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.

Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively nontranslating mRNA. Here we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur for these transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation.

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

It is well established that mRNAs encoding secretory or membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). The extent to which mRNAs that encode cytosolic proteins associate with the ER, however, remains controversial. To address this question, we quantified the number of cytosolic protein-encoding mRNAs that co-localize with the ER using single-molecule RNA imaging in fixed and living cells. We found that a small but significant number of mRNAs that encode cytosolic proteins associate with the ER and show that this interaction is translation dependent. Furthermore, we demonstrate that cytosolic protein-encoding transcripts can remain on the ER with dwell times consistent with multiple rounds of translation and have higher ribosome occupancies than transcripts translated in the cytosol. These results advance our understanding of the diversity and dynamics of localized translation on the ER.

Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.