Long-range impact of peripheral joining elements on structure and function of the hepatitis delta virus ribozyme

The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function.     

Energetic contribution of non-essential 5' sequence to catalysis in a hepatitis delta virus ribozyme

Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid-base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5' sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5' sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb's free energy of substrate binding revealed a linear relationship with a slope of -1. This relationship is consistent with a model in which components of the substrate immediately 5' to the cleavage site in the HDV ribozyme-substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme.     

A circular trans-acting hepatitis delta virus ribozyme.

A circular trans-acting ribozyme designed to adopt the motif of the hepatitis delta virus (HDV) trans-acting ribozyme was produced. The circular form was generated in vitro by splicing a modified group I intron precursor RNA in which the relative order of the 5' and 3' splice sites, flanking the single HDV-like ribozyme sequence-containing exon, is reversed. Trans-cleavage activity of the circular HDV-like ribozyme was comparable to linear permutations of HDV ribozymes containing the same core sequence, and was shown not to be due to linear contaminants in the circular ribozyme preparation. In nuclear and cytoplasmic extracts from HeLa cells, the circular ribozyme had enhanced resistance to nuclease degradation relative to a linear form of the ribozyme, suggesting that circularization may be a viable alternative to chemical modification as a means of stabilizing ribozymes against nuclease degradation.     

Tertiary structure stabilization promotes hairpin ribozyme ligation.

The hairpin ribozyme catalyzes a reversible RNA cleavage reaction that participates in processing intermediates of viral satellite RNA replication in plants. A minimal hairpin ribozyme consists of two helix-loop-helix segments. These segments associate noncoaxially in the active folded structure in a way that brings catalytically important loop nucleotides into close proximity. The hairpin ribozyme in the satellite RNA of Tobacco Ringspot Virus assembles in the context of a four-way helical junction. Recent physical characterization of hairpin ribozyme structures using fluorescence resonance energy transfer demonstrated enhanced stability of the folded structure in the context of a four-way helical junction compared to minimal hairpin ribozyme variants. Analysis of the functional consequences of this modification of the helical junction has revealed two changes in the hairpin ribozyme kinetic mechanism. First, ribozymes with a four-way helical junction bind 3' cleavage products with much higher affinity than minimal hairpin ribozymes, evidence that tertiary interactions within the folded structure contribute to product binding energy. Second, the balance between ligation and cleavage shifts in favor of ligation. The enhanced ligation activity of hairpin ribozymes that contain a four-way helical junction supports the notion that tertiary structure stability is a major determinant of the hairpin ribozyme proficiency as a ligase and illustrates the link between RNA structure and biological function.     

HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.     

The antisense sequence of the HIV-1 TAR stem-loop structure covalently linked to the hairpin ribozyme enhances its catalytic activity against two artificial substrates

This work is an in vitro study of the efficiency of catalytic antisense RNAs whose catalytic domain is the wild-type sequence of the hairpin ribozyme, derived from the minus strand of the tobacco ringspot virus satellite RNA. The sequence in the target RNA recognized by the antisense molecule was the stem-loop structure of the human immunodeficiency virus-1 (HIV-1) TAR region. This region was able to form a complex with its antisense RNA with a binding rate of 2 x 10(4) M(-1)s(-1). Any deletion of the antisense RNA comprising nucleotides of the stem-loop resulted in a decrease in binding rate. Sequences 3' of the stem in the sense RNA also contributed to binding. This stem-loop TAR-antisense segment, covalently linked to a hairpin ribozyme, enhanced its catalytic activity. The highest cleavage rate was obtained when the stem-loop structure was present in both ribozyme and substrate RNAs and they were complementary. Similarly, an extension at the 5'-end of the hairpin ribozyme increased the cleavage rate when its complementary sequence was present in the substrate. Inclusion of the stem-loop at the 3'-end and the extension at the 5'-end of the hairpin ribozyme abolished the positive effect of both antisense units independently. These results may help in the design of hairpin ribozymes for gene silencing.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

Addition of an extra substrate binding site and partial destabilization of stem structures in HDV ribozyme give rise to high sequence-specificity for its target RNA

