Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms' tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both −KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.

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In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine residue by both kinases. V8 phosphopeptide maps revealed that the target site(s) of in vitro phosphorylation are located in the C-terminal region of A1. Phosphoamino acid sequence analysis and site directed mutagenesis identified Ser 199 as the sole phosphoamino acid in the protein phosphorylated by PKA. Phosphorylation introduced by PKA resulted in the suppression of the ability of protein A1 to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity. This finding indicates that phosphorylation of a single serine residue in the C-terminal domain may significantly alter the properties of protein A1.

We demonstrate that transcription of the gene swrAA, required for swarming migration in Bacillus subtilis, is driven by two promoters: a sigD-dependent promoter and a putative sigA-dependent promoter, which is inactive during growth in liquid Luria-Bertani medium and becomes active in the presence of the phosphorylated form of the response regulator DegU or on semisolid surfaces. Since sigD transcription is enhanced by SwrAA, this finding reveals that swrA expression is controlled by a positive feedback loop. We also demonstrate that the positive action of SwrAA in swimming and swarming motility is prevented in strains carrying a deletion of the two-component system degS-degU and that this effect is independent of swrAA transcription. Therefore, both DegU and SwrAA must be present to achieve full motility in B. subtilis.A wild-type copy of the swrAA gene is necessary for swarming motility in both undomesticated and laboratory strains of Bacillus subtilis (3,12). Laboratory strains (e.g., 168) that carry an sfp 0 allele and a frameshift mutation in the swrAA gene have a nonswarmer (Swr Ϫ ) phenotype (3,12,13,14,31). The role played by swrAA in swarming is to enhance transcription of the operon fla/che, which contains sigD, the gene coding for the alternative sigma factor D , as well as genes necessary for flagellum biosynthesis and chemotaxis (14). This notwithstanding, SwrAA does not resemble a DNA binding protein and does not show any particular feature by in silico analysis, nor does it display any similarity to characterized entries in protein databases, hampering the elucidation of its mechanism of action.In order to gain insights into the biological role that it plays in the activation of the swarming behavior, our efforts were concentrated on the expression profile of the swrA dicistronic operon which contains swrAA (3).The pleiotropic effects on the synthesis of degradative enzymes, competence, sporulation, and motility of mutations in the two-component system DegS-DegU have been extensively described previously (16,23). In particular, motility is negatively affected by degS(Hy) and degU(Hy) mutations. These mutations increase the half-life of the phosphorylated form of DegU (DegUϳP) (1,6,23). In contrast, the necessity of a low level of DegUϳP, for motility in general (36) and for swarming in particular (15, 37), has been pointed out for undomesticated and laboratory strains, but its role has yet to be established. Albeit in previous reports swrAA was never identified as a DegU-regulated gene (18, 26), it has been recently shown that swrAA transcription is negatively affected by deletions of degU or degS (15).Here we demonstrate that swrAA has two promoters: a Ddependent promoter, active in planktonic growth, and a putative A -dependent promoter triggered by DegUϳP, in concordance with published results (15). Furthermore, we show that in the absence of the functional alleles of either DegU or SwrAA B. subtilis is unable to fully swim as well as to swarm (12, 37). Although DegU activates swrAA transcripti...