A systems view of spliceosomal assembly 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 map spliceosome engagement with pre-mRNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RBPs that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs, and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.