Functional splicing network reveals extensive regulatory plasticity of the core Spliceosomal machinery. Homo sapiens

Pre-mRNA splicing relies on the still poorly understood dynamic interplay between more than 150 protein components of the Spliceosome, and the steps at which splicing can be regulated remain largely unknown. Here we systematically analyze the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and use this information to reconstruct a network of functional interactions. The network accurately captures well-established physical and functional associations and identifies new, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during Spliceosome assembly. In contrast with standard models of regulation at early events of splice site recognition, factors involved in catalytic activation of the Spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF and on targets of anti-tumor drugs, and can be widely used to identify mechanisms of splicing regulation. Overall design: RNA from 3 biological replicates of 72 hours knockdowns of human IK or SMU1 and a control set were used. Changes between the control and knockdowns were measured based on using a splice-junction array (Affymetrix HJAY).

前信使RNA（pre-mRNA）剪接依赖于剪接体（Spliceosome）150余种蛋白质组分之间尚不明晰的动态互作，而剪接调控可发生的步骤在很大程度上仍未被阐明。本研究系统性分析了敲降剪接机器组分对与细胞增殖、凋亡相关的可变剪接事件的影响，并基于此重构了功能互作网络。该网络精准捕捉了已被证实的物理与功能关联，并发现了全新的互作关系，揭示了核心剪接体组分在剪接体组装过程中，其招募时序与持续时长所对应的显著调控潜能。与剪接位点识别早期事件的经典调控模型不同，参与剪接体催化激活的因子展现出独特的调控特性。该网络还阐明了异质性核糖核蛋白C（hnRNP C）与U2辅助因子（U2AF）之间的拮抗作用，以及抗肿瘤药物的作用靶点，且可被广泛用于解析剪接调控的分子机制。实验设计：本研究使用了针对人类IK或SMU1进行72小时敲降的3个生物学重复样本，以及对照组样本的RNA。基于剪接连接芯片（Affymetrix HJAY），检测对照组与敲降组之间的转录组变化。