Evaluation of mTOR-regulated mRNA translation

Iadevaia, Valentina, Wang, Xuemin, Yao, Zhong, Foster, Leonard J. and Proud, Christopher G (2012) Evaluation of mTOR-regulated mRNA translation Methods in Molecular Biology, 821, pp. 171-185. (doi:10.1007/978-1-61779-430-8_10). (PMID:22125065).


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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.

Item Type: Article
Digital Object Identifier (DOI): doi:10.1007/978-1-61779-430-8_10
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Organisations: Molecular and Cellular
ePrint ID: 337303
Date :
Date Event
Date Deposited: 24 Apr 2012 14:21
Last Modified: 17 Apr 2017 17:17
Further Information:Google Scholar
URI: http://eprints.soton.ac.uk/id/eprint/337303

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