Evaluation of mTOR-regulated mRNA translation

Valentina Iadevaia, Xuemin Wang, Zhong Yao, Leonard J. Foster, Christopher G. Proud

Research output: Chapter in Book/Report/Conference proceedingChapter

17 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationmTOR
Subtitle of host publicationMethods and Protocols
EditorsThomas Weichhart
Pages171-185
Number of pages15
DOIs
Publication statusPublished - 2 Jan 2012

Publication series

NameMethods in Molecular Biology
Volume821
ISSN (Print)1064-3745

Keywords

  • Polyribosome analysis
  • Protein synthesis
  • Pulsed SILAC
  • Stable isotope labelling
  • Target of rapamycin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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