RNA mediated formation of a phosphorothioate diester bond

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.     

Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA.

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.     

Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration.

Both polarities of the satellite RNA of tobacco ringspot virus occur in infected cells in multimeric forms which are capable of autolytic processing, using different sequences and structures [Feldstein, P.A., et al., Proc. Nat. Acad. Sci. USA (1990) 87 (in press)]. These transesterification reactions generate a 2',3'-cyclophosphate and a 5'-hydroxyl as the two new end groups. Cleavage is at a CpA for the (+) polarity RNA and at an ApG for the (-) polarity RNA. We enzymically synthesized oligoribonucleotides with processing capability and with specific 35S-labeled phosphorothioate diesters in the Rp configuration. After processing had occurred, the terminal nucleoside-2',3'-cyclophosphorothioate diester residues were recovered from the appropriate product by digestion with nuclease and phosphatase. Comparisons with specially prepared endo- and exoisomer reference compounds by thin layer chromatography and autoradiography revealed that the [35S]cytidine- and [35S]adenosine-2',3'-cyclophosphorothioate both were endo-isomers. The results are consistent with transesterification occurring by an inline SN2(P) attack of the 2'-hydroxyl group in the autolytic processing reactions of both polarities of the satellite RNA.     

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.     

Binding and cleavage of nucleic acids by the "hairpin" ribozyme

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.     

Evolution and replication of tobacco ringspot virus satellite RNA mutants.

The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.     

The introduction of reporter groups at multiple and/or specific sites in DNA containing phosphorothioate diesters

DNA fragments containing phosphorothioate diesters provide nucleophilic sites which are amenable to labeling by spin labels or fluorophores. Selecting the position for an individual phosphorothioate diester allows highly specific placement of the reporter group. The substitution of a phosphorothioate diester for each and every internucleotidic phosphodiester allows the incorporation of multiple reporter groups; ideally one for each nucleoside residue. With the use of multiple fluorophores a post-assay fluorescent labeling technique has been developed which allows the detection of DNA fragments with the "naked-eye" in the low femtomolar (10(-15) moles) range.     

Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease

Cleavage of phosphodiester bonds by small ribonuclease mimics within different bulge-loops of RNA was investigated. Bulge-loops of different size (1–7 nt) and sequence composition were formed in a 3′ terminal fragment of influenza virus M2 RNA (96 nt) by hybridization of complementary oligodeoxynucleotides. Small bulges (up to 4 nt) were readily formed upon oligonucleotide hybridization, whereas hybridization of the RNA to the oligonucleotides designed to produce larger bulges resulted in formation of several alternative structures. A synthetic ribonuclease mimic displaying Pyr–Pu cleavage specificity cleaved CpA motifs located within bulges faster than similar motifs within the rest of the RNA. In the presence of 10 mM MgCl₂, 75% of the cleavage products resulted from the attack of this motif. Thus, selective RNA cleavage at a single target phosphodiester bond was achieved by using bulge forming oligonucleotides and a small ribonuclease A mimic.     

Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: different energetic contributions from a ribose zipper motif

The hairpin catalytic motif in tobacco ringspot virus satellite RNA consists of two helix-loop-helix elements on two adjacent arms of a four-way helical junction. The bases essential for catalytic activity are located in the loops that are brought into proximity by a conformational change as a prerequisite for catalysis. The two loops interact via a ribose zipper motif involving the 2'-hydroxyls of A10, G11, A24, and C25 [Rupert, P. B., and Ferre d'Amare, A. R. (2001) Nature 401, 780-786]. To quantify the energetic importance of the ribose zipper hydrogen bonds, we have incorporated deoxy modifications at these four positions and determined the resulting destabilization of the docked conformer by means of time-resolved fluorescence resonance energy transfer. In a minimal form of the ribozyme, in which the loops are placed on the arms of a two-way helical junction, all modifications lead to a significant loss in tertiary structure stability and altered Mg2+ binding. Surprisingly, no significant destabilization was seen with the natural four-way junction ribozyme, suggesting that hydrogen bonding interactions involving the 2'-hydroxyls do not contribute to the stability of the docked conformer. These results suggest that the energetic contributions of ribose zipper hydrogen bonds are highly context dependent and differ significantly for the minimal and natural forms of the ribozyme.     

Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus

The nucleotide sequence of the satellite of arabis mosaic virus was determined using the satellite RNA encapsidated in virions. The 300-nucleotide long sequence showed extensive homology (50%) with that of the 359-nucleotide satellite RNA of tobacco ringspot virus, which occurs both in a linear and a circular form. This homology also revealed the presence of conceived sequences believed to mediate self-cleavage of the latter as well as other viral satellite RNAs. A circular form of the arabis mosaic virus satellite can be isolated from infected tissues and partially converts to the linear form upon elution from denaturing gels.     

Cross-ligation and exchange reaction of RNA catalyzed by hairpin ribozymes.

The catalytic domain in the minus strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) assumes a hairpin-like secondary structure. This ribozyme catalyzes a cross-ligation reaction between substrate RNAs of different lengths. We constructed ribozymes to probe the activities of ligation and RNA fragment exchange.     

