Post-Transcriptional Regulation of Tissue Factor Expression in Human Monocytic Cells: Identification of Novel Exonic Splicing Enhancers for the Spliceosomal Protein SRp40 and Intronic Elements Critical for Exon 5 Definition

Abstract Background. Circulating monocytes play a critical role in the pathophysiology of many thrombotic disorders. These cells are known to exhibit complex post-transcriptional regulation of Tissue Factor (TF), the principal trigger of coagulation, which they express in two forms – full length TF(flTF), a highly thrombogenic integral membrane protein, and alternatively spliced TF (asTF), a secreted hypomorphic TF form. Biosynthesis of these two forms is achieved by inclusion or, alternatively, exclusion of TF exon 5 during pre-mRNA processing. Little is known about molecular mechanisms controlling alternative pre-mRNA splicing in human monocytes. We recently developed a mini-gene splicing reporter system pGL-hTF to evaluate the dynamics of exon 5 splicing, and determined that SR proteins ASF/SF2 and SRp55, which are abundantly expressed in human monocytes, take part in TF pre-mRNA processing by promoting exon 5 definition. Objective. To expand our exploration of cis-acting elements and spliceosomal proteins governing regulated biosynthesis of human TF in monocytic cells. Results. In silico analysis of exon 5 revealed six putative binding motifs termed exonic splicing enhancers (ESE) for SRp40, an SR protein whose expression pattern in human tissues is largely unknown. SRp40 mRNA and protein were detected in monocytic cell lines THP-1 and SC, as well as freshly isolated peripheral blood mononuclear cells (PBMC). Notably, the apparent molecular weight of the SRp40 protein in PBMC was substantially larger than that observed in monocytic cell lines, indicating that SRp40 in PBMC is likely to be hyperphosphorylated and may thus exhibit increased activity. To assess functionality of the putative SRp40 ESE in TF exon 5, base substitutions were designed to weaken each ESE and introduced into pGL-hTF by site directed mutagenesis. Resultant mutants were expressed in THP-1 cells, and the expression patterns analyzed by RT-PCR. Weakening of the ESE at position 44 potentiated inclusion of exon 5, while weakening of the ESE at position 86 resulted in increased exon 5 skipping. This suggests that SRp40 plays a complex, positional role in exon 5 definition. To further confirm functionality of the identified SRp40 ESE, combinatorial mutants were generated featuring weakened ESE for the SR proteins SRp55 (position 39, in close proximity to SRp40 ESE 44) and ASF/SF2 (positions 87–117): inactivation of these ESE was shown to promote skipping of exon 5. As expected, weakened SRp40 ESE 86, when combined with weakened SRp55 ESE, resulted in more severe skipping of exon 5. In contrast to that, weakened SRp40 ESE 44, when combined with weakened SRp55 ESE, potently restored inclusion of exon 5, whereas combining weakened SRp40 ESE 44 with weakened ASF/SF2 ESE yielded only a partial restoration of exon 5 inclusion. This indicates that SRp40 ESE 44 uniquely promotes production of the TF mRNA species encoding asTF, whereas SRp40 ESE 86 appears to behave similarly to classic ESE for spliceosomal proteins SRp55 and ASF/SF2. Using pGL-hTF, we also evaluated major regulatory elements of intron 4: this relatively short intron precedes the variable exon 5 and is thus likely to be critical for its spliceosomal definition. To identify a branchpoint (BP) adenosine (A), we replaced each A within the last 100 bp of the intron with a non-canonical BP base (G); surprisingly, these mutations did not affect the degree of exon 5 inclusion, indicating that intron 4 features a highly unusual BP motif. In contrast to that, mutations within the polypyrimidine tract (PT) of intron 4 revealed that the entire PT sequence is critical to exon 5 definition: compared to wild type pGL-hTF, a pGL-hTF mutant featuring extensive purine substitutions within the PT generated ~11.4 times fewer exon 5 containing amplicons (p=0.0001), as detected by our sensitive molecular beacon qPCR assay. Conclusions. We show for the first time that the spliceosomal protein SRp40 is expressed in human monocytes and participates in TF biosynthesis. Using our reporter system, we identified two functional ESE for SRp40 in the variable exon 5. SRp40 appears to play a complex, positional role in production of TF protein variants with a distinct coagulant potential. Furthering our knowledge about the post-transcriptional mechanisms of TF biosynthesis in circulating monocytes is very likely to facilitate development of qualitatively novel classes of anti-thrombotic therapeutic agents.