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.

活细胞转录率的定量测量对于揭示转录调控机制非常重要。当测量 RNA 聚合酶 III (Pol III)（转录促进生长的小 RNA）的活性时，这一点尤其具有挑战性。为了解决这个问题，我们开发了 Corn，一种基因编码的荧光 RNA 报告基因，适用于量化细胞中的 RNA 转录。玉米结合并诱导 3,5-二氟-4-羟基苯亚甲基-咪唑啉酮-2-肟的荧光，该荧光类似于红色荧光蛋白 (RFP) 中发现的荧光团。值得注意的是，玉米表现出高光稳定性，能够对 mTOR 依赖性 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 抑制剂会导致单个细胞中的异质转录抑制。玉米标记的 Pol III 转录物水平的定量成像揭示了 mTOR 抑制引起的不同 Pol III 转录“轨迹”。总之，这些研究提供了一种通过对含有光稳定 RNA-荧光团复合物的 Pol III 转录本进行直接成像来定量测量 Pol III 转录的方法。