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Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant
P L Bernstein1, D J Herrick, R D Prokipcak
1McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Abstract:
Polysome-associated c-myc mRNA is degraded relatively rapidly in cells and in an in vitro mRNA decay system containing extracts from cultured mammalian cells. Using this system, a competition/screening assay was devised to search for factors that bind to specific regions of polysome-associated c-myc mRNA and thereby alter its half-life. mRNA stability was first assayed in reactions containing exogenous competitor RNAs corresponding to portions of c-myc mRNA itself. The addition of a 182-nucleotide sense strand fragment from the carboxy-terminal portion of the c-myc-coding region destabilized c-myc mRNA by at least eightfold. This RNA fragment had no effect on the stability of other mRNAs tested. Moreover, c-myc mRNA was not destabilized in reactions containing unrelated competitor RNAs or sense strand RNA from the c-myc 5' region. Polysome-associated globin mRNA containing the c-myc-coding region segment in-frame was also destabilized in vitro by the 182-nucleotide RNA. As determined by UV-cross-linking experiments, the 182-nucleotide RNA fragment was recognized by and bound to an approximately 75-kD polysome-associated protein. On the basis of these data plus Northern blotting analyses of c-myc mRNA decay products, we suggest that the approximately 75-kD protein is normally bound to a c-myc-coding region determinant and protects that region of the mRNA from endonuclease attack. Possible links between the protective protein, translation, ribosome pausing, and c-myc mRNA turnover are discussed.
Insights
A specific protein binds to c-myc messenger RNA (mRNA), protecting it from rapid degradation. This discovery sheds light on the regulation of c-myc mRNA stability and turnover in cells.
Area of Science:
- Molecular Biology
- Gene Regulation
- RNA Metabolism
Background:
- Polysome-associated c-myc mRNA undergoes rapid degradation in cells.
- Understanding the mechanisms controlling c-myc mRNA stability is crucial for comprehending gene expression regulation.
Purpose of the Study:
- To identify factors that bind to c-myc mRNA and influence its half-life.
- To investigate the role of specific mRNA regions in c-myc mRNA decay.
Main Methods:
- Development of a competition/screening assay using in vitro mRNA decay systems.
- Utilizing competitor RNA fragments to probe mRNA-protein interactions.
- UV-cross-linking experiments to identify binding proteins.
- Northern blotting to analyze mRNA decay products.
Main Results:
- A 182-nucleotide RNA fragment from the c-myc coding region significantly destabilized c-myc mRNA.
- This destabilization effect was specific to c-myc mRNA.
- A ~75 kD polysome-associated protein was found to bind the destabilizing RNA fragment.
- The protein appears to protect c-myc mRNA from endonuclease attack.
Conclusions:
- A ~75 kD protein binds to a specific determinant in the c-myc coding region.
- This protein likely protects c-myc mRNA from degradation, influencing its turnover.
- Potential connections between this protective protein, translation, and mRNA decay are proposed.