A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells

Abstract

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular ‘Riboglow’ platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescence turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small noncoding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye-binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Covalent attachment of fluorophores to cobalamin (Cbl) results in fluorescence quenching, inducing fluorescence turn-on of the probe upon binding to riboswitch RNA.
Fig. 2: Cbl riboswitch RNAs induce fluorescence turn-on in Cbl-fluorophore probes in vitro.
Fig. 3: Monitoring ACTB mRNA localization to stress granules (SG) via Cbl-fluorophore probe binding to the RNA tag A.
Fig. 4: Comparison of ACTB mRNA imaging in stress granules (SG) by RNA tagging systems with four fluorophores per RNA.
Fig. 5: Monitoring cytosolic U-bodies via AT-tagged U1.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Change history

  • 05 August 2019

    In the version of this article originally published, numbered compounds were not linked correctly to their respective compound pages. The error has been corrected in the HTML version of this paper.

References

  1. 1.

    Gerstberger, S., Hafner, M. & Tuschl, T. A census of human RNA-binding proteins. Nat. Rev. Genet. 15, 829–845 (2014).

    CAS  Article  Google Scholar 

  2. 2.

    Buchan, J. R. & Parker, R. Eukaryotic stress granules: the ins and outs of translation. Mol. Cell 36, 932–941 (2009).

    CAS  Article  Google Scholar 

  3. 3.

    Decker, C. J. & Parker, R. P-bodies and stress granules: possible roles in the control of translation and mRNA degradation. Cold Spring Harb. Perspect. Biol. 4, a012286 (2012).

    Article  Google Scholar 

  4. 4.

    Matera, A. G. & Wang, Z. A day in the life of the spliceosome. Nat. Rev. Mol. Cell Biol. 15, 108–121 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Tsalikis, J. et al. Intracellular bacterial pathogens trigger the formation of U small nuclear RNA bodies (U bodies) through metabolic stress induction. J. Biol. Chem. 290, 20904–20918 (2015).

    CAS  Article  Google Scholar 

  6. 6.

    Fusco, D. et al. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161–167 (2003).

    CAS  Article  Google Scholar 

  7. 7.

    Wu, B., Chen, J. & Singer, R. H. Background free imaging of single mRNAs in live cells using split fluorescent proteins. Sci. Rep. 4, 3615 (2014).

    Article  Google Scholar 

  8. 8.

    Tutucci, E. et al. An improved MS2 system for accurate reporting of the mRNA life cycle. Nat. Methods 15, 81–89 (2018).

    CAS  Article  Google Scholar 

  9. 9.

    Garcia, J. F. & Parker, R. MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA 21, 1393–1395 (2015).

    CAS  Article  Google Scholar 

  10. 10.

    Wu, B., Eliscovich, C., Yoon, Y. J. & Singer, R. H. Translation dynamics of single mRNAs in live cells and neurons. Science 352, 1430–1435 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Katz, Z. B. et al. Mapping translation ‘hot-spots’ in live cells by tracking single molecules of mRNA and ribosomes. eLife 5, 1–17 (2016).

    Article  Google Scholar 

  12. 12.

    Nguyen, D. H., DeFina, S. C., Fink, W. H. & Dieckmann, T. Binding to an RNA aptamer changes the charge distribution and conformation of malachite green. J. Am. Chem. Soc. 124, 15081–15084 (2002).

    CAS  Article  Google Scholar 

  13. 13.

    Babendure, J. R., Adams, S. R. & Tsien, R. Y. Aptamers switch on fluorescence of triphenylmethane dyes. J. Am. Chem. Soc. 125, 14716–14717 (2003).

    CAS  Article  Google Scholar 

  14. 14.

