Multicolour single-molecule tracking of mRNA interactions with RNP granules (original) (raw)

Nature Cell Biology volume 21, pages 162–168 (2019)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Access Nature and 54 other Nature Portfolio journals

Get Nature+, our best-value online-access subscription

$29.99 / 30 days

cancel any time

Subscribe to this journal

Receive 12 print issues and online access

$209.00 per year

only $17.42 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

Code availability

Data availability

References

  1. Buchan, J. R. & Parker, R. Eukaryotic stress granules: the ins and outs of translation. Mol. Cell 36, 932–941 (2009).
    Article CAS Google Scholar
  2. Kedersha, N., Ivanov, P. & Anderson, P. Stress granules and cell signaling: more than just a passing phase? Trends Biochem. Sci. 38, 494–506 (2013).
    Article CAS Google Scholar
  3. Schisa, J. A. Effects of stress and aging on ribonucleoprotein assembly and function in the germ line. Wiley Interdiscip. Rev. RNA 5, 231–246 (2014).
    Article CAS Google Scholar
  4. Sudhakaran, I. P. et al. FMRP and ataxin-2 function together in long-term olfactory habituation and neuronal translational control. Proc. Natl Acad. Sci. USA 111, E99–E108 (2014).
    Article CAS Google Scholar
  5. El-Naggar, A. M. & Sorensen, P. H. Translational control of aberrant stress responses as a hallmark of cancer. J. Pathol. 244, 650–666 (2018).
    Article CAS Google Scholar
  6. Grabocka, E. & Bar-Sagi, D. Mutant KRAS enhances tumor cell fitness by upregulating stress granules. Cell 167, 1803–1813 (2016).
    Article CAS Google Scholar
  7. Zhang, K. et al. Stress granule assembly disrupts nucleocytoplasmic transport. Cell 173, 958–971 (2018).
    Article CAS Google Scholar
  8. Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133 (2006).
    Article CAS Google Scholar
  9. Li, Y. R., King, O. D., Shorter, J. & Gitler, A. D. Stress granules as crucibles of ALS pathogenesis. J. Cell Biol. 201, 361–372 (2013).
    Article CAS Google Scholar
  10. Morisaki, T. et al. Real-time quantification of single RNA translation dynamics in living cells. Science 352, 1425–1429 (2016).
    Article CAS Google Scholar
  11. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).
    Article CAS Google Scholar
  12. Kedersha, N., Tisdale, S., Hickman, T. & Anderson, P. Real-time and quantitative imaging of mammalian stress granules and processing bodies. Methods Enzymol. 448, 521–552 (2008).
    Article CAS Google Scholar
  13. Khong, A. et al. The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol. Cell 68, 808–820 (2017).
    Article CAS Google Scholar
  14. Wheeler, J. R., Matheny, T., Jain, S., Abrisch, R. & Parker, R. Distinct stages in stress granule assembly and disassembly. eLife 5, e18413 (2016).
  15. Kedersha, N. et al. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J. Cell Biol. 151, 1257–1268 (2000).
    Article CAS Google Scholar
  16. Ohshima, D., Arimoto-Matsuzaki, K., Tomida, T., Takekawa, M. & Ichikawa, K. Spatio-temporal dynamics and mechanisms of stress granule assembly. PLoS Comput. Biol. 11, e1004326 (2015).
    Article Google Scholar
  17. Namkoong, S., Ho, A., Woo, Y. M., Kwakm, H. & Lee, J. H. Systematic characterization of stress-induced RNA granulation. Mol. Cell 70, 175–187 (2018).
    Article CAS Google Scholar
  18. Wilbertz, J. H. et al. Single-molecule imaging of mRNA localization and regulation during the integrated stress response. Mol. Cell https://doi.org/10.1016/j.molcel.2018.12.006 (2019).
  19. Kedersha, N. et al. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 169, 871–884 (2005).
    Article CAS Google Scholar
  20. Simpson, C. E., Lui, J., Kershaw, C. J., Sims, P. F. G. & Ashe, M. P. mRNA localization to P-bodies in yeast is biphasic with many mRNAs captured in a late Bfr1p-dependent wave. J. Cell Sci. 127, 1254–1262 (2014).
    Article CAS Google Scholar
  21. Wei, M.-T. et al. Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. Nat. Chem. 9, 1118–1125 (2017).
    Article CAS Google Scholar
  22. Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697 (2016).
    Article CAS Google Scholar
  23. Molliex, A. et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123–133 (2015).
    Article CAS Google Scholar
  24. Sfakianos, A. P., Whitmarsh, A. J. & Ashe, M. P. Ribonucleoprotein bodies are phased. Biochem. Soc. Trans. 44, 1411–1416 (2016).
    Article CAS Google Scholar
  25. Niewidok, B. et al. Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules. J. Cell Biol. 217, 1303–1318 (2018).
    Article CAS Google Scholar
  26. Banani, S. F. et al. Compositional control of phase-separated cellular bodies. Cell 166, 651–663 (2016).
    Article CAS Google Scholar
  27. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).
    Article CAS Google Scholar
  28. Edelstein, A. D. et al. Advanced methods of microscope control using μManager software. J. Biol. Methods 1, e10 (2014).
    Article Google Scholar
  29. Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using µManager. Curr. Protoc. Mol. Biol. 29, 14.20.1–14.20.17 (2010).
  30. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
    Article CAS Google Scholar
  31. Stellaris® RNA FISH: Protocol for Sequential IF + FISH in Adherent Cells (Biosearch Technologies, 2015); https://biosearchassets.blob.core.windows.net/assets/bti_custom_stellaris_immunofluorescence_seq_protocol.pdf.

Download references

Acknowledgements

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.

Author information

Author notes

  1. These authors contributed equally: Stephanie L. Moon, Tatsuya Morisaki.

Authors and Affiliations

  1. Department of Biochemistry, University of Colorado, Boulder, CO, USA
    Stephanie L. Moon, Anthony Khong & Roy Parker
  2. Howard Hughes Medical Institute, University of Colorado, Boulder, CO, USA
    Stephanie L. Moon, Anthony Khong & Roy Parker
  3. Department of Biochemistry, Colorado State University, Fort Collins, CO, USA
    Tatsuya Morisaki, Kenneth Lyon & Timothy J. Stasevich
  4. World Research Hub Initiative, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
    Timothy J. Stasevich

Authors

  1. Stephanie L. Moon
    You can also search for this author inPubMed Google Scholar
  2. Tatsuya Morisaki
    You can also search for this author inPubMed Google Scholar
  3. Anthony Khong
    You can also search for this author inPubMed Google Scholar
  4. Kenneth Lyon
    You can also search for this author inPubMed Google Scholar
  5. Roy Parker
    You can also search for this author inPubMed Google Scholar
  6. Timothy J. Stasevich
    You can also search for this author inPubMed Google Scholar

Contributions

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 toRoy Parker or Timothy J. Stasevich.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary information

Rights and permissions

About this article

Cite this article

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). https://doi.org/10.1038/s41556-018-0263-4

Download citation

This article is cited by

Associated content