A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs

Hepatitis δ virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5′OH and 2′,3′-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5′ hydroxyl and 2′,3′- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.

Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5′ end of the substrate to the 3′ end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA

The protein component of ribonuclease P (RNase P) binds to the RNA subunit, forming a functional ribonucleoprotein complex in vivo and enhancing the affinity of the precursor tRNA (pre-tRNA) substrate. Photocrosslinking experiments with pre-tRNA bound to RNase P reconstituted with the protein component of Bacillus subtilis ribonuclease P (P protein) site specifically modified with a crosslinking reagent indicate that: (i) the central cleft of P protein directly interacts with the single-stranded 5′ leader sequence of pre-tRNA, and (ii) the orientation and register of the pre-tRNA leader sequence in the central cleft places the protein component in close proximity to the active site. This unique mode of interaction suggests that the catalytic active site in RNase P occurs near the interface of RNA and protein. In contrast to other ribonucleoprotein complexes where the protein mainly stabilizes the active tertiary fold of the RNA, a critical function of the protein component of RNase P is to alter substrate specificity and enhance catalytic efficiency.

A ribozyme-mediated, gene “knockdown” strategy for the identification of gene function in zebrafish

The zebrafish system offers many unique opportunities for the study of molecular biology. To date, only random mutagenesis, and not directed gene knockouts, have been demonstrated in this system. To more fully develop the potential of the zebrafish system, an approach to effectively inhibit the expression of any targeted gene in the developing zebrafish embryo has been developed. This approach uses a transient, cytoplasmic, T7 expression system, injected into the fertilized zebrafish egg to rapidly produce high levels of a ribozyme directed against the mRNA encoded by the targeted gene to inhibit its expression. In a demonstration of this strategy, expression of the recessive dominant zebrafish no tail gene was effectively inhibited by using this strategy to yield a phenotype identical to that resulting from a known defective mutation in this same gene. This, ribozyme-mediated, message deletion strategy may have use in determining the function of genetic coding sequences of unknown function.

Oligonucleotide-directed peptide synthesis in a ribosome- and ribozyme-free system

Peptide bond formation by the ribosome requires 23S rRNA and its interaction with the 3′-CCA end of tRNA. To investigate the possible evolutionary development of the peptidyl transfer reaction, we tried to obtain peptide bond formation without the ribosome or rRNA simply by using a piece of tRNA—an aminoacyl-minihelix—mixed with sequence-specific oligonucleotides that contained puromycin. Peptide bond formation was detected by gel electrophoresis, TLC analysis, and mass spectrometry. Peptide synthesis depended on sequence complementarity between the 3′-CCA sequence of the minihelix and the puromycin-bearing oligonucleotide. However, proximity of the reacting species was not by itself sufficient for peptide bond formation. In addition, imidazole as a catalyst was required. Its role may be similar to the recently proposed mechanism, wherein A2451 of 23S rRNA works as a general base. Thus, peptide bond formation can be achieved with a simple, minimized system that captures the essence of an interaction seen in the ribosome.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under kcat/KM conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

Formation of a GNRA tetraloop in P5abc can disrupt an interdomain interaction in the Tetrahymena group I ribozyme

The secondary structure of a truncated P5abc subdomain (tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron ribozyme changes when its tertiary structure forms. We have now used heteronuclear NMR spectroscopy to determine its conformation in solution. The tP5abc RNA that contains only secondary structure is extended compared with the tertiary folded form; both forms coexist in slow chemical exchange (the interconversion rate constant is slower than 1 s−1) in the presence of magnesium. Kinetic experiments have shown that tertiary folding of the P5abc subdomain is one of the earliest folding transitions in the group I intron ribozyme, and that it leads to a metastable misfolded intermediate. Previous mutagenesis studies suggest that formation of the extended P5abc structure described here destabilize a misfolded intermediate. This study shows that the P5abc RNA subdomain containing a GNRA tetraloop in P5c (in contrast to the five-nucleotide loop P5c in the tertiary folded ribozyme) can disrupt the base-paired interdomain (P14) interaction between P5c and P2.

Cooperative binding of effectors by an allosteric ribozyme

An allosteric ribozyme that requires two different effectors to induce catalysis was created using modular rational design. This ribozyme construct comprises five conjoined RNA modules that operate in concert as an obligate FMN- and theophylline-dependent molecular switch. When both effectors are present, this ‘binary’ RNA switch self-cleaves with a rate enhancement of ∼300-fold over the rate observed in the absence of effectors. Kinetic and structural studies implicate a switching mechanism wherein FMN binding induces formation of the active ribozyme conformation. However, the binding site for FMN is rendered inactive unless theophylline first binds to its corresponding site and reorganizes the RNA structure. This example of cooperative binding between allosteric effectors reveals a level of structural and functional complexity for RNA that is similar to that observed with allosteric proteins.

Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20–50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to γ-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of ∼2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the α parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

The thermodynamic origin of the stability of a thermophilic ribozyme

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg2+ to fold to their native structures that are very similar. The stability is measured as a function of Mg2+ and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range.

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra- and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stem-loops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.

Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers

Accurately identifying accessible sites in RNA is a critical prerequisite for optimising the cleavage efficiency of hammerhead ribozymes and other small nucleozymes. Here we describe a simple RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible sites in gene length RNAs. Twelve semi-randomised RNA–DNA–RNA chimeric oligonucleotide probes, known as ‘gapmers’, were used to direct RNase H cleavage of transcripts with the specificity expected for hammerhead ribozymes, i.e. after NUH sites (where H is A, C or U). Cleavage sites were identified simply by the mobility of RNase H cleavage products relative to RNA markers in denaturing polyacrylamide gels. Sites were identified in transcripts encoding human interleukin-2 and platelet-derived growth factor. Thirteen minimised hammerhead ribozymes, miniribozymes (Mrz), were synthesised and in vitro cleavage efficiency (37°C, pH 7.6 and 1 mM MgCl2) at each site was analysed. Of the 13 Mrz, five were highly effective, demonstrating good initial rate constants and extents of cleavage. The speed and accuracy of this method commends its use in screening for hammerhead-accessible sites.

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.

Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.