RNA degradation plays a fundamental role in regulating gene expression. In order to characterize the spatiotemporal dynamics of RNA turnover in single cells, we developed a fluorescent biosensor based on dual-color, single-molecule RNA imaging that allows intact transcripts to be distinguished from stabilized degradation intermediates. Using this method, we measured mRNA decay in single cells and found that individual degradation events occur independently within the cytosol and are not enriched within processing bodies. We show that slicing of an mRNA targeted for endonucleolytic cleavage by the RNA-induced silencing complex can be observed in real time in living cells. This methodology provides a framework for investigating the entire life history of individual mRNAs from birth to death in single cells.

Highlights d A minority of mRNAs localize to stress granules (SGs) and P-bodies (PBs) d Very few mRNAs move directly from SGs into PBs d LARP1 anchors 5 0 TOP transcripts within SGs and PBs d Granule localization in stress does not affect translation or decay during recovery

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