Production of Circular Recombinant RNA in Escherichia coli Using Viroid Scaffolds.

Viroids are small circular, noncoding, highly base-paired RNAs able to infect higher plants. Recently, it has been shown that viroids can be used as very stable scaffolds to produce recombinant RNA in Escherichia coli. Coexpression of an RNA precursor consisting of a viroid monomer, in which the RNA of interest is inserted, flanked by domains of the viroid hammerhead ribozyme, along with a host plant tRNA ligase, the enzyme that catalyzes viroid circularization in infected plants, allows for accumulation of large amounts of the chimeric viroid-RNA of interest in E. coli. Since viroids do not replicate in E. coli, high accumulation most probably results from viroid scaffold stability, resistance to exonucleases due to circularity, and accumulation as a ribonucleoprotein complex with tRNA ligase. Purification of the recombinant RNA from total E. coli RNA is also facilitated by the circular structure of the product.

High-pressure analysis of a hammerhead ribozyme from Chrysanthemum chlorotic mottle viroid reveals two different populations of self-cleaving molecule.

The activity of the full-length hammerhead ribozyme requires a tertiary interaction between its distal loops leading to the closure of the molecule and its stabilization in the active conformation. In this study, the conformational changes accompanying the cis-cleavage reaction of Chrysanthemum chlorotic mottle viroid hammerhead ribozyme were investigated by high-pressure experiments on the complete cleavage reaction. Two activation volumes (ΔV(≠)) were measured, pointing to the presence of two different populations of molecules corresponding to fast-cleaving and slow-cleaving ribozymes in the reaction mixture. The fast population, with a small ΔV(≠) of 2.6 mL·mol(-1), most likely represents molecules in the near-active conformation, whereas the slow population, with a larger ΔV(≠) of 11.6 mL·mol(-1 , represents molecules that need a larger conformational change to induce activity. In addition, pH-dependence experiments suggest that the group whose deprotonation is required for activity intervenes in the formation of the transition state or in the chemistry of the reaction, but not in the conformational change that precedes it.

Structure-function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop-loop interaction motif conserved in most natural hammerheads.

Loop-loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (-) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5' side and an extrahelical U at its 3'-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3'-side. These structural features promote a specific loop-loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5' U of loop 1 and the 3' A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.

Catalytic activity of hammerhead ribozymes in a clay mineral environment: implications for the RNA world.

The hypothesized RNA-based world would have required the presence of a protected environment in which RNA, or an RNA-like molecule, could originate and express its biological activity. Recent studies have indicated that RNA molecules adsorbed/bound on clay minerals are able to persist in the presence of degrading agents, to interact with surrounding molecules, and to transmit the information contained in their nucleotide sequences. In this study, we assessed the ability of RNA molecules with catalytic activity to perform a specific reaction in a mineral environment. For this purpose, we investigated the self-cleavage reaction of the hammerhead ribozyme of the Avocado Sun Blotch Viroid (ASBVd), both in the monomeric and in dimeric forms. The monomeric transcript was tightly bound on the clay mineral montmorillonite to form a stable complex, while the behaviour of the dimeric transcript was studied in the presence of the clay particles in the reaction mixture. The results indicated that the hammerhead ribozyme was still active when the monomeric transcript was adsorbed on the clay surface, even though its efficiency was reduced to about 20% of that in solution. Moreover, the self-cleavage of clay-adsorbed molecule was significantly enhanced ( approximately four times) by the presence of the 5' reaction product. The self-cleavage reaction of the dimeric transcript in the presence of montmorillonite indicated that the mineral particles protected the RNA molecules against aspecific degradation and increased the rate of cleavage kinetics by about one order of magnitude. These findings corroborate the hypothesis that clay-rich environments would have been a good habitat in which RNA or RNA-like molecules could originate, accumulate and undergo Darwinian evolutionary processes, leading to the first living cells on Earth.

Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads.

Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (+) and (-) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (+) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (-) hammerhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (+) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(+)-AUC approximately ELVd-(+)-GUC>ELVd-(+)-GUA> ELVd-(+)-AUA. However, the ELVd-(+)-AUC hammerhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive metastable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.

Comparative single-turnover kinetic analyses of trans-cleaving hammerhead ribozymes with naturally derived non-conserved sequence motifs.

trans-Cleaving hammerhead ribozyme variants were generated with mimicked non-conserved internal loop motifs derived from five structurally diverse natural cis-cleaving ribozymes. Most modified trans-cleaving variants showed enhanced single-turnover cleavage rates relative to minimal counterparts that lack tertiary interactions between internal loop motifs I and II, and relative to controls with sequence changes in loop I. The trans-cleaving ribozyme derived from the positive strand of peach latent mosaic viroid had the highest observed cleavage rate, suggesting a structurally optimized motif that facilitates rapid formation of the ribozyme catalytic center in a trans-reaction.

An extra nucleotide in the consensus catalytic core of a viroid hammerhead ribozyme: implications for the design of more efficient ribozymes.

Hammerhead ribozymes catalyze self-cleavage of oligomeric RNAs generated in replication of certain viroid and viroid-like RNAs. Previous studies have defined a catalytic core conserved in most natural hammerheads, but it is still unknown why some present deviations from the consensus. We have addressed this issue in chrysanthemum chlorotic mottle viroid (CChMVd), whose (+) hammerhead has an extra A (A10) between the conserved A9 and the quasi-conserved G10.1. Effects of insertions at this position on hammerhead kinetics have not hitherto been examined. A10 caused a moderate decrease of the trans-cleaving rate constant with respect to the CChMVd (+) hammerhead without this residue, whereas A10-->C and A10-->G substitutions had major detrimental effects, likely because they favor catalytically inactive foldings. By contrast, A10-->U substitution induced a 3-4-fold increase of the rate constant, providing an explanation for the extra U10 present in two natural hammerheads. Because A10 also occupies a singular and indispensable position in the global CChMVd conformation, as revealed by bioassays, these results show that some hammerheads deviate from the consensus due to the involvement of certain residues in critical function(s) other than self-cleavage. Incorporation of the extra U10 into a model hammerhead also caused a similar increase in the rate constant, providing data for a deeper understanding of the hammerhead structural requirements and for designing more efficient ribozymes.

The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants.

Four ribozyme and antisense genes targeting citrus exocortis viroid (CEVd) positive- and negative-strand RNA molecules were constructed and used to transform the tomato Lycopersicon lycopersicum cv. UC82B. The tomato is a readily transformable plant and will support replication of CEVd following mechanical inoculation. The ribozyme genes contained three hammerhead catalytic motifs with long hybridizing arms and synthetic RNA transcripts were shown to cleave the target CEVd RNA molecule in vitro. Homozygous transgenic plants were produced from independent transformants expressing either ribozymes or antisense constructs. Inoculation of transgenic seedlings expressing antisense constructs targeting the negative-strand CEVd RNA molecule with CEVd resulted in a moderate reduction in the accumulation of CEVd RNA. In contrast, similarly inoculated transgenic plants expressing constructs targeting the positive-strand CEVd RNA molecule resulted in an increase in the rate of CEVd RNA accumulation. Addition of the ribozyme motifs to the antisense genes did not enhance their efficiency in the suppression of viroid replication and a moderation or elimination of the observed antisense effects was seen in plants expressing the corresponding catalytic RNA-encoding genes.