Translational suppression by hammerhead ribozymes and inactive variants in S. cerevisiae

The activity of hammerhead ribozymes in S. cerevisiae was assessed using two ribozymes that were designed to intramolecularly attack the hepatitis B viral X mRNA. The ribozymes effectively suppressed the expression of the X-lacZ fusion gene, when they were inserted at the 5′ end of the X mRNA. The ribozymes cleaved the target RNA efficiently at the targeted phosphodiester bond, but the inactive mutants carrying G5-to-A substitution in the core did not, as the total RNA preparations of yeast extracts was assayed by primer extension. These G5A mutants, however, exerted the suppression as effectively as the wild-type ribozymes. The results, with several mutations introduced to a ribozyme, suggested that either mere formation of hammerhead-like structures with the three stems, or the formation of any two stems, could inhibit translation. Thus, the hammerhead-like structures, leading to cleavage or not, could effectively suppress translation, especially when formed around the initiation codon. The G5-to-A and U7-to-G mutations and replacement of the stem-II hairpin tetraloop did not appear to affect the formation of the inhibitory structure(s). The inhibition that was observed when stems I and III were directly connected without a loop or with a stem II hairpin was completely relieved when they were connected with only the loop of stem II (not containing the stem portion).     

Functional dissection of adenovirus VAI RNA.

During the course of adenovirus infection, the VAI RNA protects the translation apparatus of host cells by preventing the activation of host double-stranded RNA-activated protein kinase, which phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2. In the absence of VAI RNA, protein synthesis is drastically inhibited at late times in infected cells. The experimentally derived secondary structure of VAI RNA consists of two extended base-paired regions, stems I and III, which are joined by a short base-paired region, stem II, at the center. Stems I and II are joined by a small loop, A, and stem III contains a hairpin loop, B. At the center of the molecule and at the 3' side, stems II and III are connected by a short stem-loop (stem IV and hairpin loop C). A fourth, minor loop, D, exists between stems II and IV. To determine sequences and domains critical for function within this VAI RNA structure, we have constructed adenovirus mutants with linker-scan substitution mutations in defined regions of the molecule. Cells infected with these mutants were analyzed for polypeptide synthesis, virus yield, and eIF-2 alpha kinase activity. Our results showed that disruption of base-paired regions in the distal parts of the longest stems, I and III, did not affect function, whereas mutations causing structural perturbations in the central part of the molecule containing stem II, the proximal part of stem III, and the central short stem-loop led to loss of function. Surprisingly, one substitution mutant, sub742, although dramatically perturbing the integrity of the structure of this central portion, showed a wild-type phenotype, suggesting that an RNA with an alternate secondary structure is functional. On the basis of sensitivity to single-strand-specific RNases, we can derive a novel secondary structure for the mutant RNA in which a portion of the sequences may fold to form a structure that resembles the central part of the wild-type molecule, which suggests that only the short stem-loop located in the center of the molecule and the adjoining base-paired regions may define the functional domain. These results also imply that only a portion of the VAI RNA structure may be recognized by the host factor(s).     

Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA-RNA interaction creates apical loop-internal loop complex. The aptamer binds the target with an apparent K(d) of 35nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA-RNA interactions.     

Binary hammerhead ribozymes with improved catalytic activity

A new design of binary hammerhead ribozymes displaying high catalytic activity and nucleolytic stability is described. These catalytic structures consist of two partially complementary oligoribonucleotides, capable of assembling into the hammerhead-like structure without tetraloop II on binding to the RNA target. A series of these binary ribozymes targeting the translation initiation region of multiple drug resistance gene mdr1 mRNA was synthesized and assessed in terms of catalytic activity under single and multiple reaction turnover conditions. Enhanced nuclease resistance of the binary ribozymes was achieved by incorporation of 2'-modified nucleotides at selected positions, along with addition of a 3'-3'-linked thymidine cap. The new binary ribozymes exhibit higher RNA cleavage activity than their full-length analogs because of faster dissociation of cleavage products. Furthermore, an excess of one of the ribozyme strands provides the possibility to unfold structured regions of the target RNA and facilitate productive complex formation.     

Sequence elements outside the catalytic core of natural hairpin ribozymes modulate the reactions differentially

Abstract Hairpin ribozymes occur naturally only in the satellite RNAs of tobacco ringspot virus (TRsV), chicory yellow mottle virus (CYMoV) and arabis mosaic virus (ArMV). The catalytic centre of the predominantly studied sTRsV hairpin ribozyme, and of sArMV is organised around a four-way helical junction. We show here that sCYMoV features a five-way helical junction instead. Mutational analysis indicates that the fifth stem does not influence kinetic parameters of the sCYMoV hairpin ribozyme in vitro reactions, and therefore seems an appendix to that junction in the other ribozymes. We report further that all three ribozymes feature a three-way helical junction outside the catalytic core in stem A, with Watson-Crick complementarity to loop nucleotides in stem B. Kinetic analyses of cleavage and ligation reactions of several variants of the sTRsV and sCYMoV hairpin ribozymes in vitro show that the presence of this junction interferes with their reactions, particularly the ligation. We provide evidence that this is not due to a presumed interaction of the afore-mentioned elements in stems A and B. The evolutionary survival of this cis-inhibiting element seems rather to be caused by the coincidence of its position with that of the hammerhead ribozyme in the other RNA polarity.     

An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in trans-translation.

Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+). Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.     

Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA.

