Spermine switches a Neurospora VS ribozyme from slow Cis cleavage to fast trans cleavage.

In keeping with the known role of polyamines as counterions in RNA folding, we have found that concentrations of spermine as low as 1 microM facilitate first-order Cis cleavage and decrease the concentration of magnesium required for optimal cleavage of the VS ribozyme. Surprisingly, under certain experimental conditions, cleavage reactions at concentrations of spermine above about 20 microM were not first-order. At 100 microM spermine, about half of the RNA cleaved in an initial very fast burst, k >/= 5 min-1, about 100-fold faster than under our previously optimized conditions; the remainder of the RNA cleaved more slowly. The extent of the burst and the initial rate of cleavage were proportional to RNA concentration, suggesting that the fast phase was due to intermolecular trans cleavage involving two or more RNAs. This inference of trans cleavage was confirmed by demonstrating spermine-dependent trans cleavage of particular combinations of mutant RNAs that were each incapable of cis cleavage. The experimental conditions required to switch the VS ribozyme into trans cleavage mode are quite stringent. The RNA must be preincubated with an adequate concentration of spermine at very low ionic strength near neutral pH. Concentrations of buffers and salts typically used in in vitro studies of ribozymes, including those used in our previous characterization of the VS ribozyme, are sufficiently high that they prevent or reverse the trans cleaving RNA conformation. The ability to switch cleavage modes from cis to trans provides an experimental system to study different active conformations of VS RNA, as well as to investigate the functional consequences of polyamine-RNA interactions.

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.

Enhancement of Neurospora VS ribozyme cleavage by tuberactinomycin antibiotics.

Several examples of inhibition of the function of a ribozyme or RNA-protein complex have shown that certain antibiotics can interact specifically with RNA. There are, however, few examples of antibiotics that have a positive, rather than a negative, effect on the function of an RNA. We have found that micromolar concentrations of viomycin, a basic, cyclic peptide antibiotic of the tuberactinomycin group, enhance the cleavage of a ribozyme derived from Neurospora VS RNA. Viomycin decreases by an order of magnitude the concentration of magnesium required for cleavage. It also stimulates an otherwise insignificant transcleavage reaction by enhancing interactions between RNA molecules. The ability of viomycin to enhance some RNA-mediated reactions but inhibit others, including translation and Group I intron splicing, demonstrates the potential for natural selection by small molecules during evolution in the 'RNA world' and may have broader implications with respect to ribozyme expression and activity in contemporary cells.

A secondary-structure model for the self-cleaving region of Neurospora VS RNA.

Neurospora VS RNA performs an RNA-mediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis delta virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region of VS RNA, supported by site-directed mutagenesis and chemical modification structure probing data. The secondary structure of VS RNA is distinct from those of the other naturally occurring RNA self-cleaving domains. In addition to a unique secondary structure, several Mg-dependent interactions occur during the folding of VS RNA into its active tertiary conformation.

Revision of the nucleotide sequence and RNA splicing pathway of the Neurospora mitochondrial gene encoding ATPase subunit 6.

Previous sequence analysis of the Neurospora oli2 (ATP6) mitochondrial gene suggested that, in addition to a typical Group-I intron, it contained an unusual, mostly-palindromic, 93-nucleotide intron. We report here revisions of the nucleotide sequence and analysis of the size and sequence of reverse-transcriptase PCR products that show: (1) the Group-I intron splice sites are located as predicted by previous DNA sequence analysis; (2) the putative 93-nt intron is not excised from the mature mRNA, and most of this sequence is actually in the 5' untranslated region. We conclude that the Neurospora ATP6 gene contains only one intron. Analysis of the cDNA sequence also confirms the non-universal nature of the Neurospora mitochondrial genetic code: a TGA codon inferred from the DNA sequence is present as UGA in the mRNA. This provides direct evidence that this codon is not altered, for example by RNA editing, to conform to the universal code.

Nucleotide sequence requirements for self-cleavage of Neurospora VS RNA.

We have used several complementary approaches to investigate the minimal contiguous sequence required for the in vitro self cleavage reaction performed by Neurospora VS RNA. Deletion analysis and site-directed mutagenesis revealed that only a single nucleotide is required upstream of the self-cleavage site, and that the identity of this nucleotide is not critical. This distinguishes VS RNA from all currently known ribozymes except hepatitis delta virus RNA. The shortest contiguous sequence capable of cleavage contains 153 nt downstream of the cleavage site. Linker insertion mutagenesis suggests that much of this downstream sequence is important for self-cleavage. Comparative sequence analysis of the VS plasmid from six natural isolates supports the importance in vivo of the minimal region determined by in vitro methods. Also, phylogenetic analysis raises the possibility of a recent horizontal transfer of the VS plasmid from Neurospora intermedia to Neurospora sitophila.

Reaction conditions and kinetics of self-cleavage of a ribozyme derived from Neurospora VS RNA.

We have investigated the self-cleavage reaction performed by a ribozyme that contains 164 nucleotides of Neurospora VS RNA. Self-cleavage requires a divalent cation, magnesium being more effective than manganese or calcium. Spermidine or monovalent cations stimulate the reaction but cannot replace magnesium. The temperature optimum is rather broad, around 40-50 degrees C. Unlike some other ribozymes, VS self-cleavage is inhibited by even low concentrations of urea or formamide. The rate of cleavage is the same from pH 5.5 to 8.9, suggesting either that hydroxide is not directly involved in the cleavage chemistry or that a step that precedes the actual cleavage event is rate-limiting.

The effect of altitude on tests of reaction time and alertness.

Psychomotor performance was assessed in 20 subjects on each of 2 mountaineering expeditions. During the first, which reached 5,008 m, simple reaction time and alertness were measured, on the second to 4,790 m these were replaced by a three-choice reaction time test. In both, mean reaction times increased significantly at altitude in subjects with marked symptoms of Acute Mountain Sickness (AMS), whereas the alertness tests showed no effects. Reaction times were not affected by other environmental factors but adverse conditions increased the number of errors. The increase in reaction time may be ascribed either to the lethargy associated with AMS or alternatively may have been a direct effect of hypoxia. The latter explanation is favoured because of reports by other workers of an increase in reaction time with altitude in the absence of AMS.

Birmingham Medical Research Expeditionary Society 1977 Expedition: Psychological aspects of acute mountain sickness.

In order to assess the importance of personality and expectations in the development of Acute Mountain Sickness (AMS), 3 standard personality questionnaires and a Mountain Sickness Anticipation Questionnaire were completed by all of the 17 BMRES expedition members before their departure for the Himalayas. AMS could not be predicted with these tests and its occurrence, when assessed either by clinical interview or by peer review, bore no significant relationship to personality. For comparison daily self-assessment of the signs and symptoms of AMS were also conducted throughout the expedition, using graduated and graphic rating scales. The results were found to be unreliable and dependent upon personality factors. These findings have implications for those assessing others at high altitude.