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 for measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this, 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. Unlike actinomycin D, we found that 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 measurements of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…Another common theme is a push to find more red‐shifted dyes to overcome background noise from autofluorescence of cells and tissues. Examples include the development of quinoline blue for use in FIT probes and the development of the Red Broccoli aptamer as an alternative for the original Spinach aptamer . Beyond this, structures that permit long‐term imaging of cellular analytes are commonly sought after.…”

“…Compared to Spinach and Broccoli which lose >50% of fluorescence within 200 ms of constant irradiation, Corn is unaffected up to 10 s. Corn was utilized for quantitative measurement of Pol III transcription in HEK293T cells. DFHO could also bind to Broccoli derivatives yielding Red Broccoli and Orange Broccoli which have red shifted excitation and emission properties; however, these complexes were susceptible to rapid photobleaching …”

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

“…[46] In addition, FLAPs are expressed on an aptamer scaffold (e.g., tRNA Lys 3 ,F 30, Figure 1) for efficient transcription and folding. [52] [52] …”

“…[52] Thefluorogen 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO,F igure 2A)i sa na nalogue of the intrinsic fluorescent moiety in red fluorescent protein (RFP). [52] Thefluorogen 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO,F igure 2A)i sa na nalogue of the intrinsic fluorescent moiety in red fluorescent protein (RFP).…”

“…[52] Thefluorogen 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO,F igure 2A)i sa na nalogue of the intrinsic fluorescent moiety in red fluorescent protein (RFP). [52] [52] …”