Natural antisense RNAs have stem-loop (hairpin) secondary structures that are important for their function. The sar antisense RNA of phage P22 is unusual: the 3' half of the molecule forms an extensive stem-loop, but potential structures for the 5' half are not predicted to be thermodynamically stable. We devised a novel method to determine the secondary structure of sar RNA by examining the electrophoretic mobility on non-denaturing gels of numerous sar mutants. The results show that the wild-type RNA forms a 5' stem-loop that enhances electrophoretic mobility. All mutations that disrupt the stem of this hairpin decrease mobility of the RNA. In contrast, mutations that change the sequence of the stem without disrupting it (e.g. change G.U to A.U) do not affect mobility. Nearly all mutations in single-stranded regions of the structure also have no effect on mobility. Confirmation of the proposed 5' stem-loop was obtained by constructing and analyzing compensatory double mutants. Combinations of mutations that restore a base-pair of the stem also restore mobility. The genetic phenotypes of sar mutants confirm that the proposed secondary structure is correct and is essential for optimal activity of the antisense RNA in vivo.     

Mechanism of binding of the antisense and target RNAs involved in the regulation of IncB plasmid replication.

The replication frequency of the IncB miniplasmid pMU720 is dependent upon the expression of the repA gene. Binding of a small, highly structured, antisense RNA (RNA I) to its complementary target in the RepA mRNA (RNA II) inhibits repA expression and thus regulates replication. Analyses of binding of RNA I to RNA II indicated that the reaction consists of three major steps. The first step, initial kissing complex formation, involves base pairing between complementary sequences in the hairpin loops of RNA I and RNA II. The second step is facilitated by interior loop structures in the upper stems of RNA I and RNA II and involves intrastand melting and interstrand pairing of the upper stem regions to form an extended kissing complex. This complex was shown to be sufficient for inhibition of repA expression. The third step involves stabilization of the extended kissing complex by pairing between complementary single-stranded tail regions of RNA I and RNA II. Thus, the final product of RNA I-RNA II binding is not a full duplex between the two molecules.     

Mutational Analysis of the mRNA Operator for T4 DNA Polymerase

Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.     

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.     

3' end processing of mouse histone pre-mRNA: evidence for additional base-pairing between U7 snRNA and pre-mRNA.

We have analysed the extent of base-pairing interactions between spacer sequences of histone pre-mRNA and U7 snRNA present in the trans-acting U7 snRNP and their importance for histone RNA 3' end processing in vitro. For the efficiently processed mouse H4-12 gene, a computer analysis revealed that additional base pairs could be formed with U7 RNA outside of the previously recognised spacer element (stem II). One complementarity (stem III) is located more 3' and involves nucleotides from the very 5' end of U7 RNA. The other, more 5' located complementarity (stem I) involves nucleotides of the Sm binding site of U7 RNA, a part known to interact with snRNP structural proteins. These potential stem structures are separated from each other by short internal loops of unpaired nucleotides. Mutational analyses of the pre-mRNA indicate that stems II and III are equally important for interaction with the U7 snRNP and for processing, whereas mutations in stem I have moderate effects on processing efficiency, but do not impair complex formation with the U7 snRNP. Thus nucleotides near the processing site may be important for processing, but do not contribute to the assembly of an active complex by forming a stem I structure. The importance of stem III was confirmed by the ability of a complementary mutation in U7 RNA to suppress a stem III mutation in a complementation assay using Xenopus laevis oocytes. The main role of the factor(s) binding to the upstream hairpin loop is to stabilise the U7-pre-mRNA complex. This was shown by either stabilising (by mutation) or destabilising (by increased temperature) the U7-pre-mRNA base-pairing under conditions where hairpin factor binding was either allowed or prevented (by mutation or competition). The hairpin dependence of processing was found to be inversely related to the strength of the U7-pre-mRNA interaction.     

Secondary structure of the self-cleaving RNA of hepatitis delta virus: applications to catalytic RNA design.

A model for the secondary structure of the self-cleaving RNA from hepatitis delta virus was tested. Specific base changes were introduced in each of four regions with the potential for base-pairing (stems I-IV), and for each variant sequence, a rate constant for cleavage was determined. In each stem, mutations that would interfere with Watson-Crick base-pairing also reduced the first-order rate constants by 10-10(4)-fold relative to the unmodified version. Within stems I and II and a shortened form of stem IV, compensatory changes resulted in rates of cleavage equal to or greater than the unaltered ribozyme sequence. Stem III compensatory mutants cleaved faster than the uncompensated mutants although they were not as active as the natural sequence, suggesting additional sequence-dependent requirements within this region. Structure probing of RNA containing the stem II mutations provided an independent confirmation of stem II in the ribozyme. The predictive value of the model was tested by designing two trans-acting ribozymes which were circularly permuted composites of genomic, antigenomic, and unique sequences. The core of these two catalytic RNAs was the same, but they otherwise differed in that, in one of them, a constraining tetraloop sequence was added to stem II. Both ribozymes catalyzed the trans cleavage of a substrate oligoribonucleotide, thus providing additional evidence for stem II and the proposed structure in general.     

Optimization of hammerhead ribozymes for the cleavage of S100A4 (CAPL) mRNA

Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6+8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6+6) and (5+5) nt. Stimulation was particularly strong in the case of the (5+5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.     

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.     

Viral escape from antisense RNA

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31–270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3–4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson–Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.     

NMR study of a synthetic DNA hairpin

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.