sCLIP – An integrated platform to study RNA-protein interactome(s) in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs [CLIP-Seq]

RNA-binding proteins (RBPs) are central for gene expression by controlling the RNA fate from birth to decay. Various disorders arising from perturbations of RNA-protein interactions document their critical function. However, deciphering their function is complex, limiting the general functional elucidation of this growing class of proteins and their contribution to (patho)physiology. Here we present sCLIP, a simplified and robust platform for genome-wide interrogation of RNA-protein interactomes based on crosslinking-immunoprecipitation and high-throughput sequencing. sCLIP exploits linear amplification of the immunoprecipitated RNA improving the complexity of the sequencing-library despite significantly reducing the amount of input material and omitting several purification steps. Additionally it permits a radiolabel-free visualization of immunoprecipitated RNA. In a proof of concept, we identify that CSTF2tau binds many previously not recognized RNAs including histone, snoRNA and snRNAs. CSTF2tau-binding is associated with internal oligoadenylation resulting in shortened snRNA isoforms subjected to rapid degradation. We provide evidence for a new mechanism whereby CSTF2tau controls the abundance of snRNAs resulting in alternative splicing of several RNAs including ANK2 with critical roles in tumorigenesis and cardiac function. Combined with a bioinformatic pipeline sCLIP thus uncovers new functions for established RBPs and fosters the illumination of RNA-protein interaction landscapes in health and disease. Overall design: Sequencing of CSTF2tau sCLIP (3 biological replicates).

RNA结合蛋白（RNA-binding proteins, RBPs）通过调控RNA从生成到降解的全过程，在基因表达中发挥核心作用。RNA-蛋白质相互作用紊乱可引发多种疾病，这也佐证了这类蛋白的关键功能。然而，解析其功能极具挑战性，这限制了对这一日益增多的蛋白质家族的全面功能阐释，以及它们在（病理）生理学中的贡献。本研究报道了sCLIP——一种基于交联免疫沉淀结合高通量测序的全基因组RNA-蛋白质相互作用组分析的简化且稳定的平台。sCLIP可对免疫沉淀得到的RNA进行线性扩增，在显著减少起始材料用量、省去多步纯化步骤的同时，提升了测序文库的复杂度。此外，该方法无需使用放射性标记即可实现免疫沉淀RNA的可视化。在概念验证实验中，我们发现CSTF2tau可结合此前未被识别的多种RNA，包括组蛋白编码RNA、核仁小RNA（small nucleolar RNA, snoRNA）与小核RNA（small nuclear RNAs, snRNAs）。CSTF2tau的结合会引发内部寡聚腺苷酸化，进而生成易被快速降解的截短snRNA亚型。我们的研究证实了一种全新的机制：CSTF2tau可通过调控snRNA的丰度，影响包括ANK2在内的多种RNA的可变剪接，而ANK2在肿瘤发生与心脏功能中发挥关键作用。结合生物信息学分析流程，sCLIP可揭示经典RBPs的新功能，助力阐明健康与疾病状态下的RNA-蛋白质相互作用图谱。实验整体设计：CSTF2tau sCLIP测序（3次生物学重复）。