The cytotoxins alpha-sarcin and ricin retain their specificity when tested on a synthetic oligoribonucleotide (35-mer) that mimics a region of 28 S ribosomal ribonucleic acid

An oligoribonucleotide (35-mer) that mimics the alpha-sarcin and the ricin region of eukaryotic 28 S rRNA was transcribed in vitro from a synthetic template with T7 RNA polymerase and was used to test whether the specificity of the hydrolysis by the toxins was retained. alpha-Sarcin, at a low concentration, cleaved a single phosphodiester bond on the 3' side of a guanosine residue in the synthetic oligomer that corresponds to G-4325 in 28 S rRNA, the site of action of the toxin in intact ribosomes. At a high concentration of alpha-sarcin, the substrate (35-mer) was hydrolyzed after each of its purines. alpha-Sarcin was without an effect on a synthetic RNA (20-mer) that reproduces the near universal sequence of nucleotides in the loop, but lacks the stem, of the toxin's domain. Thus, the specificity of the attack of alpha-sarcin on a precise region of 28 S rRNA appears to be contingent on the sequence of the nucleotides and the structure of the domain. Ricin depurinated a nucleotide in the synthetic oligomer (35-mer), and in the presence of aniline the phosphoribose backbone was cleaved at a position that conforms to A-4324 in 28 S rRNA, the site of action of the toxin in vivo.     

NMR analysis of tertiary interactions in HDV ribozymes.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems designed on the basis of the "pseudoknot" model were synthesized and their tertiary interactions were analyzed by NMR spectroscopy. Rz-1 is a cis-acting ribozyme system (the cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. Rz-2 is a trans-acting ribozyme system (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-2 was partially labeled with stable isotopes in guanosine residues of enzyme 35mer. Rz-4 is a trans-acting ribozyme system (substrate: 8mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme 53mer) which was designed by Perrotta and Been. Rz-4 has the same sequence and an extra loop closing stem IV. From 2D-NOESY and 2D-HSQC (except for Rz-4) spectra, it was suggested each ribozyme forms "pseudoknot" type structure in solution. Additionally, it was found that G38 of Rz-1, G28 and G29 of Rz-2 and Rz-4 form base-pairs. These novel base-pairs are observed in the crystal structure of a modified genomic HDV RNA. From temperature change experiment of Rz-2, the imino proton signal of G28 disappeared at 50 degrees C earlier than the other corresponding signals. Upon MgCl2 titration of Rz-2, this signal showed the largest shift.     

Microinjected oligonucleotides complementary to the alpha-sarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes

The integrity of the alpha-sarcin loop in 28 S ribosomal RNA is critical during protein synthesis. The toxins alpha-sarcin, ricin, Shiga toxin, and Shiga-like toxin inhibit protein synthesis in oocytes by attacking specific nucleotides within this loop (Ackerman, E.J., Saxena, S. K., and Ulbrich, N. (1988) J. Biol. Chem. 263, 17076-17083; Saxena, S.K., O'Brien, A.D., and Ackerman, E.J. (1989) J. Biol. Chem. 264, 596-601). We injected Xenopus oocytes with deoxyoligonucleotides complementary to the 17-nucleotide alpha-sarcin loop of Xenopus 28 S rRNA. Only injected oligonucleotides fully covering the alpha-sarcin loop or slightly beyond inhibited oocyte protein synthesis. Shorter alpha-sarcin domain deoxyoligonucleotides complementary to the alpha-sarcin and ricin sites but not spanning the entire loop were less effective inhibitors of protein synthesis. The alpha-sarcin domain oligonucleotides covering the entire loop were more effective inhibitors of protein synthesis than injected cycloheximide at equivalent concentrations. Control oligonucleotides complementary to nine other regions of Xenopus 28 S rRNA as well as universal M13 DNA sequencing primers had no effect on oocyte protein synthesis. Oligonucleotides complementary to the highly conserved alpha-sarcin domain therefore represent an alternative to catalytic toxins for causing cell death and may prove effective in immunotherapy.     

