class="head no_bottom_margin" id="sec1title">IntroductionPrecise tuning of the expression of each gene in the genome is critical for many aspects of cell function. The level of gene expression is regulated at multiple distinct steps, including transcription, mRNA degradation, and translation (). Regulation of all of these steps in gene expression is important, though the relative contribution of each control mechanism varies for different biological processes (, , , , ).Measuring the translation rate from individual mRNAs over time provides valuable information on the mechanisms of translation and translational regulation. In vitro experiments, mainly using bacterial ribosomes, have revealed exquisite information on ribosome translocation dynamics at the single molecule level (, , , , , ), but such methods have not yet been applied in vivo. In contrast, a genome-wide snapshot of the translational efficiency of endogenous mRNAs in vivo can be obtained through the method of ribosomal profiling (, ). However, this method requires averaging of many cells and provides limited temporal information because of the requirement to lyse cells to make these measurements. Single cell imaging studies have succeeded in measuring average protein synthesis rates (, href="#bib6" rid="bib6" class=" bibr popnode">Brittis et al., 2002, href="#bib16" rid="bib16" class=" bibr popnode">Han et al., 2014, href="#bib26" rid="bib26" class=" bibr popnode">Leung et al., 2006, href="#bib37" rid="bib37" class=" bibr popnode">Tanenbaum et al., 2015, href="#bib47" rid="bib47" class=" bibr popnode">Yu et al., 2006), observing the first translation event of an mRNA (href="#bib15" rid="bib15" class=" bibr popnode">Halstead et al., 2015), localizing sub-cellular sites of translation by co-localizing mRNAs and ribosomes (href="#bib23" rid="bib23" class=" bibr popnode">Katz et al., 2016, href="#bib45" rid="bib45" class=" bibr popnode">Wu et al., 2015), and staining nascent polypeptides with small molecule dyes (href="#bib30" rid="bib30" class=" bibr popnode">Rodriguez et al., 2006).While ribosomal profiling and other recently developed methods have provided many important new insights into the regulation of translation, many questions cannot be addressed using current technologies. For example, it is unclear to what extent different mRNA molecules produced in a single cell from the same gene behave similarly. Many methods to study translation in vivo require averaging of many mRNAs, masking potential differences between individual mRNA molecules. Such differences could arise from differential post-transcriptional regulation, such as nucleotide modifications (href="#bib9" rid="bib9" class=" bibr popnode">Choi et al., 2016, href="#bib42" rid="bib42" class=" bibr popnode">Wang et al., 2015), differential transcript lengths through use of alternative transcriptional start sites (href="#bib31" rid="bib31" class=" bibr popnode">Rojas-Duran and Gilbert, 2012) or polyadenylation site selection (href="#bib12" rid="bib12" class=" bibr popnode">Elkon et al., 2013, href="#bib14" rid="bib14" class=" bibr popnode">Gupta et al., 2014), differences in ribonucleic protein (RNP) composition (href="#bib45" rid="bib45" class=" bibr popnode">Wu et al., 2015), distinct intracellular localization (href="#bib18" rid="bib18" class=" bibr popnode">Hüttelmaier et al., 2005), or different states of RNA secondary structure (href="#bib2" rid="bib2" class=" bibr popnode">Babendure et al., 2006, href="#bib24" rid="bib24" class=" bibr popnode">Kertesz et al., 2010). Heterogeneity among mRNA molecules could have a profound impact on the total amount of polypeptide produced, as well as the localization of protein synthesis, but remains poorly studied. Furthermore, the extent to which translation of single mRNA molecules varies over time is also largely unknown. For example, translation may occur in bursts, rather than continuously (href="#bib38" rid="bib38" class=" bibr popnode">Tatavarty et al., 2012, href="#bib47" rid="bib47" class=" bibr popnode">Yu et al., 2006), and regulation of protein synthesis may occur by modulating burst size and/or frequency, which could occur either globally or on each mRNA molecule individually. In addition, the ability of an mRNA molecule to initiate translation may vary with time or spatial location, for example as cells progress through the cell cycle (href="#bib34" rid="bib34" class=" bibr popnode">Stumpf et al., 2013, href="#bib37" rid="bib37" class=" bibr popnode">Tanenbaum et al., 2015) or undergo active microtubule-based transport to particular cellular destinations (href="#bib17" rid="bib17" class=" bibr popnode">Holt and Schuman, 2013). Such regulation could involve changes in the rates of translation initiation and/or the ribosome elongation. To address these questions, new methods are required for visualizing translation of single mRNA molecules in live cells over time.Here, we present a method, based on the SunTag fluorescence tagging system that we recently developed (href="#bib36" rid="bib36" class=" bibr popnode">Tanenbaum et al., 2014), for measuring the translation of single mRNA molecules over long periods of time. Using this system, we have measured initiation, elongation, and stalling on individual mRNA molecules and have uncovered unexpected heterogeneity among different mRNA molecules encoded by the same gene within a single cell. Our system will be widely applicable to the study of mRNA translation in live cells.
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