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Multicolour single-molecule tracking of mRNA interactions with RNP granules


Ribonucleoprotein (RNP) granules are non-membrane-bound organelles that have critical roles in the stress response1,2, maternal messenger RNA storage3, synaptic plasticity4, tumour progression5,6 and neurodegeneration7,8,9. However, the dynamics of their mRNA components within and near the granule surface remain poorly characterized, particularly in the context and timing of mRNAs exiting translation. Herein, we used multicolour single-molecule tracking to quantify the precise timing and kinetics of single mRNAs as they exit translation and enter RNP granules during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionally between them. Although translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable, and sometimes rigid, associations with RNP granules with stability increasing with both mRNA length and granule size. Live and fixed cell imaging demonstrated that mRNAs can extend beyond the protein surface of a stress granule, which may facilitate interactions between RNP granules. Thus, the recruitment of mRNPs to RNP granules involves dynamic, stable and extended interactions affected by translation status, mRNA length and granule size that collectively regulate RNP granule dynamics.

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Fig. 1: mRNAs are translationally repressed before entering SGs and resume translation following SG disassembly.
Fig. 2: mRNAs interact transiently and stably with SGs.
Fig. 3: mRNAs interact transiently and stably with PBs in stressed cells and traffic bidirectionally between PBs and SGs.
Fig. 4: mRNAs can be rigidly positioned within SGs and/or tethered to them.

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Code availability

Custom Mathematica (version code was deposited on GitHub and can be accessed at

Data availability

Source data for Figs. 1b−f, 2c–e, 3a–d, 4a,c–e and Supplementary Figs. 1,2,4 are provided in Supplementary Table 2. The raw images were deposited to figshare and can be accessed at: and All other data supporting the findings of this study are available from the corresponding authors on reasonable request.


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We thank N. Kedersha for providing GFP–G3BP1/mRFP–DCP1a U-2 OS cells, L. Lavis for providing JF dyes, and E. Braselmann and T. Nahreini for isolating GFP–G3BP1 U-2 OS cells at the BioFrontiers Institute Flow Cytometry Core facility. We thank B. Dodd and lab members for assistance and helpful suggestions. S.L.M. was funded by the Anna and John J. Sie Foundation; A.K. by a Banting postdoctoral fellowship; R.P. by the HHMI; T.J.S. by the NIH (grant no. R35GM119728) and the Boettcher Foundation’s Webb-Waring Biomedical Research Program.

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Authors and Affiliations



S.L.M. and R.P. conceptualized the study; S.L.M., T.M., T.J.S., A.K. and K.L. developed and designed methodology; T.M. and T.J.S. developed and implemented software; S.L.M. validated findings; T.M., T.J.S., S.L.M. and A.K. performed formal analyses; S.L.M., A.K., K.L., T.M. and T.J.S. performed experiments and/or collected data; R.P., T.J.S., T.M., A.K., K.L. and S.L.M. provided resources; T.M., T.J.S. and S.L.M. provided data curation; S.L.M. and R.P. wrote the original draft; R.P., S.L.M., T.J.S., T.M. and A.K. reviewed and edited the drafts; T.M., T.J.S., S.L.M. and A.K. visualized data; R.P., T.J.S., T.M. and S.L.M. supervised the project; S.L.M. managed and coordinated the project; T.J.S., R.P. and S.L.M. acquired funding.

Corresponding authors

Correspondence to Roy Parker or Timothy J. Stasevich.

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Supplementary Figure 1 Translating mRNAs can (rarely) interact with stress granules for several minutes.

A representative long-term interaction between a translating KDM5B mRNA and a SG (>2 min) from 2 mRNAs tracked from 2 cells from 2 independent experiments. The mRNA translation signal persists throughout the interaction with the SG and after exiting the SG. This mRNA is shown as “mRNA2” in Supplementary Movie 3

Supplementary Figure 2 Translation resumes following complete disassembly of stress granules in a single cell during recovery from stress.

Single-cell translation resumption during the recovery from arsenite stress (representative cell shown from 4 cells collected from 2 independent experiments). Translation resumes just a few minutes after SG disassembly. Number of translation foci (green) and SGs (blue) during stress (0–60 min) and following arsenite washout (70–130 min)

Supplementary Figure 3 The distance and angle between mRNAs within a single stress granule can remain consistent over time.

Quantification of the relative locations of KDM5B mRNAs shown in Fig. 4b and c, and Supplementary Movies 11 and 12. The distances (a) and the angles (b) between two KDM5B mRNAs interacting with the same SG are shown. The pair of mRNAs used for distance and angle calculations are shown with arrows (right). The average distances and angles are shown in the left top insets (± SD) from n = 300 time points, from 2 cells collected from 2 independent experiments. (c) Distance of one mRNA (red dot) from the surface of the stress granule (blue). This mRNA exits and re-enters the SG

Supplementary Figure 4 mRNAs can be rigidly adhered within a stress granule and may be tethered to the granule surface.

Representative time series showing three KDM5B mRNAs (depicted as red, blue or green arrows and dots) positioned within SGs over time (from 300 times points, representing one of 2 SGs tracked from 2 cells collected from 2 independent experiments). Yellow arrows indicate where a KDM5B mRNA (red dots) exited and re-entered the SG

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Moon, S.L., Morisaki, T., Khong, A. et al. Multicolour single-molecule tracking of mRNA interactions with RNP granules. Nat Cell Biol 21, 162–168 (2019).

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