Core sequences and a cleavage site wobble pair required for HDV antigenomic ribozyme self-cleavage.

The secondary structures proposed for the cis-acting hepatitis delta virus (HDV) ribozymes contain four duplex regions, three sequences joining the duplexes and two hairpin loops. The core and active site of the ribozyme could be formed by portions of the joining sequences, J1/4 and J4/2, together with one of the hairpin loops, L3. To establish the core region and define essential bases within this putative active site 28 single base changes at 15 positions were made and tested for effects on ribozyme cleavage. At 14 of the 15 positions all of the changes resulted in detectable decreased rates of cleavage. At seven of the positions one or more of the changes resulted in a 500-fold or greater decrease in the observed rate constant for cleavage. Mutations that resulted in 10(3)-fold effects were found in all three regions hypothesized to form the core. At the cleavage site substitutions of the cytosine 5' of the site of cleavage did not provide strong support for a sequence-specific interaction involving this nucleotide. In contrast, an A-C combination was the most effective substitution for a potential G-U pair 3' of the cleavage site, suggesting a requirement for a wobble pair at that position.     

A sequence element necessary for self-cleavage of the antigenomic hepatitis delta RNA in 20 M formamide.

The genomic and antigenomic RNAs of hepatitis delta virus are capable of self-cleavage and show no significant sequence similarities to other known self-cleaving RNAs. We have derived an antigenomic delta RNA which cleaves to completion in 15 s in 9 mM magnesium at 37 degrees C and is capable of efficient self-cleavage in concentrations of formamide as high as 20 M. Cleavage in high concentrations of denaturant is dependent upon the presence of a polypurine sequence element, GGAGA, located between 81 and 85 nucleotides downstream of the cleavage site. Mutation of the initial G81G82 to C81C82, or removal of the sequence element, results in a loss of the ability to cleave in high formamide concentrations. Changing the final U-2C-1 of a pyrimidine-rich region, UCUUC, just upstream of the cleavage site, to G-2G-1 severely affects the self-cleavage, but introducing the two mutations, GG to CC and UC to GG, into the same molecule, restoring potential base pairing, partially restores the formamide stability. Relocating the GGAGA sequence upstream of the cleavage site also results in partial restoration of the formamide cleavage. Although the GGAGA sequence is important for self-cleavage under denaturing conditions, it does not appear to be necessary for HDV RNA cleavage in normal buffer conditions.     

Unbiased in vitro selection reveals the unique character of the self-cleaving antigenomic HDV RNA sequence

In order to revisit the architecture of the catalytic center of the antigenomic hepatitis delta virus (HDV) ribozyme we developed an unbiased in vitro selection procedure that efficiently selected novel variants from a relatively small set of sequences. Using this procedure we examined all possible variants from a pool of HDV ribozymes that had been randomized at 25 positions (4²⁵). The isolated set of sequences shows more variability than do the natural variants. Nucleotide variations were found at all randomized positions, even at positions when the general belief was that the specific base was absolutely required for catalytic activity. Covariation analysis supports the presence of several base pairs, although it failed to propose any new tertiary contacts. HDV ribozyme appears to possess a greater number of constraints, in terms of sequences capable of supporting the catalysed cleavage, than do other catalytic RNAs. This supports the idea that the appearance of this catalytic RNA structure has a low probability (i.e. is a rare event), which may explain why to date it has been found in nature only in the HDV. These contrasts with the hammerhead self-cleaving motif that is proposed to have multiple origins, and that is widespread among different organisms. Thus, just because a self-cleaving RNA motif is small does not imply that it occurs easily.     

Characterization of the trans Watson-Crick GU base pair located in the catalytic core of the antigenomic HDV ribozyme

The HDV ribozyme's folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U(23) and G(28) nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U(23) and G(28) can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction.     

The Role of the poly(A) sequence in mammalian messenger RNA

The poly(A) sequence is added to 3' termini of nuclear RNA segments destined to become part of the mRNA, and may play an essential role in the selection of these segments. It appears to be required for at least some of the splicing events involved in mRNA processing. In the cytoplasm, the poly(A) segment is the target of a degradation process which causes its gradual shortening, and leads to a heterogeneous steady-state poly(A)-size distribution. Complete loss of the poly(A) is probably followed by inactivation of the mRNA, since chains depleted of poly(A) do not accumulate in the cells. A role for this sequence in the promotion of mRNA stability is suggested by the behavior of globin mRNA depleted of poly(A) after injection into frog oocytes. The poly(A) shortening process may be part of the mRNA inactivation mechanism, as indicated by the greater sensitivity to degradation of the poly(A) of some short-lived mRNAs. However, the stochastic mRNA decay implies that new and old mRNA chains, with long and short poly(A) segments, respectively are equally susceptible to inactivation. The poly(A)-lacking histone mRNAs are stable only in cells engaged in DNA replication. Present knowledge favors a role for poly(A) in the control of mRNA stability. Loss of this sequence could be controlled through modulation of poly(A)-protein interactions or through masking of a sequence directly adjacent to the poly(A). In the nucleus, the poly(A) sequence could also serve as stabilizing agent, but, in addition, it might interact with the splicing machinery.     

