Abstract
Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that inhibits urokinase. Constitutive and regulated PAI-2 gene expression involves post-transcriptional events, and an AU-rich mRNA instability motif within the 3'-untranslated region of PAI-2 mRNA is required for this process (Maurer, F., Tierney, M., and Medcalf, R. L. (1999) Nucleic Acids Res. 27, 1664-1673). Here we show that instability determinants are present within various exons of the PAI-2 coding region, most notably within exon 4. Deletion of exon 4 from the full-length PAI-2 cDNA results in a doubling in the half-life of PAI-2 mRNA, whereas a 28-nucleotide region within exon 4 contains binding sites for cytoplasmic proteins. Inducible stabilization of PAI-2 mRNA in HT-1080 cells treated with phorbol ester and tumor necrosis factor does not alter the binding of proteins to the exon 4 instability determinant, but resulted in a transient increase in the binding of factors to the AU-rich RNA instability element. Hence, PAI-2 mRNA stability is influenced by elements located within both the coding region and the 3'-untranslated region and that cytoplasmic mRNA binding factors may influence steady state and inducible PAI-2 mRNA expression. Finally a 10-nucleotide region flanking the exon 4 protein-binding site is homologous to instability elements within five other transcripts, suggesting that a common coding region determinant may exist.
Highlights
The plasminogen activator system is an important proteolytic cascade that plays a role in the removal of blood clots from the circulation and the turnover of a variety of extracellular matrix proteins [2]
These results suggest that instability and stability determinants are located throughout the Plasminogen activator inhibitor type 2 (PAI-2) coding region, with powerful instability elements associated with the presence of exon 4 and within exons 7 and 8
We focused our efforts to assess the role of exon 4 in the control of plasminogen activator inhibitors (PAIs)-2 mRNA stability
Summary
Inducible stabilization of PAI-2 mRNA in HT1080 cells treated with phorbol ester and tumor necrosis factor does not alter the binding of proteins to the exon 4 instability determinant, but resulted in a transient increase in the binding of factors to the AU-rich RNA instability element. A 10-nucleotide region flanking the exon 4 protein-binding site is homologous to instability elements within five other transcripts, suggesting that a common coding region determinant may exist. The PAI-2 gene is one of the most tumor necrosis factor- (TNF) [10] and lipopolysaccaride (LPS) [11]-responsive genes described For the latter, this has been further confirmed by serial analysis of gene expression analysis of primary human monocytes, whereby PAI-2 mRNA levels were shown to be increased 105fold by LPS [12], being the third most LPS-induced transcript produced in these cells. Lowercase extensions represent nucleotides added to facilitate improved digestion with restriction enzymes
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