title: Parallel measurement of dynamic changes in translation rates in single cells.
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.
authors.affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.
authors.forename: Kyuho
authors.initials: K
authors.lastname: Han
authors.affiliation: 1] Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA. [2] Department of Biochemistry, Stanford University, Stanford, California, USA.
authors.forename: Ariel
authors.initials: A
authors.lastname: Jaimovich
authors.affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.
authors.forename: Gautam
authors.initials: G
authors.lastname: Dey
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Davide
authors.initials: D
authors.lastname: Ruggero
authors.affiliation: Department of Biochemistry and Molecular Biology, The Institute for Medical Research, Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: Oded
authors.initials: O
authors.lastname: Meyuhas
authors.affiliation: 1] Department of Biochemistry, McGill University Montreal, Quebec, Canada. [2] Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada.
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
authors.affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.
authors.forename: Tobias
authors.initials: T
authors.lastname: Meyer
mesh_terms: D020121:5' Untranslated Regions; D020816:Amino Acid Motifs; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008164:Luminescent Proteins / Q000737:chemistry; D051379:Mice; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D014295:Trimethoprim / Q000737:chemistry
chemical_list: D020121:5' Untranslated Regions; D000596:Amino Acids; D008164:Luminescent Proteins; D011506:Proteins; D011743:Pyrimidines; D012333:RNA, Messenger; D014295:Trimethoprim; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases