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Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex

Nature Chemical Biology volume 13, pages 1187–1194 (2017)Cite this article

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Abstract

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA–fluorophore complex.

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Figure 1: The Corn aptamer activates the fluorescence of the DFHO fluorophore.
Figure 2: Corn exhibits remarkable photostability in vivo and in vitro.
Figure 3: Orange Broccoli and Red Broccoli activate and tune the fluorescence of the DFHO fluorophore.
Figure 4: Imaging Pol III activity in single cells using Corn.
Figure 5: The mTOR inhibitor temsirolimus shows a variety of Pol III activity trajectories.

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References

  1. White, R.J. RNA polymerase III transcription and cancer. Oncogene 23, 3208–3216 (2004).

    Article  CAS  Google Scholar 

  2. White, R.J., Gottlieb, T.M., Downes, C.S. & Jackson, S.P. Mitotic regulation of a TATA-binding-protein-containing complex. Mol. Cell. Biol. 15, 1983–1992 (1995).

    Article  CAS  Google Scholar 

  3. Felton-Edkins, Z.A. et al. The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB. EMBO J. 22, 2422–2432 (2003).

    Article  CAS  Google Scholar 

  4. Moir, R.D. & Willis, I.M. Regulation of pol III transcription by nutrient and stress signaling pathways. Biochim. Biophys. Acta 1829, 361–375 (2013).

    Article  CAS  Google Scholar 

  5. Felton-Edkins, Z.A. et al. Direct regulation of RNA polymerase III transcription by RB, p53 and c-Myc. Cell Cycle 2, 181–184 (2003).

    Article  CAS  Google Scholar 

  6. Shor, B. et al. Requirement of the mTOR kinase for the regulation of Maf1 phosphorylation and control of RNA polymerase III-dependent transcription in cancer cells. J. Biol. Chem. 285, 15380–15392 (2010).

    Article  CAS  Google Scholar 

  7. Kantidakis, T., Ramsbottom, B.A., Birch, J.L., Dowding, S.N. & White, R.J. mTOR associates with TFIIIC, is found at tRNA and 5S rRNA genes, and targets their repressor Maf1. Proc. Natl. Acad. Sci. USA 107, 11823–11828 (2010).

    Article  CAS  Google Scholar 

  8. Li, X. et al. Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975 (1998).

    Article  CAS  Google Scholar 

  9. Sisodia, S.S., Sollner-Webb, B. & Cleveland, D.W. Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol. Cell. Biol. 7, 3602–3612 (1987).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Strack, R.L., Disney, M.D. & Jaffrey, S.R. A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat. Methods 10, 1219–1224 (2013).

    Article  CAS  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Wang, P.C. et al. Photochemical properties of Spinach and its use in selective imaging. Chem. Sci. 4, 2865–2873 (2013).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. You, M. & Jaffrey, S.R. Structure and mechanism of RNA mimics of green fluorescent protein. Annu. Rev. Biophys. 44, 187–206 (2015).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. Huang, H. et al. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore. Nat. Chem. Biol. 10, 686–691 (2014).

    Article  CAS  Google Scholar 

  19. Gross, L.A., Baird, G.S., Hoffman, R.C., Baldridge, K.K. & Tsien, R.Y. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11990–11995 (2000).

    Article  CAS  Google Scholar 

  20. Wall, M.A., Socolich, M. & Ranganathan, R. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat. Struct. Biol. 7, 1133–1138 (2000).

    Article  CAS  Google Scholar 

  21. Yarbrough, D., Wachter, R.M., Kallio, K., Matz, M.V. & Remington, S.J. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Proc. Natl. Acad. Sci. USA 98, 462–467 (2001).

    Article  CAS  Google Scholar 

  22. Song, W., Strack, R.L., Svensen, N. & Jaffrey, S.R. Plug-and-play fluorophores extend the spectral properties of Spinach. J. Am. Chem. Soc. 136, 1198–1201 (2014).

    Article  CAS  Google Scholar 

  23. Chudakov, D.M., Matz, M.V., Lukyanov, S. & Lukyanov, K.A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90, 1103–1163 (2010).

    Article  CAS  Google Scholar 

  24. Stoltenburg, R., Reinemann, C. & Strehlitz, B. SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 24, 381–403 (2007).

