Fluorescent reporters for real-time single-cell Pol III transcription measurement

摘要 RNA聚合酶III(Pol III)是一种重要的药物靶点，发展迅速 学术研究领域。POL III负责产生对细胞生长至关重要的RNA，例如 TRNAs、某些rRNAs和各种非编码RNAs参与了诸如信使核糖核酸剪接等过程。这些 RNA是细胞生长、增殖和分化所必需的，并扰乱了控制Pol的途径 III现在被认为是针对癌症和肥胖的有价值的药物。尽管Pol III很重要，但几乎没有 已知调控Pol III转录的途径。这在很大程度上是因为没有简单和 Pol III活动的强势记者。这与Pol II启动子的信使核糖核酸的报告分析形成对比 转录-这些记者由控制报告基因(如绿色荧光)的POL II启动子组成 蛋白质，并且一直是发现mRNA转录抑制物药物的主要驱动力。然而，Pol III 生成无法翻译的RNA。因此，几乎所有对POL III的研究都依赖于Northern印迹， 这是缓慢的，不适合高通量的筛选工作。在Lucerna，我们开发了一种 模拟红色荧光蛋白(简称玉米)的光稳定RNA，并证明该RNA可用作 Pol III启动子的强大荧光记者。通过简单地创建Pol III启动子来控制 合成玉米的RNA，荧光可以很容易地检测到，而不需要合成蛋白质。我们 已经使用POLIII启动子的5S亚型演示了这一概念。有趣的是，我们发现有标记的 在细胞暴露于血清和其他促生长信号后，Pol III转录的细胞间差异。 因此，这一期SBIR项目的目标是开发这种简单的、可遗传编码的玉米 报道活体细胞成像和测量Pol III转录的所有三种亚型。此外，我们还将 开发这一报告系统，以便它可以用于流式细胞术分析，使Pol III转录 随着时间的推移，在数十万个牢房中被监测。在第二阶段，我们将把我们的Pol III记者改编成 适销对路的工具包，在疾病相关细胞系中验证POL III报告，并使用CAS9/CRISPR 成像内源POLIII转录活性的技术。总而言之，在这个过程中产生的质粒和试剂盒 项目将产生Pol III的第一个记者，并因此将构成一个重要的商业产品 将在学术RNA研究领域具有广泛的实用价值，并将使小分子的发现成为可能 这类以前很难检测的药物靶点的抑制剂。

SUMMARY RNA polymerase III (Pol III) is increasingly recognized as an important drug target and a rapidly developing area of academic research. Pol III is responsible for generating RNAs that are critical for cell growth, such as tRNAs, certain rRNAs, and various noncoding RNAs involved in processes such as mRNA splicing. These RNAs are required for cell growth, proliferation, and differentiation and disrupting the pathways that control Pol III is now thought to be valuable for targeting cancer and obesity. Despite the importance of Pol III, little is known about the pathways that regulate Pol III transcription. This is largely due to the absence of simple and robust reporters for Pol III activity. This is in contrast to reporter assays for Pol II promoters for mRNA transcription—these reporters comprise Pol II promoters controlling reporter genes such as green fluorescent protein, and have been the major driver for drug discovery for mRNA transcription inhibitors. However, Pol III generates RNAs that cannot be translated. As a result, nearly all studies of Pol III rely on Northern blotting, which is slow and not suited to high-throughput screening efforts. At Lucerna, we have developed a photostable RNA mimic of red fluorescent protein (termed Corn) and demonstrated that this RNA can serve as a robust fluorescent reporter for Pol III promoters. By simply creating a Pol III promoter that controls the synthesis of the Corn RNA, fluorescence can be readily detected without the need to synthesize a protein. We have demonstrated this concept using the 5S subtype of a Pol III promoter. Interestingly, we found marked cell-to-cell variation in Pol III transcription after cells are exposed to serum and other growth-promoting signals. Therefore, the goal of this Phase I SBIR project is to develop this simple and genetically encodable Corn reporters for live cell imaging and measurement of all three subtypes of Pol III transcription. Further, we will develop this reporter system so that it can be used in flow cytometry analysis, allowing Pol III transcription to be monitored in hundreds of thousands of cells over time. For phase II, we will adapt our Pol III reporters into marketable toolkits, validate the Pol III reporters in disease-relevant cell lines, and use the Cas9/CRISPR technology to image endogenous Pol III transcription activity. Together, the plasmids and kits generated in this project will result in the first Pol III reporters, and will therefore constitute an important commercial product that will have wide utility in the academic RNA research community and will enable discovery of small molecule inhibitors of this previously hard-to-assay class of drug targets.