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Evaluation of mTOR-regulated mRNA translation.

Evaluation of mTOR-regulated mRNA translation.
Evaluation of mTOR-regulated mRNA translation.
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
171-185
Springer
Iadevaia, Valentina
1124252e-5709-4a5e-8a4b-956ced0c9611
Wang, Xuemin
d6bb4eb2-5687-46ed-b770-cceb22fd792e
Yao, Zhong
d8e96a4a-a98d-47d3-96b8-efd459aa7a62
Foster, Leonard J
e50f6e4f-71c5-43d6-a37a-3bba49e55e91
Proud, Christopher G
5832db56-9069-4617-a80d-08b069093dba
Weichhart, Thomas
Iadevaia, Valentina
1124252e-5709-4a5e-8a4b-956ced0c9611
Wang, Xuemin
d6bb4eb2-5687-46ed-b770-cceb22fd792e
Yao, Zhong
d8e96a4a-a98d-47d3-96b8-efd459aa7a62
Foster, Leonard J
e50f6e4f-71c5-43d6-a37a-3bba49e55e91
Proud, Christopher G
5832db56-9069-4617-a80d-08b069093dba
Weichhart, Thomas

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

Record type: Book Section

Abstract

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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Published date: 2012
Organisations: Molecular and Cellular

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Local EPrints ID: 337303
URI: http://eprints.soton.ac.uk/id/eprint/337303
PURE UUID: f39f670c-cb4c-43b4-a0b4-0581b6e61604

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Date deposited: 24 Apr 2012 14:21
Last modified: 11 Nov 2019 17:46

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