psiCLIP of SmB, Prp16 and Prp22 in C, C* and P spliceosomal complexes. psiCLIP of SmB, Prp16 and Prp22 in C, C* and P spliceosomal complexes

Despite the major progress made into spliceosome mechanisms through CryoEM, a major obstacle of this approach is its inability to map the positioning of helicases on the RNA substrate. Here we present a method ‘psiCLIP’ which probes protein-RNA interactions in specific spliceosomal states. The method is based on iCLIP, but is different to previous iCLIP studies in that it is performed on step-specific spliceosomes that are prepared from in vitro splicing extracts, similarly to those prepared for CryoEM. We applied psiCLIP to SmB (C complex), Prp16 (C complex) and Prp22 (C* and P complexes), using pre-mRNA substrates based on UBC4 and ACT1. We also used ATPase deficient dominant negative (dn) mutants of Prp16 and Prp22 to determine the contribution of ATPase activity to binding patterns.