- Open Access
Retrozymes are a unique family of non-autonomous retrotransposons with hammerhead ribozymes that propagate in plants through circular RNAs
© The Author(s). 2016
- Received: 25 January 2016
- Accepted: 7 June 2016
- Published: 23 June 2016
The Erratum to this article has been published in Genome Biology 2017 18:40
Catalytic 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.
We 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.
Our 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.
- Circular RNA
- LTR retrotransposons
- Satellite RNA
Genomic HHRs in plants are embedded in the LTRs of a new form of non-autonomous retroelement: retrozymes
The occurrence of HHRs has been previously reported in some plant genomes [9, 16, 17, 22]. In this work, we performed extensive bioinformatic searches for HHR motifs in plant genomes (see Methods section), which were mostly found in eudicots (42 species), notably among rosids, together with isolated examples in monocots, ferns and algae (Additional file 1). Type III HHRs were the most frequent motifs found, whereas only a few examples corresponded to type I HHRs. The number of ribozyme motifs detected per genome varied from the absence of any recognizable HHR in many species to more than 100 bona fide ribozymes in some others (Additional file 1).
As previously noticed , we confirmed that plant HHRs most frequently occur as isolated motifs, but also as close tandem repeats of two, three or, rarely, even four HHRs. Sequence repeats between HHR motifs were sized from 400 to 1000 bp, lacked any detectable protein-coding capacity and did not show clear sequence identity among different plant species. For each plant genome, isolated HHR motifs were usually found embedded within sequences of about 300–400 bp.
We deduced that transcription of genomic retrozymes followed by self-processing through HHR motifs would result in RNA transposition intermediates (hereafter, retrozyme RNAs) of about 600–1000 nt, depending on the plant species (Fig. 2b). These retrozyme RNAs lack the characteristic repeated regions (R, Fig. 2b and d) of the transposition intermediates of LTR-retrotransposons required for retrotranscription of the full retroelement [34–37].
Homology of retrozyme sequences was evident between plants within the same genus, despite some clear heterogeneity. However, sequence identity was almost absent between retrozymes of less related plant species, with the exception of two small boxes of about 25 nt, referred to as 3′ and 5′ boxes (Fig. 2c), and the HHR motifs (Fig. 2e). To identify potential autonomous LTR-retrotransposons responsible for retrozyme mobilization, these small conserved boxes were used as queries to search against autonomous retroelements. We found that the conserved 3′ box in retrozymes is almost identical to the LTR 3′ end and the primer binding site (PBS, tRNAMet) of the Ty3-gypsy retrotransposons (Fig. 2c). The retrozyme 5′ box, in turn, is also very similar to the polypurine tract (PPT) and the LTR 5′ end of the same family of retrotransposons  (Fig. 2c). No other sequence similarities were detected between retrozymes and Ty3-gypsy or any other family of retrotransposons.
Overall, these data indicate that retrozymes constitute a new group of non-autonomous LTR retroelements that may use the machinery of plant Ty3-gypsy retrotransposons for their genomic mobilization in the same way as other non-autonomous retrotransposons do.
Genomic retrozymes in the physic nut Jatropha curcas
Jatropha curcas or physic nut plant has a genome of about 410 Mb that has been recently sequenced [39–41]. In silico analysis of the available J. curcas sequences (70 % of the total genome) with RNAMotif revealed up to 30 bona fide type III HHRs, which showed some sequence heterogeneity for the same secondary structure (Additional file 3). Blast homology searches resulted in more than 70 HHR-like sequences (about 90 % identity for 90 nt), with 48 of these motifs occurring as tandem dimeric copies. These dimeric arrangements corresponded to 24 different retrozymes like the ones described above, whereas the rest of the HHRs motifs mostly corresponded to LTR sequences with no adjacent internal sequence (solo LTRs).
J. curcas retrozymes were flanked by TSDs of 4 bp with the consensus sequence WWRR (where W stands for A or T and R for a purine). LTRs were about 330 bp long, and the two HHR self-cleavage sites encompassed a non-coding region of 697–776 bp. At least four genomic retrozyme sequences were detected embedded within 18S rRNA gene sequences, whereas four others were found close (less than 2 kb) to LTR-retrotransposon sequences and the rest were detected within intergenic regions, in a similar way as described for other non-autonomous retroelements like TRIMs and SMARTs [32, 33].
To ascertain the activity of the HHRs contained in these retrozymes, in vitro transcription of a cloned J. curcas retrozyme fragment covering the 5′ LTR and the internal region was carried out. RNA self-cleaving activity was observed in the polarity containing the ribozyme motif (hereafter, the plus polarity of the retrozyme RNA) but not in the complementary (hereafter, the minus polarity) (Additional file 4). The amount of transcript processed by the HHR during transcription was 60 %, an efficiency similar to that reported for viroid and satellite HHRs .
