Spliceosome iCLIP in multiple cell lines. Spliceosome iCLIP in multiple cell lines

The studies of spliceosomal interactions are challenging due to their dynamic nature. Here we developed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs) to simultaneously map the spliceosomal binding to human snRNAs and pre-mRNAs. This identified 9 distinct regions on pre-mRNAs, which overlap with position-dependent binding patterns of 15 RBPs. Using spliceosome iCLIP, we additionally identified >50,000 branchpoints (BPs) that have canonical features, unlike those identified by RNA-seq. The iCLIP BPs generally overlap with the computationally predicted BPs, and alternative BPs are associated with extended regions of structurally accessible RNA. We find that the position and strength of BPs defines the binding patterns of SF3 and U2AF complexes, whereas the RNA structure around BPs affects the sensitivity of exons to perturbation of these complexes. Our findings introduce spliceosome iCLIP as a new method for transcriptomic studies of BPs and splicing mechanisms.

剪接体相互作用的研究因其动态特性而极具挑战性。本研究开发了剪接体iCLIP（spliceosome iCLIP）技术，该技术可将SmB与小核核糖核蛋白颗粒（snRNPs）及辅助RNA结合蛋白（RBPs）共同免疫沉淀，从而同时定位剪接体与人类小核RNA（snRNAs）及前体mRNA（pre-mRNAs）的结合位点。本研究通过该技术在前体mRNA（pre-mRNAs）上鉴定出9个独特区域，这些区域与15种RBPs的位置依赖性结合模式相互重叠。借助剪接体iCLIP技术，我们还鉴定出超过50000个具备经典特征的剪接分支点（BPs），这与通过RNA测序（RNA-seq）鉴定得到的分支点有所不同。iCLIP鉴定得到的BPs通常与计算机预测的BPs相重合，而选择性BPs则与结构可及的RNA延伸区域相关联。研究发现，BPs的位置与强度决定了SF3复合物与U2AF复合物的结合模式，而BPs周围的RNA结构则会影响外显子对这些复合物扰动的敏感性。本研究成果将剪接体iCLIP技术确立为一种用于剪接分支点及剪接机制转录组学研究的全新方法。