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.

活细胞中转录速率的定量测量对于揭示转录调控机制很重要。当测量RNA聚合酶III（Pol III）的活性时，这尤其具有挑战性，该酶可转录促进生长的小RNA。为了解决这个问题，我们开发了Corn，一种遗传编码的荧光RNA报告基因，适用于定量细胞中的RNA转录。玉米结合并诱导3,5-二氟-4-羟基亚苄基-咪唑啉酮-2-肟的荧光，类似于红色荧光蛋白（RFP）中的荧光团。值得注意的是，玉米显示出很高的光稳定性，从而可以对mTOR依赖的Pol III转录进行定量荧光成像。我们发现，与放线菌素D不同，mTOR抑制剂导致单个细胞中的异源转录抑制。玉米标记的Pol III转录水平的定量成像揭示了mTOR抑制引起的独特的Pol III转录“轨迹”。总之，这些研究为通过直接成像包含光稳定的RNA-荧光团复合体的Pol III转录本提供了定量测量Pol III转录的方法。