Different genomic retrozymes are transcribed, processed and accumulated as circRNAs in J. curcas tissues
When the same RNA extracts from J. curcas leaves were analysed by denaturing PAGE followed by Northern blot hybridization, the bands in the 700 nt region appeared as a clear doublet (Fig. 3b), possibly corresponding to transcribed and HHR-processed RNAs from genomic retrozymes of different size. Interestingly, an additional doublet that did not appear in native gels was observed in denaturing gels with an apparent higher molecular weight (>3 kb) (Fig. 3b). This behaviour suggested the existence of a mixture of circular and linear RNAs that co-migrate in native gels, as has been observed during plant infection by pathogenic circRNAs such as viroids and virus satellites . In order to confirm this finding, RNA samples were analysed by double PAGE: RNA extracts were first run in a native gel, and then the region of 600–800 nt was cut out and placed on a denaturing gel followed by Northern blot hybridization (Additional file 5A). This experiment confirmed that circular and linear RNAs co-migrated in the native gel and only became separated under denaturing conditions.
The transcriptional activity of genomic retrozymes in different tissues and developmental stages of J. curcas was analysed by Northern blot (Fig. 3c and Additional file 5B). RNA extracts from young seedlings and leaves showed the presence of two circular and their corresponding two linear bands. In flowers and seeds extracts, however, only the faster migrating circular and linear RNAs were observed. This result indicates that retrozymes are differentially transcribed in different tissues of J. curcas.
Purified circRNAs from J. curcas seeds, young seedlings and leaves were retrotranscribed, cloned and sequenced (Additional file 6). Two types of variants were detected in leaves, one of 753 nt approximately and a second one of about 708 nt, which are in agreement with the size estimated for the two linear bands detected by denaturing Northern blots. Prediction of minimum free energy secondary structures for the cloned retrozyme RNAs revealed a highly structured architecture with an elevated degree of self-complementarity (about 70 % of nucleotides are paired), similar to that reported for circRNA pathogens with HHRs  (Fig. 3d and Additional file 7). In the predicted structures, the LTR region adopts a long and stable hairpin structure with most of the HHR motif paired with a highly complementary sequence that prevents the hammerhead fold from forming and, consequently, its self-cleavage.
The obtained cDNA clones showed sequence variability between them (Fig. 3d), but also with respect to any of the genomic copies detected in the databases (Additional file 6). Such a high sequence heterogeneity together with the similarities between retrozyme and plant pathogenic RNAs (circular and highly structured RNA molecules, small size and presence of HHRs) suggested the possibility that retrozyme circRNAs may follow RNA-RNA replication through a rolling-circle mechanism similar to that described for viroids and viral satellite RNAs . If that were the case, there would exist replication intermediates of negative polarity, either as circular or multimeric linear RNAs. To explore this possibility, we carried out RT-PCR experiments with adjacent primers outside of the LTRs to avoid the amplification of negative polarity RNAs resulting from transcription of genomic retrozyme copies (see Methods). Positive results were obtained with RNA extracts from J. curcas seeds (Additional file 8A). Northern blot analysis of RNA-enriched extracts from J. curcas tissues, however, did not reveal the presence of negative polarity RNAs of the retrozyme.
Retrozyme-derived circRNAs accumulate to high levels in strawberry
HHR motifs have been previously reported in diverse genomic sequences of strawberry (Fragaria x ananassa) . Thanks to the recently published genome of F. ananassa , our bioinformatic searches for HHRs revealed the presence of about 90 bona fide ribozyme motifs (Additional file 1) and up to 6 potential retrozyme RNAs of sizes ranging from 673 to 701 nt.
To ascertain the presence of a mixture of circular and linear retrozyme RNAs in the plant, we carefully checked the migration properties of strawberry RNAs under native and denaturing conditions in the presence of appropriate markers obtained by in vitro transcription of a full genomic retrozyme of F. ananassa (Additional file 5C). The retrozyme RNA resulting from double self-cleavage (679 nt) was purified and circularized in vitro using a Solanum melongena tRNA ligase as previously described  (Additional file 5D). Purified linear and circularized retrozyme RNAs were run in a native PAGE together with an RNA extract of F. ananassa. Northern blot hybridization revealed a single band of about 700 nt in the three cases (Additional file 5E). When these three samples were run in a denaturing PAGE, the RNA extract of F. ananassa showed the typical duplet of bands, whereas the linear retrozyme RNA migrated as a 679-nt band and the circular RNA run with an apparent size of 3 kb. Circularized and linear RNA markers perfectly matched the two bands detected in the RNA extract, which confirms the presence of a mixed population of circular and linear retrozyme RNAs in the plant.
