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.

Data variance and statistical significance in 2D-gel electrophoresis and DIGE experiments: comparison of the effects of normalization methods.

Identifying changes in the relative abundance of proteins between different biological samples is often confounded by technical noise. In this work, we compared eight normalization methods commonly used in two-dimensional gel electrophoresis and difference gel electrophoresis (DIGE) experiments for their ability to reduce noise and for their influence on the list of proteins whose difference in abundance between two samples is determined to be statistically significant. With respect to reducing noise we find that, while all methods improve upon unnormalized data, cyclic linear normalization is the least well suited to gel-based proteomics and the performances of the other methods are similar. We also find in DIGE data that the choice of normalization method has less of an impact on the noise than does the decision to use an internal reference in the experimental design and that both normalization and standardization using the internal reference are required to maximally reduce variance. Despite the similar noise reduction achieved by most normalization methods, the list of proteins whose abundance was determined to differ significantly between biological groups differed depending on the choice of normalization method. This work provides a direct comparison of the impact of normalization methods in the context of common experimental designs.

Evidence for the phenotypic neutrality of the Neurospora Varkud (V) and Varkud satellite (VS) plasmids.

The Varkud satellite (VS) plasmid, which requires the Varkud (V) plasmid for replication, is found in the mitochondria of several natural isolates of Neurospora. The VS transcript is sufficiently abundant that it might be expected to alter the function of mitochondria; however, previous limited characterization revealed no effect. In this work we have used genetic, biochemical and proteomic approaches to search for effects of the V and VS plasmids. We observed differences in the relative abundance of several mitochondrial proteins between plasmid-containing and plasmid-lacking natural isolates, but subsequently found these not to be due to the plasmids. We constructed a pair of iso-nuclear and iso-mitochondrial strains that differed only by the presence or absence of V and VS, and observed only subtle differences in the abundance of several mitochondrial proteins. We further attempted to detect a cryptic plasmid-related phenotype by growing this pair of strains in the presence of a variety of inhibitors of mitochondrial function or other stress conditions: this also revealed no effect of the plasmids. These observations suggest that, despite the high concentration of VS RNA in the mitochondrion, the V and VS plasmids do not cause substantial changes in the host.

Gel-based mass spectrometric and computational approaches to the mitochondrial proteome of Neurospora.

We have used gel electrophoretic techniques including isoelectric focusing, blue native, acid-urea, 16-benzyldimethyl-n-hexadecylammonium chloride or SDS first dimensions and SDS Laemmli or tricine second dimensions to separate the proteins from highly-purified Neurospora mitochondria and sub-mitochondrial fractions (membrane, soluble, protein complexes and ribonucleoproteins). The products of 260 genes, many of them in multiple processed or modified forms, were identified by MALDI-TOF mass spectrometry. This work confirms the existence, expression, and mitochondrial localization of the products of 55 Neurospora genes previously annotated only as predicted or hypothetical, and of 101 genes not identified in previous mass spectrometry studies. Combined with previous mass spectrometry studies, and re-evaluation of genome annotations, we have compiled a curated list of 358 proteins identified in proteomic studies that are likely to be bona fide mitochondrial proteins plus 80 other identified proteins that may be mitochondrial. Literature data mining and computational predictions suggest that Neurospora mitochondria also contain another 299 proteins not yet identified in proteomics projects. Taken together, these data suggest that the products of at least 738 genes comprise the Neurospora mitochondrial proteome.

Use of ribozyme cleavage kinetics to measure salt-induced changes in solution pH.

Several ribozymes are active in molar concentrations of monovalent salts, and pH rate curves under such conditions are consistent with mechanisms involving general acid-base catalysis. Interpreting the apparent pK(a) values requires an accurate estimation of solution pH, which can be difficult to obtain using a typical glass pH electrode in the presence of high salt concentrations. In the current work we have used the VS ribozyme as a "biological pH meter" to evaluate the effects of molar concentrations of monovalent salts on changes in solution pH. We find that almost all of the measured change in pH observed in high concentrations of LiCl is due to a real change in solution pH. In contrast, high concentrations of NaCl cause errors in pH measurement in addition to changes in actual pH. Different buffer systems affect both the direction and the magnitude of pH changes: we observed changes in measured pH of up to 1 pH unit, which require proper interpretation to avoid adverse effects on the conclusions drawn from pH rate and other experiments that utilize very high salt concentrations.

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.

A systematic evaluation of payback of publicly funded health and health services research in Hong Kong.

BACKGROUND: The Health and Health Services Research Fund (HHSRF) is dedicated to support research related to all aspects of health and health services in Hong Kong. We evaluated the fund's outcomes and explored factors associated with the translation of research findings to changes in health policy and provider behaviour. METHODS: A locally suitable questionnaire was developed based on the "payback" evaluation framework and was sent to principal investigators of the completed research projects supported by the fund since 1993. Research "payback" in six outcome areas was surveyed, namely knowledge production, use of research in the research system, use of research project findings in health system policy/decision making, application of the research findings through changed behaviour, factors influencing the utilization of research, and health/health service/economic benefits. RESULTS: Principal investigators of 178 of 205 (87%) completed research projects returned the questionnaire. Investigators reported research publications in 86.5% (mean = 5.4 publications per project), career advancement 34.3%, acquisition of higher qualifications 38.2%, use of results in policy making 35.4%, changed behaviour in light of findings 49.4%, evidence of health service benefit 42.1% and generated subsequent research in 44.9% of the projects. Payback outcomes were positively associated with the amount of funding awarded. Multivariate analysis found participation of investigators in policy committees and liaison with potential users were significantly associated with reported health service benefit (odds ratio [OR]participation = 2.86, 95% confidence interval [CI] 1.28-6.40; ORliaison = 2.03, 95% CI 1.05-3.91), policy and decision-making (ORparticipation = 10.53, 95% CI 4.13-26.81; ORliaison = 2.52, 95% CI 1.20-5.28), and change in behavior (ORparticipation = 3.67, 95% CI 1.53-8.81). CONCLUSION: The HHSRF has produced substantial outcomes and compared favourably with similar health research funds in other developed economies. Further studies are needed to better understand the factors and pathways associated with the translation of research findings into practice.

The contribution of 2'-hydroxyls to the cleavage activity of the Neurospora VS ribozyme.

We have used nucleotide analog interference mapping and site-specific substitution to determine the effect of 2'-deoxynucleotide substitution of each nucleotide in the VS ribozyme on the self-cleavage reaction. A large number of 2'-hydroxyls (2'-OHs) that contribute to cleavage activity of the VS ribozyme were found distributed throughout the core of the ribozyme. The locations of these 2'-OHs in the context of a recently developed helical orientation model of the VS ribozyme suggest roles in multi-stem junction structure, helix packing, internal loop structure and catalysis. The functional importance of three separate 2'-OHs supports the proposal that three uridine turns contribute to local and long-range tertiary structure formation. A cluster of important 2'-OHs near the loop that is the candidate region for the active site and one very important 2'-OH in the loop that contains the cleavage site confirm the functional importance of these two loops. A cluster of important 2'-OHs lining the minor groove of stem-loop I and helix II suggests that these regions of the backbone may play an important role in positioning helices in the active structure of the ribozyme.