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.     

A nested double pseudoknot is required for self-cleavage activity of both the genomic and antigenomic hepatitis delta virus ribozymes.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3' cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G.U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.     

The poly(A)(+)RNA sequence complexity is also represented in poly(A)(-)RNA in sea-urchin embryos

The extent to which the poly(A)(+)RNA sequence complexity from sea-urchin embryos is also represented in poly(A)(-)RNA was determined by cDNA cross-hybridization. Eighty percent or more of both the cytoplasmic poly(A)(+)RNA and polysomal poly(A)(+)RNA sequences appeared in a poly(A)(-) form. In both cases, the cellular concentrations of the poly(A)(-)RNA molecules that reacted with the cDNA were similar to the concentrations of the homologous poly(A)(+) sequences. Additionally, few, if any, abundant poly(A)(+)mRNA molecules were quantitatively discriminated by polyadenylation, since the abundant poly(A)(+)sequences were also abundant in poly(A)(-)RNA. Neither degradation nor inefficient binding to oligo (dT)-cellulose can account for the observed cross-reactivity. These data indicate that, in sea-urchin embryos, the poly(A) does not regulate the utilization of mRNA by demarcating an mRNA subset that is specifically and completely polyadenylated.     

A pH-sensitive RNA tertiary interaction affects self-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion

Self-cleavage of the genomic and antigenomic ribozymes from hepatitis delta virus (HDV) requires divalent cation for optimal activity. Recently, the HDV genomic ribozyme has been shown to be active in NaCl in the absence of added divalent metal ion at low pH (apparent pKa 5.7). However, we find that the antigenomic ribozyme is 100 to 1000-fold less active under similar conditions. With deletion of a three-nucleotide sequence (C41-A42-A43) unique to the genomic ribozyme, the rate constant for cleavage decreased substantially, while activity of the antigenomic ribozyme was enhanced by introducing a CAA sequence. From the crystal structure, it has been proposed that C41 in this sequence is protonated. To investigate a possible connection between activity at low pH and protonation of C41, mutations were made that were predicted to either eliminate protonation or alter the nature of the tertiary interaction upon protonation. In the absence of added Mg2+, these mutations reduced activity and eliminated the observed pH-rate dependence. Thermal denaturation studies revealed a pH-sensitive structural feature in the genomic ribozyme, while unfolding of the mutant ribozymes was pH-independent. We propose that, in the absence of added Mg2+, protonation of C41 contributes to enhanced activity of the HDV genomic ribozyme at low pH.     

A conserved bulged adenosine in a peripheral duplex of the antigenomic HDV self-cleaving RNA reduceskinetic trapping of inactive conformations.

In the ribozyme of hepatitis delta virus antigenomic RNA, two short duplexes, P2 and P2a, stabilize the active self-cleaving structure. However, P2a also promotes kinetic trapping of non-native structures. A bulged adenosine (A14) separates P2a and P2; this bulged A is conserved in clinical isolates of HDV but is unlikely to be physically close to the cleavage site phosphate in the ribozyme structure. Removing the bulge did not significantly slow the rate of cleavage but slowed the conversion of inactive to active conformations. In the absence of the bulged A, inactive conformations required higher urea concentrations or higher temperatures to be activated. Thus, the bulged-nucleotide in the P2-P2a duplex did not provide an essential kink or hinge between P2 and P2a that was required for cleavage activity but, rather, increased the rate of refolding from an inactive to an active ribozyme structure. These data also suggest a model in which P2 and P2a form a coaxial stacked helix of 9 bp, the most likely arrangement being one in which P2-P2a is roughly parallel to P1.     

Metal binding motif in the active site of the HDV ribozyme binds divalent and monovalent ions

The hepatitis delta virus (HDV) ribozyme uses both metal ion and nucleobase catalysis in its cleavage mechanism. A reverse G·U wobble was observed in a recent crystal structure of the precleaved state. This unusual base pair positions a Mg(2+) ion to participate in catalysis. Herein, we used molecular dynamics (MD) and X-ray crystallography to characterize the conformation and metal binding characteristics of this base pair in product and precleaved forms. Beginning with a crystal structure of the product form, we observed formation of the reverse G·U wobble during MD trajectories. We also demonstrated that this base pair is compatible with the diffraction data for the product-bound state. During MD trajectories of the product form, Na(+) ions interacted with the reverse G·U wobble in the RNA active site, and a Mg(2+) ion, introduced in certain trajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G·U wobble with bound Mg(2+) remained intact during MD simulations. When we removed Mg(2+) from the starting precleaved structure, Na(+) ions interacted with the reverse G·U wobble. In support of the computational results, we observed competition between Na(+) and Mg(2+) in the precleaved ribozyme crystallographically. Nonlinear Poisson-Boltzmann calculations revealed a negatively charged patch near the reverse G·U wobble. This anionic pocket likely serves to bind metal ions and to help shift the pK(a) of the catalytic nucleobase, C75. Thus, the reverse G·U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme.     

Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA that resides in the HDV genome and regulates its replication. The native fold of the ribozyme is complex, having two pseudoknots. Earlier work implicated four non-native pairings in slowing pseudoknot formation: Alt 1, Alt 2, Alt 3, and Alt P1. The goal of the present work was design of a kinetically simplified and maximally reactive construct for in vitro mechanistic and structural studies. The initial approach chosen was site-directed mutagenesis in which known alternative pairings were destabilized while leaving the catalytic core intact. Based on prior studies, the G11C/U27Delta double mutant was prepared. However, biphasic kinetics and antisense oligonucleotide response trends opposite those of the well-studied G11C mutant were observed suggesting that new alternative pairings with multiple registers, termed Alt X and Alt Y, had been created. Enzymatic structure mapping of oligonucleotide models supported this notion. This led to a model wherein Alt 2 and the phylogenetically conserved Alt 3 act as "folding guides", facilitating folding of the major population of the RNA molecules by hindering formation of the Alt X and Alt Y registers. Attempts to eliminate the strongest of the Alt X pairings by rational design of a quadruple mutant only resulted in more complex kinetic behavior. In an effort to simultaneously destabilize multiple alternative pairings, studies were carried out on G11C/U27Delta in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs approximately 60 min(-1)) kinetics to be realized. To account for this, a model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.     

An RNA polymerase pause site is associated with the immunoglobulin mus poly(A) site

Immunoglobulin mu alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (mum) and cleavage-polyadenylation (mus) reactions. When we deleted sequences 50 to 200 nucleotides beyond the mus poly(A) site, the mus/mum mRNA ratio decreased three- to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the mus poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the mu fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the mus poly(A) site, even when the poly(A) site was inactivated. When this mu fragment and another pause site were inserted 1 kb downstream from the mus poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.     

Unique nucleotide sequence adjacent to the poly (A) region in yeast RNA

Yeast cells growing in a low phosphate medium were labeled with a pulse of ³²P_i or [³H]adenine and harvested after 15 minutes. Total RNA was extracted and digested with ribonuclease T₁. Poly(A)-rich fragments were isolated from the digest by hybridization to poly(U) impregnated fiberglass filters. Gel filtration showed the fragments to have a uniform chain length of about sixteen. Analysis of the composition gave (A₁₁, C₄, U). Complete pancreatic ribonuclease and partial spleen phosphodiesterase digests gave the sequence of the 5′ end of the fragment as CpApApUp-. Since the fragment was a ribonuclease T₁ product, the data points to a unique sequence of at least five residues, -GpCpApApUp-, adjacent to the poly(A)-rich terminus of pulse-labeled yeast mRNA. The remainder of the poly(A)-rich fragment consists of A residues with a few randomly interspaced C residues. The known specificity of yeast poly(A) polymerase can account for the presence of C residues in poly(A) tracts.     

Alternative tertiary structure attenuates self-cleavage of the ribozyme in the satellite RNA of barley yellow dwarf virus.

A self-cleaving satellite RNA associated with barley yellow dwarf virus (sBYDV) contains a sequence predicted to form a secondary structure similar to catalytic RNA molecules (ribozymes) of the 'hammerhead' class (Miller et al., 1991, Virology 183, 711-720). However, this RNA differs from other naturally occurring hammerheads both in its very slow cleavage rate, and in some aspects of its structure. One striking structural difference is that an additional helix is predicted that may be part of an unusual pseudoknot containing three stacked helices. Nucleotide substitutions that prevent formation of the additional helix and favor the hammerhead increased the self-cleavage rate up to 400-fold. Compensatory substitutions, predicted to restore the additional helix, reduced the self-cleavage rate by an extent proportional to the calculated stability of the helix. Partial digestion of the RNA with structure-sensitive nucleases supported the existence of the proposed alternative structure in the wildtype sequence, and formation of the hammerhead in the rapidly-cleaving mutants. This tertiary interaction may serve as a molecular switch that controls the rate of self-cleavage and possibly other functions of the satellite RNA.     

Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells

The deviant poly(A) signal of hepatitis B virus (HBV) not only controls the formation of the 3' end of all the viral RNA, but is also crucial for HBV replication. Hence, a cis-releasing hammerhead ribozyme (RzA) targeted to the poly(A) signal region of HBV subtype adr was investigated for its antiviral effects. In vitro, RzA cleaved HBV RNA at its target site up to 70%, while the disabled ribozyme (dRzA), which had a one-base mutation in the catalytic site, did not cleave the target RNA at all. When the ribozymes were cotransfected into HepG2 cells with the HBV genome-containing plasmid p3.6II, the wild-type ribozyme RzA could effectively decrease HBV RNA levels and inhibit HBV replication, whereas its disabled form, dRzA, had much weaker effects, indicating that the active catalytic domain of the hammerhead ribozyme could markedly increase the extent of antisense-mediated inhibition. In addition, there was a gradient of effectiveness: the higher the amount of released ribozyme, the more the reduction in target HBV RNA in cells as well as progeny DNA reduction. These results suggest the possibility of the hammerhead ribozyme RzA to be used for the gene therapy of HBV infection.