RNA stem stability in the formation of a self-cleaving hammerhead structure.

The proposed single-hammerhead structure of the self-cleaving newt RNA is unstable due to a weak stem III and therefore is unable to mediate self-cleavage. A double-hammerhead structure with greater theoretical stability has been shown to mediate the self-cleavage of this RNA (Forster et al., 1988, Nature 334, 265). We have found that the double-hammerhead mediated self-cleavage reaction of a 40 base RNA containing the newt sequence (termed nCG) can be converted to a single-hammerhead reaction by increasing the size of stem III and/or of its loop, thereby enabling a single-hammerhead structure to form. In addition, the 5'-self-cleavage fragment of the nCG RNA can act in trans to mediate the self-cleavage of a full-length RNA by the formation of a partial double-hammerhead structure.     

Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA.

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.     

Plant pathogenic RNAs and RNA catalysis.

The rolling circle replication of small circular plant pathogenic RNAs requires a processing step to convert multimeric intermediates to monomers which are then circularized. Eleven such RNAs are known so far, two are viroids, one is viroid-like and the remainder are satellite RNAs dependent on a helper virus for replication. The processing step is RNA-catalysed in all cases, at least in vitro. All plus forms of these RNAs self-cleave via the hammerhead structure whereas only eight of the minus RNAs self-cleave, five via the hammerhead structure and three via the hairpin structure. There are about 20 other viroids where the processing mechanism has yet to be determined but they are likely candidates for a new type of self-cleavage reaction which is predicted to be conserved in all these viroids. Hepatitis delta RNA is the only circular pathogenic RNA known to self-cleave in the animal kingdom. It is feasible that more single-stranded circular pathogenic RNAs are waiting to be discovered and these could be prospective for new types of self-cleavage reactions.     

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.     

A sequence element necessary for self-cleavage of the antigenomic hepatitis delta RNA in 20 M formamide.

The genomic and antigenomic RNAs of hepatitis delta virus are capable of self-cleavage and show no significant sequence similarities to other known self-cleaving RNAs. We have derived an antigenomic delta RNA which cleaves to completion in 15 s in 9 mM magnesium at 37 degrees C and is capable of efficient self-cleavage in concentrations of formamide as high as 20 M. Cleavage in high concentrations of denaturant is dependent upon the presence of a polypurine sequence element, GGAGA, located between 81 and 85 nucleotides downstream of the cleavage site. Mutation of the initial G81G82 to C81C82, or removal of the sequence element, results in a loss of the ability to cleave in high formamide concentrations. Changing the final U-2C-1 of a pyrimidine-rich region, UCUUC, just upstream of the cleavage site, to G-2G-1 severely affects the self-cleavage, but introducing the two mutations, GG to CC and UC to GG, into the same molecule, restoring potential base pairing, partially restores the formamide stability. Relocating the GGAGA sequence upstream of the cleavage site also results in partial restoration of the formamide cleavage. Although the GGAGA sequence is important for self-cleavage under denaturing conditions, it does not appear to be necessary for HDV RNA cleavage in normal buffer conditions.     

A long-range pseudoknot is required for activity of the Neurospora VS ribozyme.

Four small RNA self-cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long-range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site-directed mutagenesis and chemical modification to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme.     

Mutagenesis analysis of a self-cleaving RNA.

The hammerhead structural model proposed for sequences that mediate self-cleavage of certain RNAs contains base-paired three stems and 13 conserved bases. Insertion, deletion and base substitution mutations were carried out on a 58 base RNA containing the sequence of the single-hammerhead structure of the plus RNA of the virusoid of lucerne transient streak virus, and the effects on self-cleavage assessed. Results showed that there is flexibility in the sequence requirements for self-cleavage in vitro, but alterations of the conserved sequence or predicted secondary structure generally reduced the efficiency of self-cleavage.     

A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria

We describe a novel DNA and RNA found in the mitochondria of the Varkud-1c natural isolate of Neurospora. The majority of the RNA, termed VSRNA, is an 881 nucleotide single-stranded circular molecule complementary to one strand of a low copy, double-stranded circular DNA, VSDNA. VSRNA combines some features of the human hepatitis delta virus, group I introns, retroelements, and plant viral satellite RNAs. VSRNA synthesized in vitro performs a self-cleavage reaction whose products terminate with a 5' hydroxyl and a 2',3' cyclic phosphate. This reaction may be involved in the natural processing pathway of multimeric VSRNA in vivo. VSRNA lacks a hammer-head structure or substantial sequence similarity to any other self-cleaving RNA, suggesting that the RNA structure involved in cleavage may be different from those in previously characterized catalytic RNAs.     

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.     

Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Recently, we showed that an in vitro-synthesized RNA containing 273 nucleotides of the 324-nucleotide virusoid of lucerne transient streak virus self-cleaves at a unique site. The reaction requires heating and snap cooling of the RNA and the subsequent addition of magnesium ions. Here, we test the 55-nucleotide, hammerhead-shaped, structural model proposed for the active site by preparing RNAs with both 5' and 3' terminal deletions. Results indicate that the hammerhead structure is sufficient and necessary for self-cleavage, that certain sequences prevent the formation of the hammerhead structure in the native virusoid RNA, and that an RNA molecule containing only 52 nucleotides is capable of an RNA-mediated reaction.