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: 10242059
• 负责人: Amanda Garner
• 金额: $ 30.81万
• 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/10242059
• 关键词: Affect; Affinity; Autoimmune Diseases; Binding; Binding Proteins; Biological Assay; Biology; Biomedical Research; Catalysis; Cells; Cellular Assay; Chemicals; Chemistry; Code; Complement; Complex; DNA; Data; Data Set; Detection; Development Plans; Drug Targeting; Emerging Technologies; Ensure; Failure; Foundations; Gene Expression; Goals; Human Biology; Human Genome Project; Knowledge; Label; Learning; Length; Link; Liquid substance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Mitochondria; Modeling; Modification; Nerve Degeneration; Nuclear; Nuclear Export; Organelles; Pharmaceutical Preparations; Play; Protein Engineering; Protein Family; Proteins; Proteomics; RNA; RNA Binding; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; Reagent; Research; Role; Site; Small Nuclear RNA; Structure; System; Technology; Transcript; Transcriptional Regulation; Translations; Untranslated RNA; Validation; Work; base; design; drug discovery; experimental study; frontier; high throughput technology; human disease; innovation; novel; novel therapeutic intervention; pre-miRNA; screening; small molecule; small molecule inhibitor; technology validation; user-friendly; validation studies

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.

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，毫不奇怪，限制性商业惯例在管制商业活动的许多方面发挥着关键作用。 编码和非编码RNA生物学，包括RNA加工，核输出，细胞转运，功能， 定位和稳定性。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经常特异性和高亲和力的相互作用;然而， 还发现了非规范限制性商业惯例。破坏这种复杂的RNA-蛋白质相互作用网络 RPIs与许多人类疾病有关。因此，出现了限制性商业惯例和限制性工业倡议的目标， 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， 支持为这些努力制定一系列目标。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分析这些相互作用的能力， 验证这些数据集仍然是一个挑战。利用化学生物学和生物正交化学 策略，我们已经开发了一种创新的新的检测方法，用于RPI的活细胞检测，RNA与 蛋白质介导的互补测定，或RiPCA。通过这种方法，我们发现了 前体miRNA与RBP的相互作用，以及小分子的抑制作用。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，我们使用了RiPCA来确认相互作用 通过蛋白质组学发现的具有新RBP的pre-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，许多关键问题和挑战仍然存在，以确保 RiPCA是一种用于检测不同细胞器中的RPI的严格且无偏倚的方法。在特定 目标1，我们将进一步发展RiPCA用于细胞器特异性检测，以确保其准确性。具体目标 2，我们将通过分析来自各种RNA和RBP家族的其他RPI来研究该测定的潜力。 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，或筛选以鉴定细胞活性小分子抑制剂， RPI。在完成拟议的研究后，我们的目标是开发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。