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Sunrise – Measuring Translation Elongation at Single-Cell Resolution by Means of Flow Cytometry Rafael J

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Sunrise – Measuring Translation Elongation at Single-Cell Resolution by Means of Flow Cytometry Rafael J © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs214346. doi:10.1242/jcs.214346 TOOLS AND RESOURCES SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry Rafael J. Argüello1,*,‡, Marisa Reverendo1,*, Andreia Mendes1, Voahirana Camosseto1,AdrianG.Torres3, Lluis Ribas de Pouplana3,4, Serge A. van de Pavert1,EvelinaGatti1,2,* and Philippe Pierre1,2,*,‡ ABSTRACT or microRNA (miRNA) expression (Petersen et al., 2006; Richter and In vitro The rate at which ribosomes translate mRNAs regulates protein Coller, 2015). translation assays and ribosome profiling have expression by controlling co-translational protein folding and mRNA recently demonstrated that elongation speed affects co-translational stability. Many factors regulate translation elongation, including tRNA protein folding and protein expression (Buhr et al., 2016; Yu et al., levels, codon usage and phosphorylation of eukaryotic elongation factor 2015). The relative contribution of the different environmental and 2 (eEF2). Current methods to measure translation elongation lack endogenous factors on translation elongation in cells directly isolated single-cell resolution, require expression of multiple transgenes and have from human blood or mice tissues is poorly understood and is still never been successfully applied ex vivo. Here, we show, by using a methodologically demanding. Approaching these questions requires combination of puromycilation detection and flow cytometry (a method a technique with high-throughput potential and that measures we call ‘SunRiSE’), that translation elongation can be measured translation elongation rates under physiological conditions in non- ex vivo accurately in primary cells in pure or heterogenous populations isolated abundant and non-transformed cells . Reduced manipulation from blood or tissues. This method allows for the simultaneous and applicability to complex mixes of cells should also be a monitoring of multiple parameters, such as mTOR or S6K1/2 signaling requirement for such a method. Among the techniques available for activity, the cell cycle stage and phosphorylation of translation factors assessing translation elongation rates, one can distinguish methods in single cells, without elaborated, costly and lengthy purification that measure an average rate within a bulk of cells (Arava et al., 2003; procedures. We took advantage of SunRiSE to demonstrate that, in Brar and Weissman, 2015; Dieterich et al., 2010; Ingolia et al., 2009; mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase Starck et al., 2004) from those that measure translation rates at the (eEF2K) mostly affects translation engagement, but has a surprisingly single-mRNA molecule scale and require multiple steps of molecular small effect on elongation, except after proteotoxic stress induction. engineering (Halstead et al., 2015; tom Dieck et al., 2015; Wang et al., 2016; Yan et al., 2016). The first group of methods includes ribosome profiling, polysome profiling and radiolabeled This article has an associated First Person interview with the first author methionine incorporation assays. Given the advantages and recent of the paper. improvements in next-generation sequencing, ribosome profiling has KEY WORDS: Puromycin, Translation elongation factor, B cell, T cell, become a gold standard to monitor translation and ribosome Fetal liver, Protein synthesis engagement (McGlincy and Ingolia, 2017). This technique allows for qualitative and quantitative measurement of translation by INTRODUCTION sequencing RNA fragments that are 28 and 29 nucleotides long Protein synthesis is a central metabolic process that is regulated at and are protected from degradation through their association with different biochemical levels. Polypeptide elongation by ribosomes ribosomes. Single-cell ribosome profiling has, however, never been proceeds discontinuously, with pauses regulating the speed of protein performed, and seems for the moment to be out of technical reach, synthesis (Petersen et al., 2006; Richter and Coller, 2015). The total leaving ribosome profiling approaches with the disadvantage of level of engaged ribosomes and the associated elongation rate are measuring translation through the averaging of many cells, affected by translation elongation and initiation factors, codon usage, sometimes from very heterogeneous populations, during which tRNA abundance and post-transcriptional modifications, as well as individual phenotypes are binned and diluted into one unique dataset. by different steric impediments, such as mRNA secondary structures Puromycin (puro) is an aminonucleoside Tyr-tRNA mimetic antibiotic that enters the A site of ribosomes, and is incorporated into nascent chains. Puro incorporation induces the termination of 1Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, peptide synthesis, and this covalent reaction between nascent proteins CNRS, 13288, Marseille Cedex 9, France. 