The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points.

We are using the rat beta-tropomyosin (beta-TM) gene as a model system to study the mechanism of alternative splicing. Previous studies demonstrated that the use of the muscle-specific exon is associated with the use of distant branch points located 147-153 nt upstream of the 3' splice site. In addition, at least one protein, the polypyrimidine tract binding protein (PTB), specifically interacts with critical cis-acting sequences upstream of exon 7 that are involved in blocking the use of this alternative exon in nonmuscle cells. In order to further study the role of PTB, monoclonal antibodies to PTB were prepared. Anti-PTB antibodies did not inhibit the binding of PTB to RNA because they were able to supershift RNA-PTB complexes. To determine if additional proteins interact with sequences within the pre-mRNA, 35S-met-labeled nuclear extracts from HeLa cells were mixed with RNAs and the RNA-protein complexes were recovered by immunoprecipitation using antibodies to PTB. When RNAs containing intron 6 were added to an 35S-met-labeled nuclear extract, precipitation with PTB antibodies showed a novel set of proteins. By contrast, addition of RNAs containing introns 5 or 7 gave the same results as no RNA, indicating that these RNAs are unable to form stable complexes with PTB. These results are in agreement with our previous studies demonstrating that PTB interacts with sequences within intron 6, but not with sequences within introns 5 and 7. When 35S-met-labeled HeLa nuclear extracts were mixed with biotinylated RNA containing intron 6 and the RNA-protein complexes were recovered using streptavidin-agarose beads, an identical pattern of proteins was observed when compared with the immunoprecipitation assay. Analysis of the proteins that assembled on introns 5, 6, or 7 using biotinylated RNA revealed a unique set of proteins that interact with each of these sequences. The composition of proteins interacting with sequences associated with the use of the 3' splice site of intron 6 included proteins of 30, 40, 55, 60, 65, 70, 80, and 100 kDa. Microsequencing identified two of the proteins to be Sam68 and the Far Upstream Element Binding Protein (FBP) from the c-myc gene. In addition, a comparison of the proteins that assemble on introns from the alpha- and beta-TM genes that utilize distant branch points revealed common as well as unique proteins that assemble on these introns. These studies identify a set of proteins, in addition to PTB, that are likely involved in the use of distant branch sites associated with the use of alternatively spliced introns.

Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts.

Jurkat T-lymphoblasts were induced to undergo apoptosis by treatment with either EGTA (5 mM/24 h) or a high concentration of lovastatin (100 microM/48 h) to identify proteins that exhibited coordinate regulation between the two treatments and thus provide candidate proteins in the common apoptotic induction pathway. A pure population of apoptotic cells, as determined by morphology, "DNA laddering," and flow cytometry, was obtained by Percoll density gradient centrifugation. Cells of increased buoyant density were clearly apoptotic by all criteria. Following this gradient centrifugation, the cells were labeled with [35S]methionine/cysteine, and lysates were separated by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the two-dimensional polyacrylamide gel electrophoresis patterns generated from the apoptotic cells did not differ dramatically from that of control cells. Thus, apoptotic Jurkat cells are able to synthesize new proteins and do not exhibit extensive proteolysis. Subsequent quantitative analysis revealed that only five proteins exhibited decreases in turnover that were common to the two treatments. No increases in protein turnover were able to be confirmed across the replicate experiments. One of the proteins that showed decreased labeling by both apoptotic inductions was an abundant nuclear protein with a pI of 5.1 and M(r) 40,000. This protein was identified as numatrin/B23/nucleophosmin (NPM) based on internal amino acid sequence, and this identity was confirmed by immunoblotting and mass spectrometry. NPM is implicated in a range of diverse cellular functions, but its role in apoptosis is unclear.