A High-Throughput Screening Platform to Discover RNA Methylation Inhibitors

总结 细胞mRNAs易受各种化学修饰的影响，这些化学修饰在调节基因表达中起重要作用。 表达和细胞功能。最丰富的内部mRNA修饰是m6A，当 腺苷残基被甲基化。m6A几乎影响mRNA生命周期的各个方面， 对于各种生理过程，例如配子发生、神经发育、学习和记忆， 免疫调节以及干细胞增殖和分化。此外，m6A已成为一个重要的 癌症进展过程中的RNA调控机制：m6A阅读器、写入器和擦除器水平的改变是 在几种人类癌症中观察到，癌基因和肿瘤抑制基因的甲基化已被证明是 影响其表达并促进癌症发展。因此，我们既需要了解 m6A如何在细胞中调节，以及开发针对m6A甲基转移酶机制的药物。 然而，一个主要的限制是缺乏简单的，具有成本效益的方法来检测m6A的变化 本发明的目的是以与高通量筛选（HTS）相容的方式检测细胞中的甲基化。在这里，我们克服了 通过开发一种新的基因编码的m6A传感器，该传感器提供了一个简单的荧光读数， m6A甲基化的研究。目标1将优化m6A传感器系统，并生成细胞工具， 便于将系统用于不同的应用。Aim 2将把m6A传感器开发成与HTS兼容的 系统，并通过执行旨在识别m6A的试点HTS来展示其在药物发现工作中的实用性 甲基转移酶抑制剂。Aim 3将创建包含该系统的分子工具， m6A传感器在体内的性能。总之，这些研究将提供一个急需的工具， m6A在细胞中的动力学，并将开发一个优化的系统，用于基于HTS的m6A甲基化研究。我们 这项技术很可能对癌症中m6A生物学的基础研究产生直接影响 作为药物发现的努力，旨在确定新的m6A甲基转移酶抑制剂。

Summary Cellular mRNAs are susceptible to a variety of chemical modifications that play important roles in regulating gene expression and cellular function. The most abundant internal mRNA modification is m6A, which occurs when adenosine residues become methylated. m6A influences nearly every aspect of the mRNA life cycle and is critical for a variety of physiological processes, such as gametogenesis, neurodevelopment, learning and memory, immune regulation, and stem cell proliferation and differentiation. Additionally, m6A has emerged as an important RNA regulatory mechanism during cancer progression: altered levels of m6A readers, writers, and erasers are observed in several human cancers, and methylation of oncogenes and tumor suppressors has been shown to impact their expression and promote cancer development. Thus, there is a great need both for understanding how m6A is regulated in cells as well as for developing drugs that target the m6A methyltransferase machinery. However, a major limitation has been the lack of simple, cost-effective methods for detecting changes in m6A methylation in cells in a manner that is compatible with high-throughput screening (HTS). Here, we overcome this barrier by developing a novel genetically encoded m6A sensor which provides a simple fluorescent readout for m6A methylation in living cells. Aim 1 will optimize the m6A sensor system and generate cellular tools to facilitate using the system for diverse applications. Aim 2 will develop the m6A sensor into a HTS-compatible system and demonstrate its utility for drug discovery efforts by performing a pilot HTS designed to identify m6A methyltransferase inhibitors. Aim 3 will create molecular tools incorporating the system and measure the performance of the m6A sensor in vivo. Altogether, these studies will provide a much-needed tool for detecting m6A dynamics in cells and will develop an optimized system for HTS-based studies of m6A methylation. Our technology is likely to have an immediate impact both for basic investigations of m6A biology in cancer as well as drug discovery efforts aimed at identifying novel m6A methyltransferase inhibitors.