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 5a €2 terminal oligopyrimidine (5a €2 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.
Original language | English |
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Pages (from-to) | 86-93 |
Number of pages | 8 |
Journal | Nature Methods |
Volume | 11 |
Issue number | 1 |
DOIs | |
State | Published - 2014 |