Retrozymes in eucalyptus and citrus trees
In order to generalize the data obtained for the physic nut and strawberry, retrozymes of several woody plants were also investigated. Our bioinformatic analysis of the genomes of Eucalyptus camaldulensis  and E. grandis  detected more than 100 copies of bona fide HHRs in each of these genomes (Additional file 1). Dozens of ribozymes occurred in tandem copies of two, three and even four HHRs (Additional file 2D), suggesting large retrozyme RNAs of about 900–1050 bp.
We also analysed the genomic HHRs present in a number of citrus species. The genome of the sweet orange (Citrus sinensis, cv. Valencia)  only showed 10 bona fide HHRs, but up to 11 retrozyme-like elements able to encode putative retrozyme RNAs of 665–694 nt (Additional file 1). Many of the HHRs within putative retrozymes, however, showed punctual mutations that are expected to deeply affect their self-cleaving activity.
Finally, comparative analysis of C. clementina (13 retrozymes) and C. sinensis (11 retrozymes) genomes gave us another indication of the mobile nature of retrozymes. Alignment of two orthologous genomic regions of around 5 kb (Additional file 9) showed that while C. clementina contains a typical retrozyme element flanked by a duplicated CTAT sequence (TSDs), the equivalent region in C. sinensis genome did not show any retrozyme sequence, but showed a single CTAT sequence at this particular position.
Other putative retrozyme elements in plant and metazoan genomes
Our bioinformatic analyses revealed the presence of putative retrozymes in the genomes of more than 40 plant species (Additional file 1). Again, the retrozyme sequences in each plant genome showed a noticeable variability. Moreover, sequence identity of retrozymes from evolutionarily distant plant species was nearly absent, with the exception of the small conserved 5′ and 3′ boxes and the HHR motif (Fig. 2c and e). However, secondary structure prediction of minimal free energy for different plant retrozymes revealed a similar architecture, with a long arm corresponding to the LTR region harbouring the HHR in a blocked conformation, and a stable but more ramified structure corresponding to the rest of the RNA (Figs. 3d, 4c, 5b, 6c and Additional file 10).
In this work, we have described the retrozymes, a new and atypical group of non-autonomous retroelements with self-cleaving ribozymes. At the genomic level, retrozymes highly resemble other small non-autonomous LTR-retrotransposons of plants like TRIMs  and SMARTs  (Fig. 2 and Additional file 2), but differ in some peculiarities that make them a unique class of retroelements. As non-autonomous retrotransposons, retrozymes do not show protein-coding regions but, in contrast, do encode active self-cleaving HHR motifs in their LTRs. These ribozymes catalyze the self-processing of the retrotransposon RNA intermediate, which accumulates in vivo as circular and linear non-coding RNAs of the precise size encompassed by the HHRs.
Genomic retrozymes show a patchy distribution among plants, occurring numerously in different species, but being absent in some others. For example, the eggplant (Solanum melongena) contains more than 150 HHRs and 18 different retrozymes, whereas the genomes of related Solanum species, like tomato or potato, do not show a single example. An illuminating case is found in the cassava genomes . There are 34 full retrozymes in the wild variety (Manihot esculenta ssp. flabellifolia) but only 9 retrozyme copies in the genome of the domesticated one (M. esculenta Crantz), which suggests a negative selection pressure over these retroelements during plant domestication.
Another prominent feature of retrozymes is the high accumulation levels of heterogeneous circular and linear RNA intermediates in most of the plant tissues analysed. Retrotransposons are mostly quiescent in somatic cells, but activate under different stress conditions [38, 53, 54]. Our results indicate that, under natural conditions, some of the genomic retrozymes are either actively transcribed or weakly transcribed into highly stable covalently closed RNA circles. There is even the intriguing possibility that these circRNAs may undergo autonomous replication by plant polymerases as suggested by (1) the evident similarity of retrozymes with small circRNA pathogens with ribozymes (Additional file 6) , (2) the presence of multimeric retrozyme RNAs of the opposite polarity (Additional file 8) and (3) the observed sequence heterogeneity at RNA level (Additional file 7) indicative of replication events by error-prone RNA polymerases . However, other explanations different from RNA replication are also possible, like a genomic origin of the minus RNAs, or sequence heterogeneity due to RNA hyperediting like that observed for some intronic circRNAs in animals . Future research will be required to clarify these observations.
In summary, our work reveals that genome-encoded circRNAs carrying a self-cleaving ribozyme like the HHR are frequent molecules in plant transcriptomes, and constitute a feasible source for the origin of some virus satellites and viroids. In this regard, host RNAs derived from Ty3-gypsy retroelements are known to be efficiently encapsidated by the coat protein of a plant virus , and also Ty1-copia retrotransposons have already been proposed as the origin of non-HHR viroids of the family Pospiviroidae . Finally, a plethora of splicing-derived circRNAs with diverse biological functions have been recently reported in eukaryotes [58–64] and, consequently, future research will be focused on deciphering the possible roles and biotechnological applications of genome-encoded circRNAs with HHRs.
