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Abstract
SummaryRegulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5′ UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.
Highlights
Precise tuning of the expression of each gene in the genome is critical for many aspects of cell function
A genome-wide snapshot of the translational efficiency of endogenous mRNAs in vivo can be obtained through the method of ribosomal profiling (Ingolia et al, 2009; Ingolia et al, 2011)
Using the SunTag system, we have developed an imaging method that measures the translation of individual mRNAs in living cells
Summary
Precise tuning of the expression of each gene in the genome is critical for many aspects of cell function. A genome-wide snapshot of the translational efficiency of endogenous mRNAs in vivo can be obtained through the method of ribosomal profiling (Ingolia et al, 2009; Ingolia et al, 2011). 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 (Aakalu et al, 2001; Brittis et al, 2002; Han et al, 2014; Leung et al, 2006; Tanenbaum et al, 2015; Yu et al, 2006), observing the first translation event of an mRNA (Halstead et al, 2015), localizing sub-cellular sites of translation by co-localizing mRNAs and ribosomes (Katz et al, 2016; Wu et al, 2015), and staining nascent polypeptides with small molecule dyes (Rodriguez et al, 2006)
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