Title | Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. |
Publication Type | Journal Article |
Year of Publication | 2017 |
Authors | Song W, Filonov GS, Kim H, Hirsch M, Li X, Moon JD, Jaffrey SR |
Journal | Nat Chem Biol |
Volume | 13 |
Issue | 11 |
Pagination | 1187-1194 |
Date Published | 2017 Nov |
ISSN | 1552-4469 |
Keywords | Aptamers, Nucleotide, Base Pairing, Base Sequence, Chromophore-Assisted Light Inactivation, Fluorescent Dyes, Gene Expression Regulation, HEK293 Cells, Humans, Luminescent Proteins, Nucleic Acid Conformation, Optical Imaging, RNA Polymerase III, Sirolimus, TOR Serine-Threonine Kinases, Transcription, Genetic |
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. |
DOI | 10.1038/nchembio.2477 |
Alternate Journal | Nat. Chem. Biol. |
PubMed ID | 28945233 |
PubMed Central ID | PMC5679246 |
Grant List | R01 EB010249 / EB / NIBIB NIH HHS / United States R01 EB018659 / EB / NIBIB NIH HHS / United States R01 NS064516 / NS / NINDS NIH HHS / United States |
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