RNAMotif  was used for the detection of canonical type I and type III HHR motifs in DNA sequences and whole genomes previously downloaded from public repositories (phytozome.jgi.doe.gov, ftp.ncbi.nlm.nih.gov). The hits obtained were inspected for the presence of tertiary interactions between helixes I and II to ensure they were bona fide HHRs. Sequence homology searches through BLAST, BLASTX  and BLAT  tools were carried out against sequences of the GenBank and Whole Genome Shotgun (WGS) sequence databases. Sequence alignments were performed with ClustalX and Jalview software . Secondary RNA structures of minimum free energy were calculated with the RNAfold program from the ViennaRNA Package  and depicted with RnaViz .
DNA and RNA extraction
DNA from leaves of the different plants analysed was extracted following the CTAB-chloroform protocol  with some modifications. Briefly, the leaves were homogenized in CTAB extraction buffer with a Polytron (Kinematica) homogenator and incubated at 60 °C for 60 min. The homogenate was mixed with an equal volume of chloroform and isoamyl alcohol (24:1 v/v). DNA in the aqueous phase was precipitated with 2.5 volumes of 100 % ethanol and 0.1 volume of 3 M sodium acetate, dissolved in MilliQ water and quantified in a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific).
For RNA extractions, the CTAB-chloroform method  was used with some modifications, followed by purification with silica . Briefly, the frozen material (seeds, seedlings, flowers, sprouts or leaves) was homogenized in CTAB extraction buffer with a Polytron homogenator (Kinematica) and incubated at 65 °C for 30 min. The homogenate was extracted twice with an equal volume of chloroform and isoamyl alcohol (24:1 v/v). RNA in the aqueous phase was purified by adding 0.5 volume of 100 % ethanol, one volume of 6 M NaI and 0.175 volume of 100 % SiO2 (pH 2). The slurry was incubated for 30 min at room temperature and then washed four times with a buffer containing 10 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl and 50 % ethanol. The RNA was eluted in MilliQ water by incubating 4 min at 70 °C, and finally it was concentrated by precipitation with ethanol and quantified as described above.
J. curcas seeds of two different origins (called Jc India and Jc Malaysia) were provided by SLF seeds (Dehradun, UL 248002 India). Molecular analyses were performed with material obtained from Jc India seeds, with the exception of those analyses shown for Jc Malaysia in Additional files 5B and 6.
PCR, RT-PCR and molecular cloning of retrozyme fragments
Genomic retrozyme fragments containing one of the HHRs and one LTR sequence plus the central variable region were amplified by PCR. The proofreading enzyme PrimeSTAR HS DNA Polymerase (Takara) was used following the manufacturer’s instructions, together with adjacent degenerate primers designed to target conserved retrozyme regions (Additional file 12). Amplification products of the adequate size were extracted from native 5 % PAGE gel slices with phenol:chloroform:isoamyl alcohol (25:24:1) and concentrated by ethanol precipitation as described above. The purified amplicons were inserted between the XbaI and BamHI restriction sites of the plasmid pBlueScript KS+, and were sequenced automatically with an ABI Prism DNA sequencer (Perkin-Elmer). The resulting plasmids were used for analysis of ribozyme self-cleavage and probe synthesis for Northern blot.
Retrozyme RNAs, of both positive and negative polarity, were reverse-transcribed and PCR-amplified with divergent (adjacent and facing away from each other) primers (Additional file 12). RNA extracts were run in native 5 % polyacrylamide gels with 1× TAE, and a gel section of the appropriate retrozyme size was excised. RNA was purified from gel slices by phenol extraction and ethanol precipitation, and was digested with DNaseI (Roche Diagnostics GmbH). The enriched RNA extracts were used for reverse transcription (typically 100 ng RNA in a 20 μl reaction with SuperScript II, Invitrogen) and PCR (5 μl of retrotranscription products in a 50 μl reaction with PrimeSTAR HS DNA Polymerase), both performed following the instructions of the manufacturers. Amplicons of the adequate size were purified, cloned and sequenced as described above.
