Quantifying Single mRNA Translation Kinetics in Living Cells

One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single messenger RNA (mRNA) translation. Here we describe how recent advances in protein tagging and imaging technologies are being used to precisely visualize and quantify the synthesis of nascent polypeptide chains from single mRNA in living cells. We focus on recent applications of repeat-epitope tags and describe how they enable quantification of single mRNA ribosomal densities, translation initiation and elongation rates, and translation site mobility and higher-order structure. Together with complementary live-cell assays to monitor translation using fast-maturing fluorophores and mRNA-binding protein knockoff, single-molecule studies are beginning to uncover striking and unexpected heterogeneity in gene expression at the level of translation. Outline 1 Introduction 2 Breaking the single-molecule barrier for live-cell imaging of translation 3 Amplifying live-cell single mRNA translation signals with repeat-epitope tags 4 Limitations of repeat-epitope tags for translation imaging 5 Accessory tags for imaging single mRNA translation in living cells 6 Tagging endogenous proteins with repeatepitope tags 7 Counting ribosomes in polysomes with repeat-epitope tags 8 Quantifying translation elongation with repeat-epitope tags 9 Quantifying translation initiation with repeatepitope tags 10 Quantifying polysome mobility and shape with repeat-epitope tags 11 Emerging consensus in the field of live-cell imaging of single RNA translation 12 Heterogeneity in single mRNA translation efficiency 13 Heterogeneity in ribosome movement along single mRNA 14 Heterogeneity in the localization and mobility of translation sites