Evaluation of mTOR-regulated mRNA translation

School of Biological Sciences, University of Southampton, Southampton, UK.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 821:171-85. DOI: 10.1007/978-1-61779-430-8_10
Source: PubMed


mTOR, the mammalian target of rapamycin, regulates protein synthesis (mRNA translation) by affecting the phosphorylation or activity of several translation factors. Here, we describe methods for studying the impact of mTOR signalling on protein synthesis, using inhibitors of mTOR such as rapamycin (which impairs some of its functions) or mTOR kinase inhibitors (which probably block all functions).To assess effects of mTOR inhibition on general protein synthesis in cells, the incorporation of radiolabelled amino acids into protein is measured. This does not yield information on the effects of mTOR on the synthesis of specific proteins. To do this, two methods are described. In one, stable-isotope labelled amino acids are used, and their incorporation into new proteins is determined using mass spectrometric methods. The proportions of labelled vs. unlabeled versions of each peptide from a given protein provide quantitative information about the rate of that protein's synthesis under different conditions. Actively translated mRNAs are associated with ribosomes in polyribosomes (polysomes); thus, examining which mRNAs are found in polysomes under different conditions provides information on the translation of specific mRNAs under different conditions. A method for the separation of polysomes from non-polysomal mRNAs is described.

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    • "Under such conditions, determining real-time rates of protein synthesis is expected to be a better predictor of protein abundance than measurements of respective mRNA levels. Stable isotope labelling by amino acids in cell culture (SILAC) is a highly robust tool for quantitatively comparing different proteomes, and pulsed SILAC approaches have been developed to directly quantify protein synthesis on a proteome-wide scale [17] [18]. However, such methods are not optimal for measuring changes in protein synthesis over very short timescales due to technical and biological variability [17], or because the signal from newly synthesized proteins are generally too low to be within the quantitative dynamic range of most mass analysers. "
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