Analysis of ribozyme self-cleavage and riboprobe synthesis
Retrozyme RNAfragments harbouring one HHR motif were synthesized by in vitro run-off transcription of pBlueScript KS+ plasmids containing the corresponding retrozyme insert previously linearized with EcoRI (for T7 RNA polymerase) or XbaI (for T3 RNA polymerase). For ribozyme self-cleavage analysis, transcription reactions contained: 40 mM Tris-HCl (pH 8), 6 mM MgCl2, 2 mM spermidine, 0.5 mg/ml RNase-free bovine serum albumin, 0.1 % Triton X-100, 10 mM dithiothreitol, 1 mM each of ATP, CTP and GTP, 0.1 mM UTP plus 0.5 μCi/μl [α-32P]UTP, 0.4 U/μl of porcine liver ribonuclease inhibitor (Takara), 20 ng/μl of plasmid DNA and 4 U/μl of T7 (Takara) or T3 (Roche Diagnostics GmbH) RNA polymerases. After incubation at 37 °C for 1–2 h, products were fractionated by polyacrylamide gel electrophoresis (PAGE) in 5 % gels with 8 M urea, and detected by phosphorimaging (FLA-5100 phosphorimager with BAS-MP 2040S imaging plates, Fujifilm). For the synthesis of DIG-labelled riboprobes of positive and negative polarity, transcription reactions were carried out in the same conditions as described above, except that radiolabelled UTP was replaced by 0.5 mM digoxigenin-11-UTP (Roche Diagnostics GmbH) and reactions were incubated at 37 °C for 4 h.
Northern blot hybridization
For Northern blot analysis, from 5 up to 100 μg of purified RNA from different plant tissues were examined in 5 % polyacrylamide gels containing 8 M urea and 1× TBE (89 mM Tris/89 mM boric acid/2.5 mM EDTA, pH 8.3). For double PAGEs, nucleic acids enriched in RNAs of the appropriate retrozyme size were obtained by cutting a section from nondenaturing 5 % polyacrylamide gels. These RNAs were examined in denaturing 5 % polyacrylamide gels containing 8 M urea and 0.25× TBE (22.5 mM Tris/22.5 mM boric acid/2.5 mM EDTA, pH 8.3). After ethidium bromide staining, RNAs were electroblotted to nylon membranes (Amersham Hybond-N, GE Healthcare) and UV-fixed with a crosslinker (UVC 500, Hoefer). Prehybridization, hybridization (at 68 °C in 50 % formamide for 16 h) and washing (twice with 0.1× SSC at 68 °C for 15 min) was done following the instructions of the manufacturer (GE Healthcare). The DIG-labelled probes were detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase (anti-digoxigenin-AP Fab fragments, 1:104 dilution in blocking solution; Roche Diagnostics GmbH). The chemiluminiscence produced in the presence of the substrate CDP-Star (1:200 dilution in 0.1 M Tris-Cl, 0.1 M NaCl, pH 9.5; Roche Diagnostics GmbH) was finally visualized in a LAS-3000 Imaging System (Fujifilm).
Cloning, transcription and circularization of a full genomic retrozyme from F. ananassa
The genomic retrozyme with the highest sequence homology to most of the cloned F. ananassa retrozyme RNAs [GenBank:BATT01039028.1:c14263-13183] was amplified by PCR using primers designed to bind outside the sequence of the retrozyme (Fa92D and Fa92R, Additional file 12). The PCR product was cloned in the XbaI and BamHI sites of pBlueScript KS+, and the resulting plasmid was XbaI-linearized for run-off transcription with T3 RNA polymerase. The full uncleaved transcript and the retrozyme RNA resulting from self-cleavage at both HHR motifs were fractioned in 5 % polyacrylamide gels containing 8 M urea and 1× TBE, and extracted from gel slices as described above.
The retrozyme RNA, with 5′-hydroxyl and 2,3′-phosphodiester termini, was circularized using the chloroplastic isoform of a tRNA ligase from Solanum melongena , kindly provided by Drs. J. A. Daròs and R. Flores. The circularization reactions contained 1 μg of retrozyme RNA, about 1 μg of purified tRNA ligase, 50 mM Tris-HCl (pH 8), 50 mM KCl, 4 mM MgCl2, 5 mM DTT and 1 mM ATP, in a final volume of 50 μl. Reactions were incubated for 2 h at 30 °C and stopped by phenol extraction followed by ethanol precipitation. The circularized RNA was then separated in 5 % polyacrylamide gels containing 8 M urea and 1× TBE, and phenol-extracted from gel slices.
circRNA, circular RNA; HHR, hammerhead ribozyme; LINE, long interspersed element; LTR, long terminal repeat; MITE, miniature inverted-repeat transposable element; PBS, primer binding site; PPT, polypurine tract; RT, retrotranscriptase; SINE, small interspersed nucleotide element; SMART, small LTR-retrotransposon; TRIM, terminal-repeat retrotransposon in miniature; TSD, target site duplication
We would like to thank A. Ahuir for her excellent technical assistance, the Instituto Valenciano de Investigaciones Agrarias (IVIA) for kindly providing us with Citrus plant materials and the Molina family and ’El campito’, who kindly grew and provided us with J. curcas and strawberry plant materials.
