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Nat Chem Biol. 2017 Nov;13(11):1187-1194. doi: 10.1038/nchembio.2477. Epub 2017 Sep 25.

Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.

Author information

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

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.

PMID:
28945233
PMCID:
PMC5679246
DOI:
10.1038/nchembio.2477
[Indexed for MEDLINE]
Free PMC Article

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