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Nat Methods. 2014 Jan;11(1):86-93. doi: 10.1038/nmeth.2729. Epub 2013 Nov 10.

Parallel measurement of dynamic changes in translation rates in single cells.

Author information

  • 1Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.
  • 21] Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA. [2] Department of Biochemistry, Stanford University, Stanford, California, USA.
  • 3Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.
  • 4Department of Biochemistry and Molecular Biology, The Institute for Medical Research, Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
  • 51] Department of Biochemistry, McGill University Montreal, Quebec, Canada. [2] Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada.

Abstract

Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5' terminal oligopyrimidine (5' TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation.

PMID:
24213167
PMCID:
PMC4039304
DOI:
10.1038/nmeth.2729
[PubMed - indexed for MEDLINE]
Free PMC Article
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