Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.

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.

Spore-forming bacteria in soil cultivated with GM white poplars: isolation and characterization.

The impact of transgenic white poplars (Populus alba L. cv. 'Villafranca') was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 10(6)-2.5 x 10(8) CFU/g dry soil and of 10(4)-5 x 10(8) CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 10(3)-5 x 10(4) CFU/g dry soil and 10(2)-2 x 10(5) CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide-resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil level.

Identification of hnRNP P2 as TLS/FUS using electrospray mass spectrometry.

Protein complexes assembled on mRNA precursors can be separated by gel filtration chromatography to yield spliceosomal and H complex fractions (Reed R, Griffith J, Maniatis T, 1988, Cell 53:949-961; Reed R, 1990, Proc Natl Acad Sci USA 87:8031-8035.). Here we use Nano electrospray mass spectrometry (Wilm M, Mann M, 1994, Int J Mass Spectrometry Ion Processes 136:167-180) to identify proteins complexed with Adeno-pre-mRNA in the H complex peak. Four of the major hnRNP proteins, A1, B1, C1, and G, were identified by database analysis based on peptide mass and sequence information. A fifth protein in the H complex peak, corresponding to hnRNP P2, is shown to be the product of the TLS/FUS gene. This was originally identified as a chimeric oncogene formed by the chromosome translocation t(12;16) that is responsible for myxoid liposarcoma. The involvement of hnRNP P2 in oncogenesis provides a clear example of the importance of hnRNP proteins in molecular disease.

Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.

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.

WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes.

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.