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Interaction of the eucaryotic peptide chain initiation factor eIF-4A with the specific elements at the
R Chakrabarti1, D Chakrabarti, W W Souba
1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
Abstract:
The resistance of certain tumor cells to the chemotherapeutic agent L-asparaginase has often been found to be associated with the presence of asparagine synthetase activity. In an attempt to study the translational regulation of the asparagine synthetase gene, the 5'-untranslated region of human asparagine synthetase cDNA was mapped by antisense oligonucleotide-mediated hybrid arrest translation in reticulocyte lysate. Three consecutive cis-acting regulatory elements, spanning from -60 to -120 bases from the initiation codon, in the 5'-untranslated region of the asparagine synthetase gene, were identified. T1 RNase footprinting analysis showed that those regulatory elements can be protected from T1 digestion when incubated with reticulocyte lysate. A 46-kDa trans-acting protein factor that interacts with the cis-acting regulatory element of asparagine synthetase mRNA was detected. This 46-kDa protein factor is most likely to be the eucaryotic peptide chain initiation factor eIF-4A as determined by immunoprecipitation experiments using a monoclonal antibody raised against reticulocyte eIF-4A.
Insights
Tumor cell resistance to L-asparaginase may involve asparagine synthetase. Researchers identified regulatory elements and a protein factor, eukaryotic initiation factor eIF-4A, involved in its translational control.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Tumor cell resistance to L-asparaginase is linked to asparagine synthetase activity.
- Understanding the translational regulation of asparagine synthetase is crucial for cancer therapy.
Purpose of the Study:
- To investigate the translational regulation of the human asparagine synthetase gene.
- To identify regulatory elements and protein factors controlling asparagine synthetase expression.
Main Methods:
- Mapping the 5'-untranslated region of human asparagine synthetase cDNA using hybrid arrest translation.
- T1 RNase footprinting analysis to identify regulatory elements.
- Immunoprecipitation to detect and identify protein factors.
Main Results:
- Identified three consecutive cis-acting regulatory elements in the 5'-untranslated region (-60 to -120 bases).
- Detected a 46-kDa trans-acting protein factor interacting with these elements.
- Identified the protein factor as eukaryotic peptide chain initiation factor eIF-4A.
Conclusions:
- The 5'-untranslated region of asparagine synthetase mRNA contains regulatory elements controlling translation.
- A 46-kDa protein, identified as eIF-4A, binds to these elements, suggesting a role in translational regulation.
- This finding provides insights into mechanisms of L-asparaginase resistance in cancer cells.