The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant.

The sequence requirements for self-cleavage of hepatitis delta virus genomic RNA were examined using precursor RNAs which were labeled at either the 5' or 3' ends and progressively deleted from the unlabeled end. In the presence of 50% formamide, which enhances self-cleavage in 2 mM MgCl2 at 37 degrees C, 84 nucleotides (nt) 3' of the break site were required. In the absence of formamide the minimum was reduced to 82 nt. Under both sets of conditions, precursors with 1 nt 5' to the break site cleaved. These results allowed two condition-dependent minimal domains for self-cleavage to be defined. However, in the absence of formamide, sequences flanking the minimal domain inhibited cleavage, possibly through involvement in the formation of non-cleaving structures. These data are consistent with the idea that cleavage in vivo could be regulated by alternative RNA structures.     

Antigenomic Hepatitis delta virus ribozymes self-cleave in 18 M formamide.

The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requirements for self-cleavage. While truncating the 1679 nucleotide antigenomic HDV RNA, we have characterized the cleavage requirements of a number of ribozymes of intermediate length. Two of these, containing 186 and 106 HDV nucleotides respectively, cleaved to completion in the presence of 18 M formamide. The 186 nucleotide ribozyme also cleaved to completion in 10 M urea. Removal of an additional 10 nts from the 3' terminus of the 106 nt ribozyme resulted in a loss of the ability to cleave in high concentrations of the denaturants. The interaction of nucleotides near the cleavage site with a sequence within this 10 base region may confer unusual stability on these ribozymes.     

The poly(A) site sequence in HDV RNA alters both extent and rate of self-cleavage of the antigenomic ribozyme

The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1–2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.     

Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA.

To determine the sequence requirements and structural features of the self-cleavage domain of hepatitis delta virus (HDV) antigenomic RNA, we constructed a series of mutants and measured the rate constant of the cleavage reaction for each. The self-cleavage activity of HDV RNA of antigenomic sense was found to reside in a region of less than 90 nucleotides in length. The catalytic domain contained a long complementary sequence which could be deleted to half of its original size. Moreover, this region could be replaced by other sequences as long as they could fold into a stem-and-loop structure. The catalytic domain also required a 6-basepair helix adjacent to the cleaving point for activity. The structural features of these two base-pairing regions are quite similar to those of the HDV genomic self-cleavage domain. The cleavage site as well as the the hinge region (the sequence between the two stems) requires specific sequences for activity.     

Evidence that genomic and antigenomic RNA self-cleaving elements from hepatitis delta virus have similar secondary structures.

The two sequences that define the self-cleaving elements from the genomic and antigenomic RNA of hepatitis delta virus were folded into secondary structures with similar features. Evidence in support of the two models was obtained from limited ribonuclease digestion of genomic and antigenomic RNA fragments containing the sequence 3' of the cleavage site. Under conditions where the rates of self-cleavage are enhanced by addition of 5 M urea (2-10 mM Mg2+ at 37 degrees C), ribonucleases T1, U2, A and V1 generated digestion patterns consistent with the proposed RNA structures. The evidence for a relatively stable structure in urea when Mg2+ is present suggests that denaturant-enhanced rates of self-cleavage could result from destabilization of competing inactive structures.     

Assessment of disparate structural features in three models of the hepatitis delta virus ribozyme.

Three models for the secondary structure of the hepatitis delta virus (HDV) antigenomic self-cleaving RNA element were tested by site-directed mutagenesis. Two models in which bases 5' to the cleavage site are paired with sequence at the 3' end of the element were both inconsistent with the data from the mutagenesis. Specifically, mutations in the 3' sequence which decrease self-cleavage activity could not be compensated by base changes in the 5' sequence as predicted by these models. The evidence was consistent with a third model in which the 3' end pairs with a portion of a loop within the ribozyme sequence to generate a pseudoknot structure. This same pairing was also required to generate higher rates of cleavage in trans with a 15-mer ribozyme, thus ruling out a proposed hammerhead-like 'axehead' model for the HDV ribozyme.     

Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage.

