Transcriptome-wide profiling of mammalian spliceosome and branchpoints with iCLIP

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to simultaneously map human spliceosome engagement with snRNAs and pre-mRNAs. We identify nine sites on pre-mRNAs that overlap with position-dependent binding profiles of 15 RBPs. We reveal over 50,000 branchpoints (BPs), indicating that most human introns use a primary BP for spliceosome assembly, whereas alternative BPs are amplified when analysing intron lariats. Notably, we find that the binding patterns of many RBPs, especially the components of SF3 complex, are affected by RNA structure and position of BPs. Moreover, the stability of RNA structures around BPs distinguishes exons regulated by RBPs involved in early exon definition from those regulated by the SF3B components and PRPF8. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of BPs and splicing mechanisms.