Design and NMR analysis of HDV ribozymes for structural investigation.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems (Rz-1 to approximately Rz-3) (Fig. 1) were designed on the basis of the "pseudoknot" structure model and synthesized. Rz-1 is a cis-acting ribozyme system (a cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. The 2D-NOESY and 2D-HSQC data for Rz-1 suggest that Rz-1 forms the pseudoknot structure and G38 which is opposite to the cleavage site makes a base-pair. Rz-2 is a trans-acting ribozyme system which consists of three RNA oligomer strands (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-3 is a ribozyme in which the three RNA strands of Rz-2 are connected. It turns out that Rz-3 forms an inactive structure with low cleavage activity (k(obs) = 0.009) and final cleavage yield (6%). Rz-3 has the highest cleavage activity at pH 5.5. The optimal activity at acidic pH is similar to that of the wild type ribozyme. We also synthesized and examined the activity and structure of Rz-4 (designed by Perrotta and Been) which consists of two RNA strands (1).     

Cleavage of specific sites of RNA by designed ribozymes

Two ribozymes were designed for site-specific cleavage of RNA. A UA site in an undecaribonucleotide was cleaved by a ribozyme consisting of two partially paired oligoribonucleotides with chain lengths of 19 and 15. The other ribozyme, which consists of a 19-mer and a 13-mer, recognized a UC sequence at positions 42 and 43 of 5 S rRNA.     

A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.     

Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence.

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.     

Structural energetics and base-pair opening dynamics in sarcin-ricin domain RNA

The sarcin-ricin domain is a universal element of the RNA from the large ribosomal subunit. The domain is part of the binding site for elongation factors and is specifically cleaved by the toxins alpha-sarcin and ricin. In this work, we have mapped the energetics and dynamics of individual structural motifs in a 29-mer RNA oligomer containing the sarcin-ricin domain. The stability of individual base pairs in the structure was characterized from measurements of the exchange rates of imino protons using nuclear magnetic resonance spectroscopy at 10 degrees C. The measurements also provided the rates of opening and closing for selected base pairs. The results reveal that the structural stabilization free energies in the sarcin-ricin domain are broadly distributed between 2.9 and 10.6 kcal/mol. One of the least stable sites in the structure is the noncanonical G-A base pair located next to the phosphodiester bond that is cleaved by alpha-sarcin. The low stability of this base pair supports the proposal that cleavage by alpha-sarcin occurs by a base flipping mechanism. The opening dynamics of other base pairs is affected by elements of the structure such as the bulged-G motif and its cross-strand stacking. Participation in these motifs increases the lifetimes of the bases in an open, solvent-accessible conformation.     

RNA-protein interaction. An analysis with RNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function

alpha-Sarcin is a cytotoxic protein that inactivates ribosomes by hydrolyzing a single phosphodiester bond on the 3' side of G-4325 in eukaryotic 28 S rRNA. We have examined the requirements for the recognition by alpha-sarcin of this domain using a synthetic oligoribonucleotide (35-mer) that reproduces the sequence and, we presume, the secondary structure (a stem, a bulged nucleotide, and a loop) at the site of modification. The wild type structure and a large number of variants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. Recognition of the substrate is strongly favored by a G at the position that corresponds to 4325. There is an absolute requirement for a helical stem; however, it can be reduced from the 7 base pairs in the natural structure to 3 without loss of specificity. The nature of the base pairs in the stem modifies but does not abolish recognition; whereas, the bulged nucleotide does not contribute to identification. Cleavage is materially affected by altering the nucleotides in the universal sequence surrounding G-4325 and changing the position in the loop of the tetranucleotide GAG(sarcin)A leads to loss of recognition by the toxin. We propose that the alpha-sarcin domain RNA participates in elongation factor catalyzed binding of aminoacyl-tRNA and of translocation; that translocation is driven by transitions in the structure of the alpha-sarcin domain RNA initiated by the binding of the factors, or the hydrolysis of GTP, or both; and that to toxin inactivates the ribosomes by preventing this transition.     

