Molecular Dissection of Human Tissue Factor Pre-mRNA Processing: Identification and Characterization of Functional Exonic Splicing Enhancers in Variable Exon 5.

Tissue Factor (TF), the regulatory co-factor of FVII/VIIa, is the physiologic trigger of blood coagulation. Two naturally occurring isoforms of TF differ in their structure: the full length TF (flTF) is a membrane-spanning molecule, whereas the recently discovered alternatively spliced TF (asTF), generated by exclusion of exon 5, lacks the transmembrane domain, and is soluble. Biosynthesis of the soluble TF variant appears to be evolutionarily advantageous, as it exists in mice. Both isoforms are present in plasma and produced predominantly by circulating monocytes. The biologic role of asTF is unknown; it has, however, been proposed that precise regulation of the TF isoform ratio in blood is critical to normal hemostasis. This study aims to identify molecular mechanisms governing the fate of variable exon 5 during the processing of the human TF pre-mRNA, and to apply these findings to generate mini-genes expressing altered human flTF/asTF isoform ratios in animal models to elucidate the role of TF isoforms in normal hemostasis and/or development of coagulopathies. In silico analysis of the human TF exon 5 revealed 14 putative exonic splicing enhancer (ESE) motifs for SF2/ASF and SC35, essential SR proteins involved in pre-mRNA splicing. A segment of the TF gene comprising the end of exon 4 through the beginning of exon 6 (including exon 5 and the entire introns 4 and 5) was inserted into the GFP open reading frame of the pGL expression vector. The resultant construct (pGL-hTF) was transfected into a human monocytic cell line (THP-1) and shown to faithfully recapitulate the pattern of TF mRNA isoform production. Site-directed mutagenesis of each putative ESE motif was then performed, and the mRNA isoform levels were assayed by RT-PCR. Removal of four ASF/SF2 sites at positions 100–117 favored exclusion of exon 5. In contrast, removal of either of the two SC35 motifs at positions 33 and 81 favored inclusion of the exon. A pSPL-3b-hTF expression vector, featuring the above mutations in the context of a different promoter and a smaller segment of the TF gene, was generated to confirm these findings: similar shifts in isoform ratio production were observed. Results were further validated by introducing both synergistic and antagonistic double mutations of the functional ESEs in pGL-hTF. Production of the two naturally occurring human TF isoforms appears to be regulated at the post-transcriptional level by SR proteins ASF/SF2 and SC35. Further elucidation of the molecular mechanisms involved in TF isoform production is very likely to provide effective means for creating state-of-the-art animal models to characterize the biologic importance of flTF/asTF ratio in circulating blood. In addition, this study is the first to show that SF2/ASF and SC35 can exert opposite effects on pre-mRNA splicing.