SR proteins ASF/SF2 and SRp55 participate in tissue factor biosynthesis in human monocytic cells.
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.


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.
Tardos
J G
JG
Division of Hematology and Medical Oncology, The Samuel Bronfman Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029-6754, USA.
Eisenreich
A
A
Deikus
G
G
Bechhofer
D H
DH
Chandradas
S
S
Zafar
U
U
Rauch
U
U
Bogdanov
V Y
VY
D017398:Alternative Splicing; D005091:Exons; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D009687:Nuclear Proteins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics
D009687:Nuclear Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C489470:SRSF6 protein, human; D000068103:Serine-Arginine Splicing Factors; D013925:Thromboplastin