Four SR Proteins Play Opposing Roles in the Regulated Biosynthesis of Tissue Factor in Human Monocytic Cells.

Abstract 2128

Poster Board II-105

Background. Circulating Tissue Factor (TF) is a major contributor to the etiology of thrombotic disorders. Blood monocytes are the primary source of circulating TF, which they express in two forms: full length TF (flTF), a transmembrane surface protein, and alternatively spliced TF (asTF), a secreted soluble protein. The presence or absence of the internal cassette exon 5 in the TF mRNA determines whether the encoded protein is flTF or asTF, respectively. While the procoagulant potential of flTF vastly exceeds that of asTF when assessed using conventional static assays, asTF exhibits unique angiogenic properties distinct from flTF. Investigation of the molecular mechanisms governing TF exon 5 processing is of much interest as it may afford novel approaches to modulate monocyte-mediated blood thrombogenicity as well as monocyte-induced angiogenesis. Using a splicing reporter system developed to study the processing of TF pre-mRNA, we previously determined that SR proteins ASF/SF2 and SRp55 are vital for TF exon 5 inclusion in human monocytes (Tardos et al, J Thromb Haemost 6:877-884, 2008). In the course of more recent experiments, we found that in contrast to ASF/SF2 and SRp55, two other SR proteins – SC35 and SRp40 – appear to promote exclusion of this variable exon: weakening of the binding motifs for SC35 (sites 33 and 81) and SRp40 (site 44), whose positions in exon 5 overlap with the binding sites for ASF/SF2 and SRp55, results in the decrease of the splicing event unique to asTF. The competition of these four SR proteins for binding to their overlapping binding sites may thus play a role in the maintenance of the asTF / flTF ratio; however, a physical association of SC35 and SRp40 with their putative sites in exon 5 was not demonstrated. Objective. To determine whether SC35 and/or SRp40 physically associate with their putative binding sites. Results. To evaluate interaction of the SR proteins SC35 and SRp40 with their putative binding sites in TF exon 5, we employed RNA mobility shift assay methodology using freshly prepared nuclear extracts of THP-1 cells, a monocytic cell line, and in vitro transcribed, uniformly labeled RNA probes comprising the two regions of interest – one spanning SC35 site 33 and SRp40 site 44, and the other spanning the SC35 site 81. As expected, these RNA probes yielded reproducible band shift products, confirming RNA-protein complex formation. We then developed three counterpart mutant RNA probes in which the SC35 and SRp40 binding motifs were selectively weakened by targeted site-directed mutagenesis, and performed RNA gel shift assays alongside the corresponding wild-type probes. Each mutant probe exhibited a significantly weaker interaction with the SR proteins compared to its wild-type counterpart: the SC35 site 33 mutant probe produced a 31.1% reduction in complex formation relative to wild-type (p = 0.011), the SRp40 site 44 mutant probe also produced a 31.1% reduction in complex formation relative to wild-type (p = 0.0001), and the SC35 site 81 mutant probe produced a 33.0% reduction in complex formation relative to wild-type (p = 0.0151). Conclusions. We show for the first time that SR proteins SC35 and SRp40 physically associate with functional binding sites within TF exon 5. The SC35 and SRp40 binding sites overlap with the binding sites for ASF/SF2 and SRp55, the SR proteins that promote exon 5 inclusion. The opposing effects of distinct SR proteins on TF exon 5 processing reveal a heretofore unknown mechanism governing regulated TF biosynthesis. Further studies of SR protein-mediated effects on the TF profile of human monocytes are likely to aid in the development of novel therapeutic strategies aimed at selective targeting of biologically distinct TF forms.