Funding for this work was provided by the Ministerio de Economía y Competitividad of Spain (grants BFU2011-23398 and BFU2014-56094-P). Support of the publication fee was provided by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).
Availability of data and materials
Sequences corresponding to cloned retrozyme circRNAs have been deposited in the GenBank database (accession numbers KX273065-KX273079 for J. curcas retrozymes and KX281154-KX281154 for F. ananassa retrozymes, respectively).
AC and MdlP designed the experiments. AC, DU and MdlP performed the experiments. AC and MdlP wrote and edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Ethics approval was not needed for this study.
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- Crick FH. The origin of the genetic code. J Mol Biol. 1968;38:367–79.View ArticlePubMedGoogle Scholar
- Orgel LE. Evolution of the genetic apparatus. J Mol Biol. 1968;38:381–93.View ArticlePubMedGoogle Scholar
- Woese CR. The fundamental nature of the genetic code: prebiotic interactions between polynucleotides and polyamino acids or their derivatives. Proc Natl Acad Sci U S A. 1968;59:110–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982;31:147–57.View ArticlePubMedGoogle Scholar
- Guerrier-Takada C, Gardiner K, Marsh T, Pace N, Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 1983;35:849–57.View ArticlePubMedGoogle Scholar
- Nissen P, Hansen J, Ban N, Moore PB, Steitz TA. The structural basis of ribosome activity in peptide bond synthesis. Science. 2000;289:920–30.View ArticlePubMedGoogle Scholar
- Valadkhan S, Manley JL. Splicing-related catalysis by protein-free snRNAs. Nature. 2001;413:701–7.View ArticlePubMedGoogle Scholar
- Webb CH, Luptak A. HDV-like self-cleaving ribozymes. RNA Biol. 2011;8:719–27.View ArticlePubMedPubMed CentralGoogle Scholar
- Hammann C, Luptak A, Perreault J, De la Peña M. The ubiquitous hammerhead ribozyme. RNA. 2012;18:871–85.View ArticlePubMedPubMed CentralGoogle Scholar
- Garcia-Robles I, Sanchez-Navarro J, De la Peña M. Intronic hammerhead ribozymes in mRNA biogenesis. Biol Chem. 2012;393:1317–26.View ArticlePubMedGoogle Scholar
- De la Peña M, Gago S, Flores R. Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity. EMBO J. 2003;22:5561–70.View ArticlePubMedPubMed CentralGoogle Scholar
- Khvorova A, Lescoute A, Westhof E, Jayasena SD. Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nat Struct Biol. 2003;10:708–12.View ArticlePubMedGoogle Scholar
- Martick M, Scott WG. Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell. 2006;126:309–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Prody GA, Bakos JT, Buzayan JM, Schneider IR, Bruening G. Autolytic processing of dimeric plant virus satellite RNA. Science. 1986;231:1577–80.View ArticlePubMedGoogle Scholar
- Hutchins CJ, Rathjen PD, Forster AC, Symons RH. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 1986;14:3627–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Daros JA, Flores R. Identification of a retroviroid-like element from plants. Proc Natl Acad Sci U S A. 1995;92:6856–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Przybilski R, Graf S, Lescoute A, Nellen W, Westhof E, Steger G, et al. Functional hammerhead ribozymes naturally encoded in the genome of Arabidopsis thaliana. Plant Cell. 2005;17:1877–85.View ArticlePubMedPubMed CentralGoogle Scholar
- Ferbeyre G, Smith JM, Cedergren R. Schistosome satellite DNA encodes active hammerhead ribozymes. Mol Cell Biol. 1998;18:3880–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Rojas AA, Vazquez-Tello A, Ferbeyre G, Venanzetti F, Bachmann L, Paquin B, et al. Hammerhead-mediated processing of satellite pDo500 family transcripts from Dolichopoda cave crickets. Nucleic Acids Res. 2000;28:4037–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Epstein LM, Gall JG. Self-cleaving transcripts of satellite DNA from the newt. Cell. 1987;48:535–43.View ArticlePubMedGoogle Scholar
- Martick M, Horan LH, Noller HF, Scott WG. A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA. Nature. 2008;454:899–902.View ArticlePubMedPubMed CentralGoogle Scholar