2Institute for Research in Biomedicine (iBiMED) and Ilidio Pinho Foundation, Department of Medical Sciences, University and puro is called puromycilation (Azzam and Algranati, 1973). of Aveiro, 3810-193 Aveiro, Portugal. 3Institute for Research in Biomedicine (IRB Puromycilation detection with ad hoc antibodies and measurement by Barcelona), The Barcelona Institute of Science and Technology, Parc Cientıfić de flow cytometry has proven to be an extremely versatile method to Barcelona, C/Baldiri Reixac 10, 08028 Barcelona, Catalonia, Spain. 4Catalan Institute for Research and Advanced Studies (ICREA), P/Lluis Companys 23, replace radiolabeled amino acid incorporation as a means to monitor 08010 Barcelona, Catalonia, Spain. protein synthesis levels (Goodman et al., 2012; Schmidt et al., 2009). *These authors contributed equally to this work Here, we propose to combine puro immuno-detection together with ‡ ‘ ’ Authors for correspondence ([email protected]; [email protected]) run-off experiments on translating ribosomes to determine the translation elongation rate in different cell populations, including M.R., 0000-0001-6999-1816; V.C., 0000-0003-1167-6279; A.G.T., 0000-0002- human blood cells and mouse embryonic cells. Importantly, accurate 5479-1325; L.R., 0000-0002-2087-0359; S.A.v., 0000-0002-7147-4380; E.G., 0000- 0002-0667-0799; P.P., 0000-0003-0863-8255 rate measurements allow the direct comparison of protein synthesis activity in different cells types and tissues, independently of the Received 13 December 2017; Accepted 4 April 2018 variations observed in cell size, amount of ribosomes or environmental Journal of Cell Science 1 TOOLS AND RESOURCES Journal of Cell Science (2018) 131, jcs214346. doi:10.1242/jcs.214346 growth conditions. This novel method called ‘SunRiSE’ (for puro-immunomonitoring and, in particular, flow cytometry to SUnSET-based Ribosome Speed of Elongation; SUnSET refers to measure translation in living cells and organisms (Schmidt et al., ‘Surface sensing of translation’) offers novel exploratory capabilities, 2009; Seedhom et al., 2016). Surface sensing of translation (SUnSET) which are illustrated here by an analysis of protein synthesis in normal allows for measuring global translation in individual cells found in and stress conditions after genetic inactivation of translation elongation mixed populations grown in vitro or ex vivo. Despite these clear factor 2 kinase (eEF2K) in mouse embryonic fibroblasts (MEFs). advantages, quantitative SUnSET measurements are not absolute and Surprisingly, eEF2K ablation mostly affects ribosome engagement, can only be performed relative to a given reference sample, and are and most likely protein synthesis initiation at steady state, rather than therefore not adapted for a direct comparison among many cellular ribosome processing speed. The impact of eEF2K on translation samples of different origins and obtained at different times. A precise elongation is, however, revealed upon ER-stress induction, since the measure of a translation elongation rate in different populations translation intensity and elongation rate in eEF2K-deficient cells should, however, solve this issue, by attributing an elongation rate remain unaffected by tunicamycin treatment. Interestingly, the levels constant to each specific cell group. Such a rate constant would allow of phosphorylated eEF2 (P-eEF2) are highly heterogeneous among a direct quantitative comparison of variations in protein synthesis single wild-type (WT) MEFs grown in the same culture conditions, activity among different cells present in heterogeneous populations or and SunRiSE reveals that eEF2 phosphorylation at steady state does tissues, and growing in different environmental conditions. not necessarily correlate with a lower protein synthesis rate. The In addition to puro, antibiotics interfering with ribosome existence of such biochemical heterogeneity further demonstrates the assembly and function have proven to be invaluable reagents to importance of developing novel techniques, like SunRiSE, to measure study translation mechanisms, and in particular initiation and protein synthesis at high resolution and study its impact on the control elongation. Harringtonine efficiently blocks the initiation of of protein expression. elongation during protein synthesis, and thus had been used with ribosome profiling in run-off experiments to establish the rate of RESULTS elongation of mammalian ribosomes (∼330 codons/min) (Ingolia A combination of puro labeling
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