Splicing machine shifts into gear Spliceosome activation involves extensive protein exchanges and RNA rearrangements that lead to the formation of a catalytically active U2/U6 RNA structure called B act . Previously, little was known about the pathway leading to the U2/U6 active site and how proteins aid in folding the U2/U6 RNA. Using cryo–electron microscopy to determine structures of two human pre-B act complexes, Townsend et al. uncovered an intricate cascade of coordinated structural changes involving mutually exclusive interactions that facilitate the directionality of the activation process. These structures reveal the assembly pathway of the U2/U6 catalytic RNA and the mechanism whereby proteins facilitate its folding. Science , this issue p. eabc3753