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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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Strack, and J. Litke for useful comments and suggestions, K.D. Warner and A. Ferré-D'Amaré (NIH) for communication of unpublished results, J. McCormick (Weill Cornell) for help with flow cytometry and J. Chiaravalli for guidance and support developing the fluorescence polarization assay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.
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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