FLAG Immunoprecipitation-Based Mapping of the In Vivo Assembled Spliceosomal C* Complex

Pre-mRNA splicing is catalyzed by the ribonucleoprotein (RNP) complex known as the spliceosome. The spliceosomes are dynamic and undergo constant rearrangement, leading to the formation of the different spliceosomal complexes A, B, B^act, C, C*, and P. Isolation of the spliceosomal complex at a specific intermediate stage requires a means to enrich it. This study describes a strategy for studying intermediate spliceosomal complexes by combining BioID with splicing assays. The MINX splicing substrate with a mutation at the 3' splice site was utilized to arrest and capture the spliceosomal C* complex before the second catalytic step of splicing. The splicing substrate also contains binding sites for the MS2 coat protein, which facilitates the pull-down of assembled complex by FLAG-MS2-tagged RNP immunoprecipitation and determines the captured proximal proteins by mass spectrometry.