Alpha-sarcin causes a specific cut in 28 S rRNA when microinjected into Xenopus oocytes

The toxin alpha-sarcin specifically cuts 28 S rRNA at a single position 393 nucleotides from its 3' end in isolated rat liver polysomes, provided the ribosomes are pretreated with EDTA or puromycin (Endo, Y. & Wool, I. G. (1982) J. Biol. Chem. 257, 9054-9060). In addition, alpha-sarcin behaves as a purine-specific RNase on deproteinized RNA, cleaving on the 3' side of purines in both single- and double-stranded RNA (Endo, Y., Huber, P. W., and Wool, I. G. (1983) J. Biol. Chem. 258, 2662-2667). Since alpha-sarcin does not readily enter tissue culture cells, we have injected it into Xenopus oocytes in order to determine whether the toxin cleaves after all purines or if it specifically makes a single cut in 28 S rRNA in intact cells. We report here that in oocytes alpha-sarcin specifically cuts 28 S rRNA 377 nucleotides from its 3' end, even when used at concentrations that would degrade deproteinized RNA. alpha-Sarcin does not behave as a general nuclease when injected into Xenopus oocytes nor does it operate by another means such as initiating proteolytic digestion of endogenous oocyte proteins. We demonstrate that injected alpha-sarcin causes a rapid decline in oocyte protein synthesis for soluble cytoplasmic proteins, similar in effect to injection of cycloheximide or puromycin.     

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+.     

Design and specificity of hammerhead ribozymes against calretinin mRNA.

We obtained a partial sequence of mouse calretinin mRNA from cDNA clones, and designed hammerhead ribozymes to cleave positions within it. With a view to optimising hammerhead ribozymes for eliminating the mRNA in vivo, we varied the length and sequence of the three duplex 'arms' and measured the cleavage of long RNA substrates in vitro at 37 degrees C (as well as 50 degrees C). Precise cleavage occurred, but it could only go to completion with a large excess of ribozyme. The evidence suggests that the rate-limiting step with a large target is not the cleavage, but the formation of the active ribozyme: substrate complex. The efficiency varied unpredictably according to the target site, the length of the substrate RNA, and the length of the ribozyme; secondary structure in vitro may be responsible. We particularly investigated the degree of sequence-specificity. Some mismatches could be tolerated, but shortening of the total basepairing with the substrate to less than 14 bp drastically reduced activity, implying that interaction with weakly-matched RNAs is unlikely to be a serious problem in vivo. These results suggest that specific and complete cleavage of a mRNA in vivo should be possible, given high-level expression of a ribozyme against a favourable target site.     

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.     

Ribozyme mediated destruction of RNA in vivo.

Previous studies have demonstrated that high ribozyme to substrate ratios are required for ribozyme inhibitory function in nuclear extracts. To obtain high intracellular levels of ribozymes, tRNA genes, known to be highly expressed in most tissues, have been modified for use as ribozyme expression cassettes. Ribozyme coding sequences were placed between the A and the B box, internal promoter sequences of a Xenopus tRNAMet gene. When injected into the nucleus of frog oocytes, the ribozyme tRNA gene (ribtDNA) produces 'hammerhead' ribozymes which cleave the 5' sequences of U7snRNA, its target substrate, with high efficiency in vitro. Oocytes were coinjected with ribtDNA, U7snRNA and control substrate RNA devoid of a cleavage sequence. It was found that the ribtRNA remained localized mainly in the nucleus, whereas the substrate and the control RNA exited rapidly into the cytoplasm. However, sufficient ribtRNA migrated into the cytoplasm to cleave, and destroy, the U7snRNA. Thus, the action of targeted 'hammerhead' ribozymes in vivo is demonstrated.     