The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the HDV genome. Because the site is located almost adjacent to the 3' end of the delta antigen-coding region, the only known functional open reading frame of HDV, we suggest that the cleavage may have a role not only in genome replication but also in RNA processing, helping to produce a functional mRNA for the translation of delta antigen.     

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation

Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3'-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short insertions/deletions at their 3' termini failed to cross-link. Because U81 and C289 are conserved in PLMVd variants and because the initiation site of PLMVd minus-strand RNA maps at a short double-stranded motif containing C289, the UV-photo-cross-linkable element of tertiary structure may be functionally significant. A second cross-linked species similar in size and sequence to the monomeric circular PLMVd form, observed in some PLMVd variants, probably derives from UV-induced ligation of the two termini resulting from self-cleavage.     

Tm studies of a tertiary structure from the human hepatitis delta agent which functions in vitro as a ribozyme control element.

Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; > 50% of the complexes remain following incubation in 1 mM EDTA at 60 degrees C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures approximately 10 degrees C lower in buffers containing 0.5 mM MgCl2 and 100 mM NaCl than in buffers containing 0.5 mM MgCl2 with no NaCl (78.1 compared with 87.7 degrees C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.     

RNA conformational requirements of self-cleavage of hepatitis delta virus RNA.

Hepatitis delta virus (HDV) RNA subfragments undergo self-cleavage at varying efficiencies. We have developed a procedure of using repeated cycles of heat denaturation and renaturation of RNA to achieve a high efficiency of cleavage. This effect can also be achieved by gradual denaturation of RNA with heat or formamide. These results suggest that only a subpopulation of the catalytic RNA molecules assumes the active conformation required for self-cleavage. This procedure could be of general use for detecting catalytic RNA activities.     

Delta ribozyme has the ability to cleave in transan mRNA.

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.     

Nucleotides -1 to -4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.     

Artificial self-cleaving molecules consisting of a tRNA precursor and the catalytic RNA of RNase P.

We synthesized two types of chimeric RNAs between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a tRNA precursor (pre-tRNA); one had pre-tRNA at the 3' side to the M1 RNA (M1 RNA-pre-tRNA). The second had pre-tRNA at the 5' side of the M1 RNA (pre-tRNA-M1 RNA). Both molecules were self-cleaving RNAs. The self-cleavage of M1 RNA-pre-tRNA occurred at the normal site (5'-end of mature tRNA sequence) and proceeded under the condition of 10 mM Mg2+ concentration. This reaction at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage), while, at 40 mM and 80 mM Mg2+, trans-cleavage partially occurred. The self-cleavage rate was strictly affected by the distance between the M1 RNA and the pre-tRNA in the molecule. The self-cleavage of pre-tRNA-M1 RNA occurred mainly at three sites within the mature tRNA sequence. This cleavage did not occur at 10 mM Mg2+. Use of M1 RNA-pre-tRNA molecule for the in vitro evolution of M1 RNA is discussed.     

Optimal self-cleavage activity of the hepatitis delta virus RNA is dependent on a homopurine base pair in the ribozyme core.

A non-Watson-Crick G.G interaction within the core region of the hepatitis delta virus (HDV) antigenomic ribozyme is required for optimal rates of self-cleavage activity. Base substitutions for either one or both G's revealed that full activity was obtained only when both G's were replaced with A's. At those positions, substitutions that generate potential Watson-Crick, G.U, heteropurine, or homopyrimidine combinations resulted in dramatically lower cleavage activity. A homopurine symmetric base pair, of the same type identified in the high-affinity binding site of the HIV RRE, is most consistent with this data. Additional features shared between the antigenomic ribozyme and the Rev binding site in the vicinity of the homopurine pairs suggest some structural similarity for this region of the two RNAs and a possible motif associated with this homopurine interaction. Evidence for a homopurine pair at the equivalent position in a modified form of the HDV genomic ribozyme was also found. With the postulated symmetric pairing scheme, large distortions in the nucleotide conformation, the sugar-phosphate backbone, or both would be necessary to accommodate this interaction at the end of a helix; we hypothesize that this distortion is critical to the structure of the active site of the ribozyme and it is stabilized by the homopurine base pair.