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SR proteins ASF/SF2 and SRp55 participate in tissue factor biosynthesis in human monocytic cells
Author(s) -
TARDOS J. G.,
EISENREICH A.,
DEIKUS G.,
BECHHOFER D. H.,
CHANDRADAS S.,
ZAFAR U.,
RAUCH U.,
BOGDANOV V. Y.
Publication year - 2008
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2008.02946.x
Subject(s) - biosynthesis , tissue factor , microbiology and biotechnology , chemistry , cancer research , biochemistry , biology , medicine , gene , coagulation
Summary. 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.