- De la Peña M, Garcia-Robles I. Ubiquitous presence of the hammerhead ribozyme motif along the tree of life. RNA. 2010;16:1943–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Seehafer C, Kalweit A, Steger G, Gräf S, Hammann C. From alpaca to zebrafish: hammerhead ribozymes wherever you look. RNA. 2011;17:21–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Perreault J, Weinberg Z, Roth A, Popescu O, Chartrand P, Ferbeyre G, et al. Identification of hammerhead ribozymes in all domains of life reveals novel structural variations. PLoS Comput Biol. 2011;7:e1002031.View ArticlePubMedPubMed CentralGoogle Scholar
- Jimenez RM, Delwart E, Luptak A. Structure-based search reveals hammerhead ribozymes in the human microbiome. J Biol Chem. 2011;286:7737–43.View ArticlePubMedPubMed CentralGoogle Scholar
- De la Peña M, Garcia-Robles I. Intronic hammerhead ribozymes are ultraconserved in the human genome. EMBO Rep. 2010;11:711–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Webb CH, Riccitelli NJ, Ruminski DJ, Luptak A. Widespread occurrence of self-cleaving ribozymes. Science. 2009;326:953.View ArticlePubMedPubMed CentralGoogle Scholar
- Roth A, Weinberg Z, Chen AG, Kim PB, Ames TD, Breaker RR. A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nat Chem Biol. 2014;10:56–60.View ArticlePubMedGoogle Scholar
- Cervera A, De la Peña M. Eukaryotic Penelope-like retroelements encode hammerhead ribozyme motifs. Mol Biol Evol. 2014;31:2941–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Eickbush DG, Eickbush TH. R2 retrotransposons encode a self-cleaving ribozyme for processing from an rRNA cotranscript. Mol Cell Biol. 2010;30:3142–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, et al. The B73 maize genome: complexity, diversity, and dynamics. Science. 2009;326:1112–5.View ArticlePubMedGoogle Scholar
- Witte CP, Le QH, Bureau T, Kumar A. Terminal-repeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes. Proc Natl Acad Sci U S A. 2001;98:13778–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Gao D, Chen J, Chen M, Meyers BC, Jackson S. A highly conserved, small LTR retrotransposon that preferentially targets genes in grass genomes. PLoS One. 2012;7:e32010.View ArticlePubMedPubMed CentralGoogle Scholar
- Sandmeyer S, Patterson K, Bilanchone V. Ty3, a position-specific retrotransposon in budding yeast. Microbiol Spectr. 2015;3:MDNA3-0057-2014.Google Scholar
- Kumar A, Bennetzen JL. Plant retrotransposons. Annu Rev Genet. 1999;33:479–532.View ArticlePubMedGoogle Scholar
- Sabot F, Schulman AH. Parasitism and the retrotransposon life cycle in plants: a hitchhiker’s guide to the genome. Heredity (Edinb). 2006;97:381–8.View ArticleGoogle Scholar
- Finnegan DJ. Retrotransposons. Curr Biol. 2012;22:R432–437.View ArticlePubMedGoogle Scholar
- Gorinsek B, Gubensek F, Kordis D. Evolutionary genomics of chromoviruses in eukaryotes. Mol Biol Evol. 2004;21:781–98.View ArticlePubMedGoogle Scholar
- Sato S, Hirakawa H, Isobe S, Fukai E, Watanabe A, Kato M, et al. Sequence analysis of the genome of an oil-bearing tree, Jatropha curcas L. DNA Res. 2011;18:65–76.View ArticlePubMedGoogle Scholar
- Wu P, Zhou C, Cheng S, Wu Z, Lu W, Han J, et al. Integrated genome sequence and linkage map of physic nut (Jatropha curcas L.), a biodiesel plant. Plant J. 2015;81:810–21.View ArticlePubMedGoogle Scholar
- Zhang L, Zhang C, Wu P, Chen Y, Li M, Jiang H, et al. Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to salt stress. PLoS One. 2014;9:e97878.Google Scholar
- De la Peña M, Flores R. An extra nucleotide in the consensus catalytic core of a viroid hammerhead ribozyme: implications for the design of more efficient ribozymes. J Biol Chem. 2001;276:34586–93.View ArticlePubMedGoogle Scholar
- Flores R, Grubb D, Elleuch A, Nohales MA, Delgado S, Gago S. Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: variations on a theme. RNA Biol. 2011;8:200–6.View ArticlePubMedGoogle Scholar
- Gago S, Elena SF, Flores R, Sanjuan R. Extremely high mutation rate of a hammerhead viroid. Science. 2009;323:1308.View ArticlePubMedGoogle Scholar
- Hirakawa H, Shirasawa K, Kosugi S, Tashiro K, Nakayama S, Yamada M, et al. Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria species. DNA Res. 2014;21:169–81.View ArticlePubMedGoogle Scholar
- Nohales MA, Molina-Serrano D, Flores R, Daros JA. Involvement of the chloroplastic isoform of tRNA ligase in the replication of viroids belonging to the family Avsunviroidae. J Virol. 2012;86:8269–76.View ArticlePubMedPubMed CentralGoogle Scholar