    Arora, A., Sunbul, M. & Jäschke, A. Dual-colour imaging of RNAs using quencher- and fluorophore-binding aptamers. Nucleic Acids Res. 43, e144 (2015).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Sunbul, M. & Jäschke, A. Contact-mediated quenching for RNA imaging in bacteria with a fluorophore-binding aptamer. Angew. Chem. Int. Edn. Engl. 52, 13401–13404 (2013).

    CAS  Article  Google Scholar 

  16. 16.

    Tan, X. et al. Fluoromodules consisting of a promiscuous RNA aptamer and red or blue fluorogenic cyanine dyes: selection, characterization, and bioimaging. J. Am. Chem. Soc. 139, 9001–9009 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).

    CAS  Article  Google Scholar 

  18. 18.

    Filonov, G. S., Moon, J. D., Svensen, N. & Jaffrey, S. R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J. Am. Chem. Soc. 136, 16299–16308 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Dolgosheina, E. V. et al. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem. Biol. 9, 2412–2420 (2014).

    CAS  Article  Google Scholar 

  20. 20.

    Autour, A. et al. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nat. Commun. 9, 656 (2018).

    Article  Google Scholar 

  21. 21.

    Filonov, G. S. & Jaffrey, S. R. RNA imaging with dimeric Broccoli in live bacterial and mammalian cells. Curr. Protoc. Chem. Biol. 8, 1–28 (2016).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Song, W. et al. Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nat. Chem. Biol. 13, 1187–1194 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Ceres, P., Trausch, J. J. & Batey, R. T. Engineering modular ‘ON’ RNA switches using biological components. Nucleic Acids Res. 41, 10449–10461 (2013).

    CAS  Article  Google Scholar 

  24. 24.

    Ceres, P., Garst, A. D., Marcano-Velázquez, J. G. & Batey, R. T. Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices. ACS Synth. Biol. 2, 463–472 (2013).

    CAS  Article  Google Scholar 

  25. 25.

    Johnson, J. E. Jr., Reyes, F. E., Polaski, J. T. & Batey, R. T. B12 cofactors directly stabilize an mRNA regulatory switch. Nature 492, 133–137 (2012).

    CAS  Article  Google Scholar 

  26. 26.

    Lee, M. & Grissom, C. B. Design, synthesis, and characterization of fluorescent cobalamin analogues with high quantum efficiencies. Org. Lett. 11, 2499–2502 (2009).

    CAS  Article  Google Scholar 

  27. 27.

    Smeltzer, C. C. et al. Synthesis and characterization of fluorescent cobalamin (CobalaFluor) derivatives for imaging. Org. Lett. 3, 799–801 (2001).

    CAS  Article  Google Scholar 

  28. 28.

    Fedosov, S. N. et al. Application of a fluorescent cobalamin analogue for analysis of the binding kinetics. A study employing recombinant human transcobalamin and intrinsic factor. FEBS J. 273, 4742–4753 (2006).

    CAS  Article  Google Scholar 

  29. 29.

    Chromiński, M. & Gryko, D. “Clickable” vitamin B12 derivative. Chemistry 19, 5141–5148 (2013).

    Article  Google Scholar 

  30. 30.

    Loska, R., Janiga, A. & Gryko, D. Design and synthesis of protoporphyrin IX/vitamin B-12 molecular hybrids via CuAAC reaction. J. Porphyr. phtalocyanines 17, 104–117 (2013).

    CAS  Article  Google Scholar 

  31. 31.

    Trachman, R. J. III et al. Structural basis for high-affinity fluorophore binding and activation by RNA Mango. Nat. Chem. Biol. 13, 807–813 (2017).

    CAS  Article  Google Scholar 

  32. 32.

    Warner, K. D. et al. Structural basis for activity of highly efficient RNA mimics of green fluorescent protein. Nat. Struct. Mol. Biol. 21, 658–663 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Jeng, S. C. Y., Chan, H. H. Y., Booy, E. P., McKenna, S. A. & Unrau, P. J. Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers. RNA 22, 1884–1892 (2016).

    CAS  Article  Google Scholar 

  34. 34.

