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.

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.

Correction of discrepancy between prescribed and actual blood flow rates in chronic hemodialysis patients with use of larger gauge needles.

The blood flow rate delivered by the dialysis machine (d-BFR) may not be accurately reflected by the blood flow rate set on the machine (sm-BFR). High negative arterial pressure may lead to deformity of the blood pump-segment tubing, resulting in a lower stroke volume and d-BFR. The Hagen-Poiseuille law predicts that the use of larger gauge needles should make arterial pressure less negative. Twenty-two patients on chronic hemodialysis therapy with a percentage of reduction in urea (PRU) levels less than 65% and/or a greater than 10% difference between sm-BFR and d-BFR underwent dialysis using one-gauge larger arterial and venous needles. d-BFR increased by 23 +/- 5 mL/min. Arterial pressure became less negative by 58 +/- 5 mmHg, and venous pressure decreased by 31 +/- 7 mm Hg. Changes in arterial and venous pressures allowed sm-BFR to be increased to 500 mL/min in all 22 patients, resulting in an increase in d-BFR of 83 +/- 7 mL/min. This translated into an increase in PRU of 5% +/- 0.01%. All results were significant at P < 0.001. A survey showed that less than 5% of needles used in our region were 14 G. This study shows that the use of larger gauge needles can significantly increase d-BFR and PRU as a result of changes in arterial and venous pressures, resulting in a significantly increased dialysis dose at no additional cost.