An efficient strategy for the synthesis of circular RNA molecules

Beaudry, Diane; Perreault, Jean‐Pierre

doi:10.1093/nar/23.15.3064

Cited by 58 publications

(35 citation statements)

References 5 publications

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“…Briefly, this construct possesses two tandemly repeated PLMVd sequences (from the Armking peach cultivar [9]) cloned into the PstI restriction site of pBluescript II KS. The strategy for the production of monomeric PLMVd RNA has been reported previously (2). A first DNA template was amplified from pPD1 with Pwo DNA polymerase (Boehringer Mannheim) using sense (5Ј-TA ATACGACTCACTATAGGGTCAAAAGTTTCGCCGC-3Ј) and antisense (5Ј-TATGAGTTTCGTCTCATTTC-3Ј) primers for RNA transcripts starting from position 337.…”

Section: Methodsmentioning

confidence: 99%

Mapping in Solution Shows the Peach Latent Mosaic Viroid To Possess a New Pseudoknot in a Complex, Branched Secondary Structure

Bussière

Ouellet

Côté

et al. 2000

J Virol

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We have investigated the secondary structure of peach latent mosaic viroid (PLMVd) in solution, and we present here the first description of the structure of a branched viroid in solution. Different PLMVd transcripts of plus polarity were produced by using the circularly permuted RNA method and the exploitation of RNA internal secondary structure to position the 5 and 3 termini and studied by nuclease mapping and binding shift assays using DNA and RNA oligonucleotides. We show that PLMVd folds into a complex, branched secondary structure. In general, this structure is similar to that reported previously, which was based on sequence comparison and computer modelling. The structural microheterogeneity is apparently limited to only some small domains. More importantly, this structure includes a novel pseudoknot that is conserved in all PLMVd isolates and seems to allow folding into a very compact form. This pseudoknot is also found in chrysanthemum chlorotic mottle viroid, suggesting that it is a unique feature of the viroid members of the PLMVd subgroup.Viroids are small (ϳ300 nucleotides), single-stranded, circular RNAs that infect higher plants, causing significant losses in the agricultural industry (see references 7 and 13 for reviews). Viroids have been classified in two groups (groups A and B) based primarily on whether or not they possess five typical structural domains found in the group B viroids (7). Further division among the group B members depends on the sequence and length of the conserved central region. Viroids that do not possess any kind of sequence or structural similarity with the group B viroids have been classified as belonging to group A. The viroids from this group possess self-cleaving hammerhead motifs that are crucial for their replication via a rolling circle mechanism.The group A viroids include the avocado sunblotch viroid (ASBVd), the peach latent mosaic viroid (PLMVd), and the chrysanthemum chlorotic mottle viroid (CChMVd) (10). Both PLMVd and CChMVd have been proposed to adopt branched secondary structures (Fig. 1A) instead of the rod-like ones proposed for most viroids, including ASBVd (10). The unusual conformations of PLMVd and CChMVd are supported by their insolubility in 2 M lithium chloride, whereas ASBVd and a number of non-self-cleaving viroids (i.e., the group B viroids) are soluble in this high salt solution (10). In general, secondary structures of viroids are predicted using computer software and are useful for the formulation of hypotheses on the structure-function relationships of these RNA molecules (4). Characterization of biological structures in vitro as well as in vivo is obviously more accurate for elucidating the structure-function relationship. The only secondary structure of a viroid that has been extensively studied in solution is that of the potato spindle tuber viroid (8). This group B species, which was shown to adopt a rod-like shape in solution, is responsible for most of our knowledge of the biology of viroids.In order to determine the secondary struct...

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Section: Methodsmentioning

confidence: 99%

Mapping in Solution Shows the Peach Latent Mosaic Viroid To Possess a New Pseudoknot in a Complex, Branched Secondary Structure

Bussière

Ouellet

Côté

et al. 2000

J Virol

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“…PSTVd circularization was performed following the method of Beaudry and Perreault (1995). Briefly, 10 mg of linear PSTVd transcripts was incubated at 37°C for 2 h in a final volume of 200 mL containing 150 units of calf intestinal alkaline phosphatase (New England Biolabs), 80 units of RNase inhibitor (New England Biolabs), and 13 calf intestinal alkaline phosphatase buffer.…”

Section: Rna Preparation In Vitromentioning

confidence: 99%

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II

Wang

et al. 2015

The Plant Cell

106

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Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (2)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (2)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes.

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“…The synthesis and purification of both the L-PLMVd and the C-PLMVd transcripts used were performed as described previously (2,3,10). Briefly, in vitro transcription was performed using recombinant plasmid pPD1, which possesses two tandemly repeated PLMVd sequences (plus and minus strands).…”

Section: Methodsmentioning

confidence: 99%

“…C-PLMVd transcripts which include exclusively 3Ј,5Ј-phosphodiester bonds (3Ј,5Ј-C-PLMVd) were synthesized by use of a procedure based on the circularly permuted RNA strategy described previously (2). C-PLMVd transcripts including a 2Ј,5Ј-phosphodiester bond at the ligation site (2Ј,5Ј-C-PLMVd) were synthesized by in vitro self-ligation of L-PLMVd as described previously 10,22).…”

Section: Methodsmentioning

confidence: 99%

Natural 2′,5′-Phosphodiester Bonds Found at the Ligation Sites of Peach Latent Mosaic Viroid

Côté

Lévesque

Perreault

2001

J Virol

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Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2,5-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2,5-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2,5-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2,5-phosphodiester bonds are still present in nature and that they are of biological importance.Viroids are the smallest nucleic acid-based pathogens known to date (see references 15 and 33 for reviews). They are small (ϳ300 nucleotides [nt]), single-stranded, circular RNAs that infect higher plants and cause significant losses in agriculture. Viroids have been classified into two groups (A and B) based primarily on whether or not they possess the five typical structural domains found in the group B viroids. Further division among the 27 group B members depends on the sequence and length of the conserved central regions (7,14,33). Viroids that do not possess any kind of sequence or structural similarity with group B viroids have been classified as belonging to group A. The latter group includes the avocado sunblotch viroid, the peach latent mosaic viroid (PLMVd), and the chrysanthenum chlorotic mottle viroid (7, 15). All group A viroids possess hammerhead self-cleaving motifs and are proposed to replicate in chloroplasts (8,11,23). This localization has received recent support from the identification of a chloroplastic RNA polymerase that has the ability to replicate avocado sunblotch viroid (26). In contrast, group B viroids are localized in the nucleus, and RNA polymerase II appears to be responsible for their replication (4,18,30).In infected cells, viroids replicate in a DNA-independent manner via a rolling circle mechanism that follows either a symmetric or an asymmetric mode (6,8,33,36). The replicational intermediates of PLMVd, a 338-nt group A viroid that is the causal agent of peach latent mosaic disease (19), were recently studied by Northern blot analysis (8). PLMVd has been shown to replicate in a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities (see Fig. 1). No multimeric conformer (i.e., intermediate) has been detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Moreover, it has been observed that monomeric linear RNAs accumulate at a high level...

show abstract

An efficient strategy for the synthesis of circular RNA molecules

Cited by 58 publications

References 5 publications

Mapping in Solution Shows the Peach Latent Mosaic Viroid To Possess a New Pseudoknot in a Complex, Branched Secondary Structure

Mapping in Solution Shows the Peach Latent Mosaic Viroid To Possess a New Pseudoknot in a Complex, Branched Secondary Structure

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II

Natural 2′,5′-Phosphodiester Bonds Found at the Ligation Sites of Peach Latent Mosaic Viroid

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