    Filonov, G. S., Kam, C. W., Song, W. & Jaffrey, S. R. In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies. Chem. Biol. 22, 649–660 (2015).

    CAS  Article  Google Scholar 

  35. 35.

    Ponchon, L. & Dardel, F. Recombinant RNA technology: the tRNA scaffold. Nat. Methods 4, 571–576 (2007).

    CAS  Article  Google Scholar 

  36. 36.

    Morisaki, T. et al. Real-time quantification of single RNA translation dynamics in living cells. Science 352, 1425–1429 (2016).

    CAS  Article  Google Scholar 

  37. 37.

    Hayashi-Takanaka, Y. et al. Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling. Nucleic Acids Res. 39, 6475–6488 (2011).

    CAS  Article  Google Scholar 

  38. 38.

    McNeil, P. L. & Warder, E. Glass beads load macromolecules into living cells. J. Cell Sci. 88, 669–678 (1987).

    PubMed  Google Scholar 

  39. 39.

    Nelles, D. A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Zurla, C., Lifland, A. W. & Santangelo, P. J. Characterizing mRNA interactions with RNA granules during translation initiation inhibition. PLoS One 6, e19727 (2011).

    CAS  Article  Google Scholar 

  41. 41.

    Grimm, J. B. et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat. Methods 14, 987–994 (2017).

    CAS  Article  Google Scholar 

  42. 42.

    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).

    CAS  Article  Google Scholar 

  43. 43.

    Wu, B., Chao, J. A. & Singer, R. H. Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys. J. 102, 2936–2944 (2012).

    CAS  Article  Google Scholar 

  44. 44.

    Ni, C. Z. et al. Crystal structure of the MS2 coat protein dimer: implications for RNA binding and virus assembly. Structure 3, 255–263 (1995).

    CAS  Article  Google Scholar 

  45. 45.

    Han, K. Y., Leslie, B. J., Fei, J., Zhang, J. & Ha, T. Understanding the photophysics of the spinach-DFHBI RNA aptamer-fluorogen complex to improve live-cell RNA imaging. J. Am. Chem. Soc. 135, 19033–19038 (2013).

    CAS  Article  Google Scholar 

  46. 46.

    McCloskey, A., Taniguchi, I., Shinmyozu, K. & Ohno, M. hnRNP C tetramer measures RNA length to classify RNA polymerase II transcripts for export. Science 335, 1643–1646 (2012).

    CAS  Article  Google Scholar 

  47. 47.

    Ishikawa, H. et al. Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis. Nucleic Acids Res. 42, 2708–2724 (2014).

    CAS  Article  Google Scholar 

  48. 48.

    Hutten, S., Chachami, G., Winter, U., Melchior, F. & Lamond, A. I. A role for the Cajal-body-associated SUMO isopeptidase USPL1 in snRNA transcription mediated by RNA polymerase II. J. Cell Sci. 127, 1065–1078 (2014).

    CAS  Article  Google Scholar 

  49. 49.

    Nahvi, A., Barrick, J. E. & Breaker, R. R. Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res. 32, 143–150 (2004).

    CAS  Article  Google Scholar 

  50. 50.

    Quadros, E. V. & Sequeira, J. M. Cellular uptake of cobalamin: transcobalamin and the TCblR/CD320 receptor. Biochimie 95, 1008–1018 (2013).

    CAS  Article  Google Scholar 

  51. 51.

    Edwards, A. L., Garst, A. D. & Batey, R. T. Determining structures of RNA aptamers and riboswitches by X-ray crystallography. Methods Mol. Biol. 535, 135–163 (2009).

    CAS  Article  Google Scholar 

  52. 52.

    Polaski, J. T., Holmstrom, E. D., Nesbitt, D. J. & Batey, R. T. Mechanistic insights into cofactor-dependent coupling of RNA folding and mRNA transcription/translation by a cobalamin riboswitch. Cell Rep. 15, 1100–1110 (2016).

