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

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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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Strack, and J. Litke for useful comments and suggestions, K.D. Warner and A. Ferré-D'Amaré (NIH) for communication of unpublished results, J. McCormick (Weill Cornell) for help with flow cytometry and J. Chiaravalli for guidance and support developing the fluorescence polarization assay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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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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Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–21 (PDF 4890 kb)

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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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