatopancreas, mantle and gills). The linear (∼350–400 nt) and circular forms of the retrozyme RNAs (apparent size of ∼600 nt) are indicated. (C) Northern blot analysis under denaturing conditions of RNA extracts from gonads of different M. galloprovincialis mussels (two females, #1F and #2F, and two males, #1M and #2M) showing the circular and, to a lesser extent, linear retrozyme RNAs. Quantified amounts of retrozyme transcripts (Mg Rtzm), size markers and loading controls in panels (B) and (C) are shown as in Figure 2. (D) Minimum free energy secondary structure predicted for a cloned retrozyme circRNA from mussel gonads, and its corresponding HHR structure (inset in purple).

Figure 4.

(A) The two families of canonical type I HHRs found in the genome of the Mexican axolotl Ambystoma mexicanum (HHR_Amex1, left and HHR_Amex2, right). The number of copies found of each motif is indicated. (B) Autoradiographies of run-off transcriptions from two genomic retrozyme copies (Rtz331 carrying HHR_Amex1 at the left, and Rtz353 carrying HHR_Amex2 at the right) of the axolotl at different times. (C) Autoradiography of 1 h run-off transcriptions of the corresponding dimeric constructs (DRtz331 and DRtz353) obtained from the monomers shown in panel B. The resulting RNA bands and their sizes after single or double HHR self-cleavage are indicated. (D) Northern blot analyses of RNA extracts from different axolotl tissues (muscle, liver and male gonads) that were run under native (left membrane) and denaturing conditions (right membrane). Monomeric, multimeric and circular RNAs with their sizes are indicated. The approximate amounts of RNAs for every sample (10 μg) are indicated in each gel line. Size markers and loading controls are shown as in Figure 2. (E) Minimum free energy secondary structure predictions for axolotl circRNAs of 331 and 353 nt derived from genomic retrozymes.

Figure 5.

(A) Denaturing PAGE showing the transcription products of the DRtz_Ax353 construct (the scheme of the construct is shown at the bottom of the panel). The linear monomeric RNA Ax353 nt resulting from double self-cleavage and the full uncleaved transcript (Ax740) were excised and purified from the gel. (B) Denaturing gel showing the resulting products of the incubation of the purified Ax353 RNA with RtcB ligase. The major product of the ligation reaction putatively corresponds to the circular molecule (circRNA Ax353, apparent size of ∼500 nt). Residual products corresponding to, most likely, the linear dimer (706 nt) and its circular form (apparent size >> 1000 nt) are also indicated. CircRNA Ax353 was excised and purified from the gel. (C) About 200 ng of the purified circRNA Ax353 were run in a native gel together with the linear form of Ax353 and the uncleaved dimeric (740 nt) transcript purified from the gel shown in panel A. (D) Denaturing PAGE running the resulting products of RNase R incubations at different times of the circular Ax353, linear Ax353 and the uncleaved dimeric (740 nt) transcripts. After 15 min incubation, only the circRNA is found to be resistant to degradation. (E) Traces of a purified linear Ax353 RNA monomer resulting from a radiolabeled transcription of the DRtz_Ax353 construct were run in a denaturing gel, either directly (lane 1), after 1 h incubation with RtcB ligase in its corresponding buffer (lane 2), in the RtcB buffer without the ligase (lane 3), and in 50 mM Mg²⁺ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the Ax_350 monomer) and lane 4 (∼3% self-circularization). The presence of minor fractions of linear and circular dimeric RNAs (Ax706, ∼700 nt and an apparent size >>1000 nt, respectively) are also indicated.