    CAS  Article  Google Scholar 

  53. 53.

    Gilbert, S. D. & Batey, R. T. Monitoring RNA-ligand interactions using isothermal titration calorimetry. Methods Mol. Biol. 540, 97–114 (2009).

    CAS  Article  Google Scholar 

  54. 54.

    Lakowicz, J. R. Quenching of Fluorescence. in Principles of Fluorescence Spectroscopy 277–330 (Springer, 2006).

  55. 55.

    Beckley, S. A. et al. Reduction of target gene expression by a modified U1 snRNA. Mol. Cell. Biol. 21, 2815–2825 (2001).

    CAS  Article  Google Scholar 

  56. 56.

    Wheeler, J. R., Matheny, T., Jain, S., Abrisch, R. & Parker, R. Distinct stages in stress granule assembly and disassembly. eLife 5, 1–25 (2016).

    Google Scholar 

  57. 57.

    Xi, L., Schmidt, J. C., Zaug, A. J., Ascarrunz, D. R. & Cech, T. R. A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression. Genome Biol. 16, 231 (2015).

    Article  Google Scholar 

  58. 58.

    Shpargel, K. B. & Matera, A. G. Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins. Proc. Natl Acad. Sci. USA 102, 17372–17377 (2005).

    CAS  Article  Google Scholar 

  59. 59.

    Shpargel, K. B., Ospina, J. K., Tucker, K. E., Matera, A. G. & Hebert, M. D. Control of Cajal body number is mediated by the coilin C-terminus. J. Cell Sci. 116, 303–312 (2003).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors would like to acknowledge financial support from the Human Frontiers Science Project and NIH Director’s Pioneer Award GM114863 (to A.E.P.). We acknowledge support from the National Science Centre, SYMFONIA DEC-2014/12/W/ST5/00589 to D. Gryko and A.J.W., the National Institutes of Health (5R01 GM073850) to R.T.B., and NSF Physics Frontier Center at JILA (PHY1734006) to R.J.. We thank L. Lavis (Janelia Research Campus) for providing fluorophores JF585, SiR594 and JF646 for Halo staining, S. Jaffrey (Cornell University) and G. Matera (UNC Chapel Hill) for contributing plasmids and S. Shukla and J. Garcia for helpful discussions; T. Stasevich, D. Muhlrad, J. Lee and M. Lo for technical expertise; and to J. Eberhard and J. Gassensmith for helpful discussions. The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core, whose Nikon A1R microscope was acquired by the generous support of the NIST-CU Cooperative Agreement award number 70NANB15H226. R.J is a staff member in the Quantum Physics Division of the National Institute of Standards and Technology (NIST). Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Author information

Affiliations

Authors

Contributions

E.B., J.T.P., R.T.B. and A.E.P. conceptualized and designed the study. J.T.P. and R.T.B. rationally designed riboswitch variants. E.B., J.T.P., R.T.B., A.J.W., D.G. and A.E.P. designed organic probes. A.J.W. and M.C. synthesized organic probes. J.T.P. and Z.E.H. purified riboswitch variants for in vitro work. E.B. performed in vitro work, designed and performed cellular work, and analyzed data with input from all authors. D.B. constructed plasmids and assisted with cellular work. S.-T.H. performed in vitro fluorescence lifetime and bleaching experiments. J.R.W. made the Halo-G3BP1 U2-OS cell line. R.P. and R.J. provided critical advice. E.B. and A.E.P. wrote the manuscript with edits from all authors.

Corresponding author

Correspondence to Amy E. Palmer.

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.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–13, Supplementary Figures 1–33

Reporting Summary

Supplementary Note 1

Synthetic Procedures

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Braselmann, E., Wierzba, A.J., Polaski, J.T. et al. A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells. Nat Chem Biol 14, 964–971 (2018). https://doi.org/10.1038/s41589-018-0103-7

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing