Translational repression of EF-1 alpha mRNA in vitro

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Mammalian cell growth arrest is associated with repressed mRNA translation.
• Specific mRNAs, like eukaryotic elongation factor 1 alpha (EF-1 alpha), are translationally silenced in vivo during growth arrest.

Purpose of the Study:

• To investigate the mechanism of mRNA translational repression in growth-arrested murine erythroleukemia (MEL) cells.
• To identify factors involved in the regulation of eukaryotic elongation factor 1 alpha (EF-1 alpha) mRNA translation.

Main Methods:

• Preparation of 10,000 x g supernatant extracts from growth-arrested MEL cells.
• Incubation of extracts to observe mRNA to messenger ribonucleoprotein (RNP) particle conversion.
• Translation assays using nuclease-treated rabbit reticulocyte lysate.
• Chromatography on oligo(dT)-cellulose and salt wash treatments.

Main Results:

• Growth-arrested MEL cell extracts rapidly convert most mRNAs into non-translating RNPs.
• RNPs are generally translatable in a reticulocyte lysate system.
• Eukaryotic elongation factor 1 alpha (EF-1 alpha) mRNA and poly(A)-binding protein mRNA are exceptions, remaining non-translating.
• Translational activity of EF-1 alpha mRNA can be restored by salt wash treatment and oligo(dT)-cellulose chromatography.
• The salt wash selectively inhibits EF-1 alpha mRNA translation, indicating a trans-acting repressor.

Conclusions:

• Growth arrest in MEL cells leads to the formation of non-translating RNPs.
• A trans-acting repressor factor present in the salt wash selectively inhibits eukaryotic elongation factor 1 alpha (EF-1 alpha) mRNA translation.