    Article  CAS  Google Scholar 

  25. Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. 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).

    Article  CAS  Google Scholar 

  28. Warner, K.D. et al. A homodimer interface without base pairs in an RNA mimic of red fluorescent protein. Nat. Chem. Biol. 13 http://dx.doi.org/10.1038/nchembio.2475 (2017).

    Article  CAS  Google Scholar 

  29. Good, P.D. et al. Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther. 4, 45–54 (1997).

    Article  CAS  Google Scholar 

  30. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  Google Scholar 

  31. Dieci, G., Fiorino, G., Castelnuovo, M., Teichmann, M. & Pagano, A. The expanding RNA polymerase III transcriptome. Trends Genet. 23, 614–622 (2007).

    Article  CAS  Google Scholar 

  32. Paul, C.P. et al. Localized expression of small RNA inhibitors in human cells. Mol. Ther. 7, 237–247 (2003).

    Article  CAS  Google Scholar 

  33. Perry, R.P. & Kelley, D.E. Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J. Cell. Physiol. 76, 127–139 (1970).

    Article  CAS  Google Scholar 

  34. Neshat, M.S. et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl. Acad. Sci. USA 98, 10314–10319 (2001).

    Article  CAS  Google Scholar 

  35. García-Martínez, J.M. et al. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem. J. 421, 29–42 (2009).

    Article  Google Scholar 

  36. Mayer, C. & Grummt, I. Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases. Oncogene 25, 6384–6391 (2006).

    Article  CAS  Google Scholar 

  37. Bonhoure, N. et al. Loss of the RNA polymerase III repressor MAF1 confers obesity resistance. Genes Dev. 29, 934–947 (2015).

    Article  CAS  Google Scholar 

  38. Dong, J. et al. Isomerization in fluorescent protein chromophores involves addition/elimination. J. Am. Chem. Soc. 130, 14096–14098 (2008).

    Article  CAS  Google Scholar 

  39. Subach, F.V. & Verkhusha, V.V. Chromophore transformations in red fluorescent proteins. Chem. Rev. 112, 4308–4327 (2012).

    Article  CAS  Google Scholar 

  40. Shaner, N.C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank members of the Jaffrey lab for helpful comments and suggestions. We thank J.S. Paige for early contributions to this project, M. You, R.S. Strack, and J. Litke for useful comments and suggestions, K.D. Warner and A. Ferré-D'Amaré (NIH) for communication of unpublished results, J. McCormick (Weill Cornell) for help with flow cytometry and J. Chiaravalli for guidance and support developing the fluorescence polarization assay at the High Throughput and Spectroscopy Resource Center at The Rockefeller University. This work was supported by NIH grants to S.R.J. (R01 NS064516 and R01 EB010249), Kwanjeong Educational Foundation to H.K., and the DFG to M.H.

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Author notes

  1. Grigory S Filonov

    Present address: Essen BioScience, Ann Arbor, Michigan, USA

  2. Wenjiao Song and Grigory S Filonov: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA

    Wenjiao Song, Grigory S Filonov, Hyaeyeong Kim, Markus Hirsch, Xing Li, Jared D Moon & Samie R Jaffrey

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Contributions

W.S., G.S.F., H.K., M.H. and S.R.J. designed experiments, analyzed the data and wrote the manuscript. W.S. devised the synthetic methodologies, developed the aptamers, and characterized their binding properties in vitro and performed live imaging trajectory analysis in living cells. G.S.F. developed and imaged the Pol III reporters using FACS and using microscopy. H.K. performed imaging with different mTOR inhibitors in living cells and studies correlating reporter expression to RNA expression. M.H. performed anisotropy, melting, FPLC, native PAGE and affinity measurements. J.D.M. performed studies of reporter dynamics and half-life. X.L. performed studies of fluorophore stability in diverse conditions.

Corresponding author

Correspondence to Samie R Jaffrey.

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Competing interests

S.R.J. is the co-founder of Lucerna Technologies and has equity in this company. Lucerna has licensed commercialization of technology related to Spinach and other RNA–fluorophore complexes.

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Song, W., Filonov, G., Kim, H. et al. Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex. Nat Chem Biol 13, 1187–1194 (2017). https://doi.org/10.1038/nchembio.2477

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