Because the substrate binding site (P1) of HDV ribozyme consists of only seven nucleotides, cleavage of undesired RNA is likely to occur when applied for a specific long RNA target such as mRNA. To overcome this problem, we designed modified trans-acting HDV ribozymes with an extra substrate-binding site (P5) in addition to the original binding site (P1). By inserting an additional seven base-pair stem (P5 stem) into the J1/2 single-stranded region of the ribozyme core system and partial destabilization of the P2 or P4 stem, we succeeded in preparation of new HDV ribozymes that can cleave the target RNA depending on the formation of P5 stem. Moreover, the ribozyme with a six-nucleotide P1 site was able to distinguish the substrate RNA with a complete match from that with a single mismatch in the P1 region. These results suggest that the HDV ribozyme system is useful for the application in vivo.     

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library.     

Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme

The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.     

Ribozymes: recent advances in the development of RNA tools

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.     

Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation.

Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication. A compensatory mutation which restores the stem structure also restores TAR decoy RNA function. Synthesis of viral RNA is drastically reduced in cells expressing a functional TAR decoy RNA, but it is unaffected in cells expressing a mutant form of TAR decoy RNA. It is therefore concluded that overexpression of TAR-containing sequences in CEM SS cells interferes with the process of Tat-mediated transactivation of viral gene expression. However, the phenotype of several mutations suggests that TAR decoy RNA does not inhibit HIV-1 gene expression by simply sequestering Tat but rather does so by sequestering a transactivation protein complex, implying that transactivation requires the cooperative binding of both Tat and a loop-binding cellular factor(s) to TAR. Expression of wild-type or mutant forms of TAR had no discernible effects on cell viability, thus reducing concerns about using TAR decoy RNAs as part of an intracellular immunization protocol for the treatment of AIDS.     

Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences.

Conventionally designed ribozymes may be unable to cleave RNA at sites which are inaccessible due to secondary structure. In addition, it may also be difficult to specifically target a conventionally designed ribozyme to some chimeric RNA molecules. Novel approaches for ribozyme targeting were developed by using the L6 bcr-abl fusion RNA as a model. Using one approach, we successfully directed ribozyme nucleation to a site on the bcr-abl RNA that is distant from the GUA cleavage site. These ribozymes bound to the L6 substrate RNA via an anchor sequence that was complementary to bcr sequences. The anchor was necessary for efficient cleavage as the anchor minus ribozyme, a conventionally designed ribozyme, was inefficient at catalyzing cleavage at this same site. The effect of anchor sequences on catalytic rates was determined for two of these ribozymes. Ribozymes generated by a second approach were designed to cleave at a CUU site in proximity to the bcr-abl junction. Both approaches have led to the development of a series of ribozymes specific for both the L6 and K28 bcr-abl chimeric RNAs, but not normal abl or bcr RNAs. The specificity of the ribozyme correlated in part with the ability of the ribozyme to bind substrate as demonstrated by gel shift analyses. Secondary structure predictions for the RNA substrate support the experimental results and may prove useful as a theoretical basis for the design of ribozymes.     

RNA double cleavage by a hairpin-derived twin ribozyme

The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.     

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.     

Properties of antigenomic hepatitis delta virus ribozyme cis- and trans- analogs

A series of permuted variants of antigenomic HDV ribozyme and trans-acting variants were constructed. The catalytic activity study of the ribozymes has shown that all the variants were capable of self-cleaving with equally biphasic kinetics. Ribonuclease and Fe(II)-EDTA cleavage have provided evidence that all designed ribozymes fold according to the pseudoknot model and the conformations of the initial and cleaved ribozyme are different. A scheme of HDV ribozyme self-cleavage reaction was suggested. The role of hydrogen bonds in the reaction was evaluated by substitution of ribose in the ribozyme for deoxyribose. It was found that the 2'-OH group of U23 and C27 is critical for the reaction to occur; the 2'-OH group of U32 and U39 is important, while 2'-OH groups of other nucleotides of loop 3, stem 4 and stem 1 are unimportant for the cleavage activity.