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. 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）通过依赖于上下文的方式靶向RNA，以调控基因表达。RBP复合物与网络的蛋白质相互作用（protein-protein interaction, PPI）图谱，对阐明RBP功能及RNA靶向机制至关重要。然而，当前PPI网络中以RBP为诱饵的样本代表性不足，且缺乏RNA介导相互作用的相关信息。为此，本研究构建了具备RNA感知能力的RBP相互作用组图谱，整合了两种基于系统性蛋白质组学方法的策略：其一为在添加或不添加核糖核酸酶（RNase）处理的条件下，针对RNA生命周期中约100种RBP开展免疫共沉淀（co-immunoprecipitation, CoIP）实验，以鉴定蛋白质相互作用；其二为通过尺寸排阻色谱法（Size Exclusion Chromatography, SEC）鉴定RNA依赖型复合物。本数据集可在蛋白质组范围内以细胞类型特异性的方式，定量鉴定多个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对应了mRNA生命周期各阶段运行的众多复合物；近千个全新相互作用则预示了RBP在RNA生命周期多个步骤中的新功能。将本网络与增强型交联免疫沉淀（eCLIP）数据整合后，我们鉴定出了相互作用蛋白间的RNA靶标，并揭示了复合物驱动的结合特征。通过介数中心性评分，我们筛选出参与多个mRNA生命周期步骤的多功能RBP，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。研究发现，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出核过程。最后，本研究证实剪接因子SNRNP200可与核出核、定位及翻译相关蛋白相互作用，且是应激状态下RNA颗粒形成的关键因子。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。本数据集包含人类SNRNP200、CAPRIN及G3BP1的eCLIP-seq实验，以及目标RBP的eCLIP实验；每个样本均设有Input对照与免疫沉淀（IP）实验组。