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Multiple instability-regulating sites in the 3' untranslated region of the urokinase-type plasminogen activator mRNA
R Nanbu1, P A Menoud, Y Nagamine
1Friedrich Miescher-Institut, Basel, Switzerland.
Abstract:
In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.
Insights
The 3' untranslated region of urokinase-type plasminogen activator (uPA) mRNA contains instability elements. These elements control uPA mRNA turnover, influenced by protein synthesis and protein kinase C (PKC).
Area of Science:
- Molecular Biology
- Gene Regulation
- RNA Metabolism
Background:
- Urokinase-type plasminogen activator (uPA) mRNA exhibits rapid degradation in LLC-PK1 cells.
- mRNA stability is modulated by protein synthesis inhibition and protein kinase C (PKC) activity.
Purpose of the Study:
- To investigate the role of the 3' untranslated region (3'UTR) of uPA mRNA in its rapid turnover.
- To identify specific sequences within the 3'UTR responsible for mRNA instability.
Main Methods:
- Insertion of the uPA 3'UTR into a stable rabbit beta-globin reporter mRNA.
- Stable transfection into LLC-PK1 cells.
- Analysis of chimeric mRNA stability under conditions of inhibited protein synthesis (cycloheximide) and PKC downregulation.
Main Results:
- The uPA 3'UTR conferred rapid degradation to the otherwise stable beta-globin mRNA.
- At least three independent instability determinants were identified within the 3'UTR, including an AU-rich element and a stem-loop structure.
- mRNA stabilization by cycloheximide was sequence-independent, while PKC downregulation stabilized mRNA via the AU-rich element.
Conclusions:
- The 3'UTR of uPA mRNA contains critical elements dictating its short half-life.
- Multiple, independent instability determinants within the 3'UTR contribute to the regulation of uPA mRNA metabolism.
- PKC signaling pathway, potentially through the AU-rich element, plays a role in stabilizing uPA mRNA.