Kinetic parameters of hydrolysis of CpA and UpA sequences in an oligoribonucleotide by compounds functionally mimicking ribonuclease A

Kinetic parameters of cleavage of CpA and UpA sequences in an oligoribonucleotide under the action of artificial ribonuclease ABL3C1 were measured. The compounds were built of RNA-binding domain B, catalytic fragment C, linker L3 comprising 3 methylene groups, and aliphatic fragment A. The rate of cleavage of phosphodiester bonds in CpA sequence within decaribonucleotide UUCAUGUAAA was shown to be 3.4 +/- 0.2 times higher than in UpA sequence. The rate of cleavage of phosphodiester bonds were found to depend on substrate length: a thousandfold increase in cleavage rate constant was observed for CpA sequence in decaribonucleotide as compared with diribonucleotide monophosphate CpA. A slight decrease in the cleavage rates was observed for the reactions proceeding in different buffers at pH 7.0: imidazole > HEPES > phosphate > cacodylate. At the same time, the ratio of cleavage rates for CpA and UpA sequences remained constant.     

Synthesis and properties of RNA with catalytic activity

Ribooligonucleotides, which form the self-cleavage domain of a satellite RNA of tobacco ringspot virus, were synthesized by the solid-phase phosphoramidite method using o-nitrobenzyl groups for 2'-hydroxyl protection. The oligomers were obtained in quantities sufficient for NMR measurement. Specific cleavage at an expected site was observed when the three RNA fragments were mixed in the presence of magnesium ions.     

Directed protein replacement in recombination full sites reveals trans-horizontal DNA cleavage by Flp recombinase.

One round of site-specific recombination between two DNA partners mediated by the Flp recombinase requires the breakage and reformation of four phosphodiester bonds. The reaction is accomplished by the combined action of four Flp monomers. Within the recombination complex, what is the relative disposition of a Flp monomer with respect to the target diester that it cleaves? To address this question, we have devised a strategy for the targeted orientation of Flp monomers within full-site recombination substrates. Our experimental design is not dependent on ''altered binding specificity'' of the recombinase. Analysis of the pattern of DNA cleavage by this method reveals no evidence for DNA cleavage in cis. A Flp monomer bound to its recognition element within the full site does not cleave the scissile phosphodiester bond adjacent to it. Our results are most consistent with ''trans-horizontal cleavage''. Cleavage by Flp occurs at the scissile phosphodiester distal to it, but within the same full site. The general experimental design employed here will be of widespread utility in mechanistic analyses of nucleic acid transactions involving multimeric DNA-protein assemblies.     

Kinetic Parameters of Cleavage of CpA and UpA Sequences in an Oligoribonucleotide by Compounds Functionally Mimicking Ribonuclease A

Kinetic parameters of cleavage of CpA and UpA sites in an oligoribonucleotide under the action of artificial ribonuclease ABL3C1 were measured. The compounds were built of RNA-binding domain B, catalytic fragment C, linker L3 comprising 3 methylene groups, and aliphatic fragment A. The rate of cleavage of phosphodiester bonds in the CpA site within decaribonucleotide UUCAUGUAAA was shown to be 3.4 ± 0.2 times higher than in UpA. The rate of cleavage of phosphodiester bonds was found to depend on substrate length: a thousandfold increase in cleavage rate constant was observed for the CpA site in decaribonucleotide as compared with diribonucleoside monophosphate CpA. A slight decrease in the cleavage rates was observed for the reactions proceeding in different buffers at pH 7.0: imidazole > HEPES > phosphate > cacodylate. At the same time, the ratio of cleavage rates for CpA and UpA sites remained constant.     

Evidence that the Low Molecular Weight RNA Associated with Chicory Yellow Mottle Virus is a Satellite

Chicory yellow mottle virus, ringspot strain (CYMV-RS), supports the replication of a low molecular weight RNA (0.17 × 10⁶ daltons) associated with CYMV-T (type stain).
Competition hybridization experiments revealed lack or nucleotide sequence homology between 0.17 × 10⁶ mol. wt. RNA (Sat RNA) and CYMV-RS genomic RNAs, and partial homology (33 %) with CYMV-T genomic RNAs. However, such apparent partial homology can be due to contamination of CYMV-T genomic RNAs with a multimeric form of Sat RNA having a similar molecular weight. On this account the hypothesis that CYMV-T Sat RNA is a true satellite RNA becomes tenable.     

Sequence elements outside the catalytic core of natural hairpin ribozymes modulate the reactions differentially

Abstract Hairpin ribozymes occur naturally only in the satellite RNAs of tobacco ringspot virus (TRsV), chicory yellow mottle virus (CYMoV) and arabis mosaic virus (ArMV). The catalytic centre of the predominantly studied sTRsV hairpin ribozyme, and of sArMV is organised around a four-way helical junction. We show here that sCYMoV features a five-way helical junction instead. Mutational analysis indicates that the fifth stem does not influence kinetic parameters of the sCYMoV hairpin ribozyme in vitro reactions, and therefore seems an appendix to that junction in the other ribozymes. We report further that all three ribozymes feature a three-way helical junction outside the catalytic core in stem A, with Watson-Crick complementarity to loop nucleotides in stem B. Kinetic analyses of cleavage and ligation reactions of several variants of the sTRsV and sCYMoV hairpin ribozymes in vitro show that the presence of this junction interferes with their reactions, particularly the ligation. We provide evidence that this is not due to a presumed interaction of the afore-mentioned elements in stems A and B. The evolutionary survival of this cis-inhibiting element seems rather to be caused by the coincidence of its position with that of the hammerhead ribozyme in the other RNA polarity.