Chemical Biology Approach for Validating and Manipulating Cellular RNA-Protein Interactions

摘要 最近的研究表明，RNA总是与RNA结合蛋白结合，并经常被RNA结合蛋白修饰 (限制性商业惯例)。因此，人们发现限制性商业惯例在规范编码的许多方面发挥着关键作用也就不足为奇了 和非编码RNA生物学，包括RNA加工，核输出，细胞运输，功能，定位， 和稳定性。这些努力是由1,500个独特的限制性商业惯例执行的，这些限制性商业惯例利用各种RNA结合域 经常实现与目标转录本的特定和高亲和力的相互作用；然而，非规范的限制性商业惯例 也已被确认。这种复杂的RNA-蛋白质相互作用网络(RPI)的破坏已经被 与许多人类疾病有关。因此，以限制性商业惯例和可持续发展指标为目标已成为一个新的前沿领域 在以RNA为靶点的药物发现中；然而，很少有相互作用被验证为支持 这些努力的目标。虽然测序和定量质谱学的出现戏剧性地 增强了我们全局描述这些交互的能力，但这些数据集的实验验证仍然是 挑战。使用基于化学生物学和生物正交化学的策略，我们开发了一种 新的活细胞检测RPI的新方法，RNA与蛋白质介导的互补作用 化验，或RiPCA。通过这种方法，我们检测到了前miRNAs与限制性商业惯例的相互作用，此外 小分子的抑制作用。此外，为了提供证据证明我们的技术在验证 新的RPI，我们使用RiPCA来确认前miRNA与通过蛋白质组学发现的新的RBP之间的相互作用。 总而言之，这些数据为这项新兴技术提供了令人鼓舞的概念验证；然而，许多关键问题 仍然存在挑战，以确保RiPCA是检测RPI的严格和不偏不倚的方法 在不同的细胞器中。在具体目标1中，我们将进一步开发用于细胞器特异性检测的RiPCA 以确保其准确性。在特定的目标2中，我们将通过分析其他 RPI来自不同的RNA和RBP家族。最后，在具体目标3中，我们将探索其对高性能的适应性。 验证大规模CLIP或蛋白质组学数据集的吞吐量实验，或用于识别 RPI的细胞活性小分子抑制剂。在完成拟议的研究后，我们的目标是生产 一种强大且用户友好的技术，用于快速验证和研究细胞RPI，以实现生物医学 研究。

ABSTRACT Recent studies have shown that RNAs are invariably bound to and often modified by RNA-binding proteins (RBPs). Thus, it is no surprise that RBPs have been found to play key roles in regulating many aspects of coding and non-coding RNA biology, including RNA processing, nuclear export, cellular transport, function, localization, and stability. These efforts are carried out by >1,500 unique RBPs that utilize a variety of RNA-binding domains to achieve oftentimes specific and high affinity interactions with target transcripts; however, non-canonical RBPs have also been identified. Disruption of this complex network of RNA-protein interactions (RPIs) has been implicated in a number of human diseases. Thus, the targeting of RBPs and RPIs has arisen as a new frontier in RNA-targeted drug discovery; however, very few interactions have been validated to support a pipeline of targets for these efforts. While the advent of sequencing and quantitative mass spectrometry has dramatically enhanced our ability to globally profile these interactions, experimental validation of these data sets remains a challenge. Using chemical biology- and bioorthogonal chemistry-based strategies, we have developed an innovative new assay for the live-cell detection of RPIs, RNA interaction with Protein-mediated Complementation Assay, or RiPCA. Through this approach, we have detected the interaction of pre-miRNAs with RBPs, in addition to inhibition with small molecules. Moreover, to provide evidence for the potential of our technology in validating new RPIs, we used RiPCA to confirm the interaction of a pre-miRNA with a novel RBP discovered via proteomics. In total, these data provide encouraging proof-of-concept for this emerging technology; yet, many key questions and challenges still remain to ensure that RiPCA is a rigorous and unbiased approach for the detection of RPIs in distinct cellular organelles. In Specific Aim 1, we will further develop RiPCA for organelle-specific detection to ensure its accuracy. In Specific Aim 2, we will investigate the potential of the assay by profiling additional RPIs from various RNA and RBP families. Finally, in Specific Aim 3, we will explore its adaptability toward high- throughput experimentation for validation of large-scale CLIP or proteomics data sets, or screening to identify cell-active small molecule inhibitors of RPIs. Upon completion of the proposed research, our goal is to produce a robust and user-friendly technology for the rapid validation and study of cellular RPIs to enable biomedical research.