ABSTRACT
BACKGROUND: Human monocytes express two naturally occurring forms of circulating tissue factor (TF) - full-length TF, a membrane-spanning protein, and alternatively spliced TF, a soluble molecule. Presence of the variable exon 5 in TF mRNA determines whether the encoded TF protein is transmembrane, or soluble. Recently, an essential SR protein ASF/SF2 was implicated in TF pre-mRNA processing in human platelets. OBJECTIVE: To examine molecular mechanisms governing regulated processing of TF pre-mRNA in human monocytic cells. METHODS AND RESULTS: In silico analysis of the human TF exon 5, present only in full-length TF mRNA, revealed putative binding motifs termed exonic splicing enhancers (ESE) for the SR proteins ASF/SF2 and SRp55, which were found to be abundantly expressed in monocytic cell lines THP-1 and SC, as well as monocyte-enriched peripheral blood mononuclear cells (PBMC). Using a splice competent mini-gene reporter system transiently expressed in monocytic cells, it was determined that weakening of either five closely positioned ASF/SF2 ESE (bases 87-117) or a single conserved SRp55 ESE (base 39) results in severe skipping of exon 5. ASF/SF2 and SRp55 were found to physically associate with the identified ESE. CONCLUSIONS: SR proteins ASF/SF2 and SRp55 appear to interact with the variable TF exon 5 through ESE at bases 39 and 87-117. Weakening of the above ESE modulates splicing of TF exon 5. This study is the first to identify and experimentally characterize cis-acting splicing elements involved in regulated biosynthesis of human TF.
