Abstract 1658: Regulated Biosynthesis of Tissue Factor: Identification of Novel cis-Acting Exonic and Intronic Regions Involved in Tissue Factor Pre-mRNA Splicing in Human Monocytic Cells
Tissue Factor (TF), the principal trigger of coagulation, occurs in two forms - full-length TF, a transmembrane protein, and alternatively spliced TF, a soluble protein formed due to exclusion of exon 5. We previously developed a reporter system to evaluate the dynamics of TF exon 5 splicing, and showed that SR proteins ASF/SF2 and SRp55 take part in TF pre-mRNA processing in human monocytes by promoting exon 5 definition. To expand exploration of cis-acting elements governing regulated processing of human TF pre-mRNA in monocytic cells, focusing on the possible role of other SR proteins, specifically SRp40. SRp40 mRNA and protein were detected in monocytic cell lines THP-1 and SC, as well as peripheral blood monocytes. Analysis of the exon 5 sequence for SR protein binding sites termed exonic splicing enhancers revealed that, in contrast to ASF/SF2 sites that are narrowly clustered within exon 5, the six sites for SRp40 are distributed more evenly. Mutations were introduced into the reporter to weaken each SRp40 site individually. Mutants were expressed in THP-1 cells, and the expression patterns evaluated by RT-PCR. Results showed that weakening the site at position 44 increases exon 5 inclusion, while weakening the site 86 increases exon 5 exclusion. Additional mutations were then generated for each SRp40 site, yielding consistent results. Effects of mutations in site 86 may not be solely due to weakened SRp40 binding: site 86 overlaps with an ASF/SF2 site that promotes exon 5 inclusion. We also investigated whether substitutions in the polypyrimidine tract (PT) of intron 4, preceding TF exon 5, would affect the splicing pattern. Two mutations, termed PT1 and PT2, were designed: PT1 causes major alterations in the PT, while PT2 introduces only minor modifications. Results showed that the PT of intron 4 is important for exon 5 definition as disrupting the PT structure results in severe skipping of this exon, yielding the pattern unique to alternatively spliced TF. SRp40 site 44 appears to promote exclusion of TF exon 5, while site 86 promotes its inclusion; thus, SRp40 may play a complex, positional role in TF pre-mRNA splicing. Further, the polypyrimidine tract of TF intron 4 appears critical for exon 5 definition.