The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

Additional roles of a peripheral loop-loop interaction in the Neurospora VS ribozyme.

Many RNAs contain tertiary interactions that contribute to folding the RNA into its functional 3D structure. In the VS ribozyme, a tertiary loop-loop kissing interaction involving stem-loops I and V is also required to rearrange the secondary structure of stem-loop I such that nucleotides at the base of stem I, which contains the cleavage-ligation site, can adopt the conformation required for activity. In the current work, we have used mutants that constitutively adopt the catalytically permissive conformation to search for additional roles of the kissing interaction in vitro. Using mutations that disrupt or restore the kissing interaction, we find that the kissing interaction contributes ~1000-fold enhancement to the rates of cleavage and ligation. Large Mg(2+)-dependent effects on equilibrium were also observed: in the presence of the kissing interaction cleavage is favored >10-fold at micromolar concentrations of Mg(2+); whereas ligation is favored >10-fold at millimolar concentrations of Mg(2+). In the absence of the kissing interaction cleavage exceeds ligation at all concentrations of Mg(2+). These data provide evidence that the kissing interaction strongly affects the observed cleavage and ligation rate constants and the cleavage-ligation equilibrium of the ribozyme.

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a.

Several small ribozymes employ general acid-base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK (a) values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK (a) values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid-base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid-base catalysis indicate that k (1), the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK (a). The inference that k (1) is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK (a) is that of the general acid and the higher pK (a) is that of the general base.

An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method.

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.

Identification of separate structural features that affect rate and cation concentration dependence of self-cleavage by the Neurospora VS ribozyme.

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.

Exceptionally fast self-cleavage by a Neurospora Varkud satellite ribozyme.

Most of the small ribozymes, including those that have been investigated as potential therapeutic agents, appear to be rather poor catalysts. These RNAs use an internal phosphoester transfer mechanism to catalyze site-specific RNA cleavage with apparent cleavage rate constants typically <2 min(-1). We have identified variants of one of these, the Neurospora Varkud satellite ribozyme, that self-cleaves with experimentally measured apparent rate constants of up to 10 s(-1) (600 min(-1)), approximately 2 orders of magnitude faster than any previously characterized self-cleaving RNA. We describe structural features of the cleavage site loop and an adjacent helix that affect the apparent rate constants for cleavage and ligation and the equilibrium between them. These data show that the phosphoester transfer ribozymes can catalyze reactions with rate constants much larger than previously appreciated and in the range of those of protein enzymes that perform similar reactions.