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

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，不足为奇的是，限制性商业惯例被发现在管理许多方面起着关键作用。 编码和非编码RNA生物学，包括RNA加工、核输出、细胞运输、功能、 本地化和稳定性。这些努力是由1,500个独特的限制性商业惯例进行的，这些限制性商业惯例利用各种RNA- 结合结构域以实现经常与目标转录本的特异性和高亲和力的相互作用；然而， 还确定了非规范的限制性商业惯例。破坏这一复杂的RNA-蛋白质相互作用网络 (RPI)与许多人类疾病有牵连。因此，出现了以限制性商业惯例和限制性商业惯例为目标的问题 作为RNA靶向药物发现的新前沿；然而，很少有相互作用被验证为 支持这些努力的一系列目标。虽然测序和定量质量的出现 光谱学极大地增强了我们在全球范围内描述这些相互作用的能力，试验性的 对这些数据集的验证仍然是一个挑战。使用基于化学生物学和生物正交化学的 策略，我们开发了一种创新的新方法，用于活细胞检测RPI，RNA与 蛋白质介导的互补试验，或称RiPCA。通过这种方法，我们检测到 前miRNAs与限制性商业惯例的相互作用，以及与小分子的抑制。此外，为了提供 为了证明我们的技术在验证新的RPI方面的潜力，我们使用RiPCA来确认相互作用 通过蛋白质组学发现具有新的RBP的前miRNA。总的来说，这些数据提供了令人鼓舞的证据： 这一新兴技术的概念；然而，仍存在许多关键问题和挑战，以确保 RiPCA是一种在不同的细胞器中检测RPI的严格和公正的方法。具体而言 目的1、进一步建立细胞器特异性检测的RiPCA方法，以保证其准确性。以特定的目标 2，我们将通过分析来自不同RNA和RBP家族的其他RPI来研究该检测的潜力。 最后，在具体目标3中，我们将探索其对高通量实验的适应性以进行验证 大规模CLIP或蛋白质组学数据集，或筛选以识别细胞活性小分子抑制物 RPI。在完成建议的研究后，我们的目标是生产出一种强大和方便用户使用的技术 用于细胞RPI的快速验证和研究，以实现生物医学研究。

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.