- Hirakawa H, Nakamura Y, Kaneko T, Isobe S, Sakai H, Kato M, et al. Survey of the genetic information carried in the genome of Eucalyptus camaldulensis. Plant Biotech. 2011;28:471–80.View ArticleGoogle Scholar
- Myburg AA, Grattapaglia D, Tuskan GA, Hellsten U, Hayes RD, Grimwood J, et al. The genome of Eucalyptus grandis. Nature. 2014;510:356–62.PubMedGoogle Scholar
- Xu Q, Chen LL, Ruan X, Chen D, Zhu A, Chen C, et al. The draft genome of sweet orange (Citrus sinensis). Nat Genet. 2013;45:59–66.View ArticlePubMedGoogle Scholar
- Copeland CS, Marz M, Rose D, Hertel J, Brindley PJ, Santana CB, et al. Homology-based annotation of non-coding RNAs in the genomes of Schistosoma mansoni and Schistosoma japonicum. BMC Genomics. 2009;10:464.View ArticlePubMedPubMed CentralGoogle Scholar
- Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, et al. A unified classification system for eukaryotic transposable elements. Nat Rev Genet. 2007;8:973–82.View ArticlePubMedGoogle Scholar
- Wang W, Feng B, Xiao J, Xia Z, Zhou X, Li P, et al. Cassava genome from a wild ancestor to cultivated varieties. Nat Commun. 2014;5:5110.View ArticlePubMedPubMed CentralGoogle Scholar
- Okamoto H, Hirochika H. Silencing of transposable elements in plants. Trends Plant Sci. 2001;6:527–34.View ArticlePubMedGoogle Scholar
- Feschotte C, Jiang N, Wessler SR. Plant transposable elements: where genetics meets genomics. Nat Rev Genet. 2002;3:329–41.View ArticlePubMedGoogle Scholar
- Ivanov A, Memczak S, Wyler E, Torti F, Porath HT, Orejuela MR, et al. Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals. Cell Rep. 2015;10:170–7.View ArticlePubMedGoogle Scholar
- Ghoshal K, Theilmann J, Reade R, Maghodia A, Rochon D. Encapsidation of host RNAs by cucumber necrosis virus coat protein during both agroinfiltration and infection. J Virol. 2015;89:10748–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Kiefer MC, Owens RA, Diener TO. Structural similarities between viroids and transposable genetic elements. Proc Natl Acad Sci U S A. 1983;80:6234–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One. 2012;7:e30733.View ArticlePubMedPubMed CentralGoogle Scholar
- Jeck WR, Sorrentino JA, Wang K, Slevin MK, Burd CE, Liu J, et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA. 2013;19:141–57.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang PL, Bao Y, Yee MC, Barrett SP, Hogan GJ, Olsen MN, et al. Circular RNA is expressed across the eukaryotic tree of life. PLoS One. 2014;9:e90859.View ArticlePubMedPubMed CentralGoogle Scholar
- Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013;495:333–8.View ArticlePubMedGoogle Scholar
- Ashwal-Fluss R, Meyer M, Pamudurti NR, Ivanov A, Bartok O, Hanan M, et al. circRNA biogenesis competes with pre-mRNA splicing. Mol Cell. 2014;56:55–66.View ArticlePubMedGoogle Scholar
- Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard CK, et al. Natural RNA circles function as efficient microRNA sponges. Nature. 2013;495:384–8.View ArticlePubMedGoogle Scholar
- Talhouarne GJ, Gall JG. Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes. RNA. 2014;20:1476–87.View ArticlePubMedPubMed CentralGoogle Scholar
- Macke TJ, Ecker DJ, Gutell RR, Gautheret D, Case DA, Sampath R. RNAMotif, an RNA secondary structure definition and search algorithm. Nucleic Acids Res. 2001;29:4724–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.View ArticlePubMedGoogle Scholar
- Kent WJ. BLAT—the BLAST-like alignment tool. Genome Res. 2002;12:656–64.View ArticlePubMedPubMed CentralGoogle Scholar
- Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2—-a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Lorenz R, Bernhart SH, Honer zu Siederdissen C, Tafer H, Flamm C, Stadler PF, et al. ViennaRNA Package 2.0. Algorithms Mol Biol. 2011;6:26.View ArticlePubMedPubMed CentralGoogle Scholar
- De Rijk P, Wuyts J, De Wachter R. RnaViz 2: an improved representation of RNA secondary structure. Bioinformatics. 2003;19:299–300.View ArticlePubMedGoogle Scholar
- Dhakshanamoorthy D, Selvaraj R. Extraction of genomic DNA from Jatropha sp. using modified CTAB method. Rom J Biol Plant Biol. 2009;54:117–25.Google Scholar
- Sangha JS, Gu K, Kaur J, Yin Z. An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L. BMC Res Notes. 2010;3:126.View ArticlePubMedPubMed CentralGoogle Scholar
- Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28:495–503.PubMedPubMed CentralGoogle Scholar