Large-scale map of RNA binding protein interactomes across the mRNA life-cycle. Large-scale map of RNA binding protein interactomes across the mRNA life-cycle

RNA-binding proteins (RBPs) target RNA in a context-dependent manner to regulate gene expression. Protein-protein interaction (PPI) maps of RBP complexes and networks are critical for defining RBP function and RNA targeting. Yet, PPI networks under-represent RBP baits and need more information about RNA-driven interactions. Therefore, we generated an RNA-aware, RBP-interactome map combining two strategies that use systematic proteomic methods to identify 1) protein interactions using co-immunoprecipitation in the presence or absence of RNase treatment of a ~100 RBPs across the RNA life-cycle and 2) RNA-dependent complexes using Size Exclusion Chromatography. Together, this dataset provides proteome-wide, cell-type specific, and quantitative identification of RNA-protein interactions across multiple RNA processing events. In the resulting PPI network, several hundred database-supported interactions establish many complexes operating at each of these mRNA life-cycle stages. Nearly a thousand novel interactions imply new functions for RBPs across multiple steps of the RNA life cycle. Overlapping our network with eCLIP data, we find RNA targets between interactors and uncover complex-driven binding signatures. Betweenness-centrality scores identify multi-functional RBPs that participate across multiple mRNA life-cycle steps. We characterize the novel interactions and functions of different classes of multi-functional proteins. We find the scaffolding protein, ERH, interacts with numerous nuclear speckle proteins and facilitates splicing and mRNA export. Finally, we show that the splicing factor, SNRNP200, interacts with nuclear export, localization, and translation proteins and is an essential factor of RNA granule formation during stress. Our large-scale RBP interaction network provides new insights and a valuable resource for exploring new RBP complexes operating to control gene expression. Overall design: eCLIP-seq of human SNRNP200, CAPRIN, and G3BP1 eCLIP of RBP of interest. Each sample has an input and IP sample

RNA结合蛋白（RNA-binding proteins, RBPs）以上下文依赖的方式靶向RNA，以调控基因表达。蛋白质相互作用（Protein-protein interaction, PPI）图谱对于明确RBP的功能与RNA靶向机制至关重要。然而，现有PPI网络对RBP诱饵蛋白的覆盖度不足，且缺乏关于RNA驱动的相互作用的相关信息。为此，本研究整合两种基于系统性蛋白质组学方法的策略，构建了RNA感知型RBP相互作用组图谱：其一为在经核糖核酸酶（RNase）处理或未处理的条件下，对涵盖RNA生命周期各阶段的约100种RBP开展免疫共沉淀（co-immunoprecipitation）实验以鉴定蛋白质相互作用；其二为利用尺寸排阻色谱（Size Exclusion Chromatography）鉴定RNA依赖性复合物。本数据集可实现蛋白质组范围、细胞类型特异性的定量鉴定，覆盖多种RNA加工事件中的RNA-蛋白质相互作用。
在最终构建的PPI网络中，数百个经数据库验证的相互作用证实了各mRNA生命周期阶段所存在的多种复合物；近千个全新相互作用则揭示了RBPs在RNA生命周期多个步骤中的新功能。将本网络与eCLIP数据进行整合分析后，我们可明确相互作用蛋白之间的RNA靶向关系，并揭示复合物驱动的结合特征。通过介数中心性（betweenness-centrality）评分，我们鉴定出可参与多个mRNA生命周期步骤的多功能RBPs，并对不同类别多功能蛋白的全新相互作用与功能进行了表征。我们发现支架蛋白ERH可与大量核斑蛋白相互作用，并参与调控剪接与mRNA输出过程。最后，我们证实剪接因子SNRNP200可与核输出、定位及翻译相关蛋白相互作用，且是细胞应激期间RNA颗粒形成的必需因子。本大规模RBP相互作用网络为探索调控基因表达的新型RBP复合物提供了全新视角与宝贵资源。
总体设计：针对目标RBP（人源SNRNP200、CAPRIN及G3BP1）开展eCLIP实验并进行eCLIP测序。每个样本均设置输入对照与免疫沉淀（IP）实验组。