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

验证和操纵细胞 RNA-蛋白质相互作用的化学生物学方法

• 批准号: 10408902
• 负责人: Amanda Garner
• 金额: $ 7.82万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408902
• 关键词: Affinity; Binding Proteins; Biological Assay; Biology; Biomedical Research; Cells; Cellular Assay; Chemicals; Chemistry; Code; Complement; Complex; DNA; Data; Data Set; Detection; Development Plans; Drug Targeting; Emerging Technologies; Ensure; Gene Expression; Goals; Mass Spectrum Analysis; Mediating; Nuclear Export; Organelles; Play; Protein Family; Proteins; Proteomics; RNA; RNA Binding; RNA Processing; RNA Recognition Motif; RNA-Binding Proteins; RNA-Protein Interaction; Research; Technology; Transcript; Untranslated RNA; Validation; base; drug discovery; frontier; human disease; innovation; novel; pre-miRNA; screening; small molecule; small molecule inhibitor; user-friendly; validation studies

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.

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调节编码的许多方面发挥着关键作用，这并不奇怪 和非编码RNA生物学，包括RNA加工，核输出，细胞转运，功能，定位， 与稳定这些努力是由超过1，500个独特的RBP进行的，这些RBP利用各种RNA结合结构域 以实现与靶转录物的特异性和高亲和力相互作用;然而， 也已被确认。破坏这种复杂的RNA-蛋白质相互作用（RPIs）网络已经被证明是一种有效的方法。 与许多人类疾病有关。因此，针对限制性商业惯例和限制性工业举措已成为一个新的前沿领域 在RNA靶向药物的发现;然而，很少有相互作用已被验证，以支持管道， 这些努力的目标。虽然测序和定量质谱的出现大大改变了 增强了我们在全球范围内分析这些相互作用的能力，这些数据集的实验验证仍然是一个问题。 挑战.使用化学生物学和生物正交化学为基础的战略，我们已经开发了一个 用于RPI活细胞检测的创新新检测方法，RNA与蛋白质介导互补作用的相互作用 测定或RiPCA。通过这种方法，我们检测了前体miRNA与RBP的相互作用，此外， 到小分子抑制。此外，为了证明我们的技术在验证 在新的RPI中，我们使用RiPCA来证实前体miRNA与通过蛋白质组学发现的新RBP的相互作用。 总的来说，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 但要确保RiPCA是一种严格且无偏见的RPI检测方法， 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。在特定目标2中，我们将通过分析额外的 来自各种RNA和RBP家族的RPI。最后，在具体目标3中，我们将探讨其对高性能的适应性， 用于验证大规模CLIP或蛋白质组学数据集的通量实验，或筛选以识别 RPIs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， research.