A pH controlled conformational switch in the cleavage site of the VS ribozyme substrate RNA

The VS ribozyme is a 154 nucleotide sequence found in certain natural strains of Neurospora. The RNA can be divided into a substrate and a catalytic domain. Here we present the solution structure of the substrate RNA that is cleaved in a trans reaction by the catalytic domain in the presence of Mg2+. The 30 nucleotide substrate RNA forms a compact helix capped by a flexible loop. The cleavage site bulge contains three non-canonical base-pairs, including an A+.C pair with a protonated adenine. This adenine (A622) is a pH controlled conformational switch that opens up the internal loop at higher pH. The possible significance of this switch for substrate recognition and cleavage is discussed.     

Effects of antisense DNA against the alpha-sarcin stem-loop structure of the ribosomal 23S rRNA.

Antisense DNAs complementary against various sequences of the alpha-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S rRNA as well as to 70S ribosomes. Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of alpha-sarcin. The RNase alpha-sarcin seems to approach its cleavage site from the 3' half of the loop of the alpha-sarcin domain. Hybridization is efficiently achieved at 37 degrees C and can extend at least into the 3' strand of the stem of the alpha-sarcin domain. However, the inhibition of alpha-sarcin activity is observed at 30 degrees C but not at 37 degrees C. For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C. The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha-sarcin domain.     

特异切割12-脂加氧酶mRNA的核酶体外活性及动力学研究

根据锤头状核酶（Ｒｉｂｏｚｙｍｅ）的作用模式,设计、合成并克隆了特异性切割１２－脂加氧酶（１２－ＬＯ）ｍＮＲＡ的核酶基因。以合成的２５个核苷酸长的１２－脂加氧酶ＲＮＡ片段为底物与转录的核酶ＲＮＡ一起保温检测其体 割活性。实验结果表明,在３７℃保温时,核酶在体外对１２－脂加氧酶具有较高的特异切割活性,其Ｋｍ值为１３００ｎｍｏｌ／Ｌ,其ｋｃａｔ值为０．０８３／ｍｉｎ,在５０℃保温时,核酶具有很高的切割     

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.     

Intracellular ribozyme-catalyzed trans-cleavage of RNA monitored by fluorescence resonance energy transfer

Small catalytic RNAs like the hairpin ribozyme are proving to be useful intracellular tools; however, most attempts to demonstrate trans-cleavage of RNA by ribozymes in cells have been frustrated by rapid cellular degradation of the cleavage products. Here, we describe a fluorescence resonance energy transfer (FRET) assay that directly monitors cleavage of target RNA in tissue-culture cells. An oligoribonucleotide substrate was modified to inhibit cellular ribonuclease degradation without interfering with ribozyme cleavage, and donor (fluorescein) and acceptor (tetramethylrhodamine) fluorophores were introduced at positions flanking the cleavage site. In simple buffers, the intact substrate produces a strong FRET signal that is lost upon cleavage, resulting in a red-to-green shift in dominant fluorescence emission. Hairpin ribozyme and fluorescent substrate were microinjected into murine fibroblasts under conditions in which substrate cleavage can occur only inside the cell. A strong FRET signal was observed by fluorescence microscopy when substrate was injected, but rapid decay of the FRET signal occurred when an active, cognate ribozyme was introduced with the substrate. No acceleration in cleavage rates was observed in control experiments utilizing a noncleavable substrate, inactive ribozyme, or an active ribozyme with altered substrate specificity. Subsequently, the fluorescent substrates were injected into clonal cell lines that expressed cognate or noncognate ribozymes. A decrease in FRET signal was observed only when substrate was microinjected into cells expressing its cognate ribozyme. These results demonstrate trans-cleavage of RNA within mammalian cells, and provide an experimental basis for quantitative analysis of ribozyme activity and specificity within the cell.