SunRiSE: measuring translation elongation at single cell resolution by flow cytometry

Author:

Argüello Rafael J.1,Simoes Marisa Reverendo1,Mendes Andreia1,Camosseto Voahirana1ORCID,Torres Adrian G.2ORCID,de Pouplana Lluis Ribas23,van de Pavert Serge A.1,Gatti Evelina14ORCID,Pierre Philippe14

Affiliation:

1. Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS, Marseille, France

2. Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Parc Científic de Barcelona, C/Baldiri Reixac 10, 08028 Barcelona, Catalonia, Spain

3. Catalan Institute for Research and Advanced Studies (ICREA), P/Lluis Companys 23, 08010 Barcelona, Catalonia, Spain

4. Institute for Research in Biomedicine (iBiMED) and Ilidio Pinho Foundation, Department of Medical Sciences, University of Aveiro, 3810-193 Aveiro, Portugal

Abstract

The processing rate of translating ribosomes along mRNAs regulates gene expression by controling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage, or phosphorylation of eukaryotic Elongation Factor 2 (eEF2). Current methods to measure translation elongation lack single cell resolution, require expression of multiple transgenes and have never been successfully applied ex-vivo. Here, we show by using a combination of puromycilation detection and flow cytometry (SunRiSE), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, cell cycle, phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, and surprisingly little elongation, except upon proteotoxic stress induction.

Funder

Fondation pour la Recherche Medicale

Agence Nationale de la Recherche

Publisher

The Company of Biologists

Subject

Cell Biology

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