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Translational repression of endogenous thymidine kinase mRNA in differentiating and arresting mouse cells
M Knöfler1, C Waltner, E Wintersberger
1Institute of Molecular Biology, Vienna Biocenter, University of Vienna, Austria.
Abstract:
We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells. In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit. High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system. Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells. Pulse labeling of these cells in vivo with [35S]methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum. This was not due to increased turnover of the protein as measured in pulse chase experiments. In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells. Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow.
Insights
Cellular growth arrest leads to faster decline in thymidine kinase (TK) enzyme activity than its messenger RNA (mRNA). This suggests TK mRNA becomes translationally repressed when mouse cells stop growing.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Thymidine kinase (TK) enzyme activity declines faster than its corresponding mRNA during cellular processes like growth arrest and differentiation.
- The molecular mechanisms underlying this discrepancy require investigation.
Purpose of the Study:
- To investigate the molecular mechanisms responsible for the faster decline in thymidine kinase (TK) enzyme activity compared to its mRNA levels.
- To determine if translational repression of TK mRNA occurs during cellular growth arrest or differentiation.
Main Methods:
- Generation of a polyclonal antiserum against mouse TK.
- High-level expression of mouse TK polypeptide in E. coli using a T7 RNA polymerase system.
- Immunoblotting to detect inactive enzyme pools.
- Pulse labeling with [35S]methionine to assess protein synthesis rates.
- Pulse-chase experiments to measure protein turnover.
- Analysis of TK mRNA association with polysomes.
Main Results:
- No evidence of inactive TK enzyme pools was found during F9 cell differentiation or 3T6 cell growth arrest.
- A significant decrease (over 6-fold) in TK protein synthesis rate was observed in F9 cells and 3T6 cells under specific conditions.
- Increased protein turnover did not account for the observed decrease in TK protein levels.
- Full-length TK mRNA remained associated with polysomes, indicating mRNA availability for translation.
Conclusions:
- The results suggest that translational repression of endogenous TK mRNA is a key mechanism regulating TK enzyme levels.
- This translational repression occurs under various conditions when mouse cells cease to grow, such as during growth arrest or differentiation.