Extended Data Fig. 4: Conformational and compositional changes from ILS′ to ILS″.

a. Close-up view of protein-protein interactions between the PRP8 RNase H (RH) ___domain, TFIP11, PAXBP1 and BRR2 in the ILS′. Protein elements thar are mobile in the ILS′-to-ILS″-transition are shown as ribbons, whereas elements thar are static are shown in addition as transparent surfaces. b. Close-up view of protein-protein interactions between the PRP8 RNase H (RH) ___domain, TFIP11, PAXBP1 and C19L2 in the ILS″. C19L2 binding requires repositioning of the PRP8 RH ___domain and TFIP11–PAXBP1, which displaces the BRR2–PRP8 JAB1/MPN domains from PAXBP1. C19L2 recruits C19L1 by binding its C-terminal CWFJ ___domain. c. Overlay of TFIP11–PAXBP1 in the ILS′ and ILS″ in a 90° rotated view. Yellow arrows connect identical residues in both states. d. Overview of the ILS′. DHX15 (transparent) is likely tethered via the TFIP11 G-patch ___domain but cannot dock onto its target. e. Overview of the ILS″. C19L1 and C19L2 binding allows docking of DHX15, and the associated conformational change in TFIP11–PAXBP1 and PRP8 displaces BRR2. Circled numbers 1 and 2 indicate regions of zoom-ins in panels f, i and j. f. Close-up view of the ILS″ U6 snRNA 3′ end, with DHX15 and the TFIP11 G-patch removed for clarity. The oligo-uridylated and single-stranded U6 snRNA 3′ end is the ideal substrate for DHX15, and SDE2 and SYF2 shield the U2/U6 helix II. g. RNA cryo-EM density in the ILS′. After dissociation of ligated mRNA and catalysis-specific splicing proteins, the RNA active site is more mobile. h. RNA cryo-EM density in the ILS″. Compared to the ILS′, RNA densities are better defined in the ILS″, presumably due to binding of C19L2 (see panel j). i. Continuous cryo-EM density between U2-U6 helix II and the DHX15 active site reveals U6 snRNA as the target for ILS disassembly. j. C19L2 binds the active site RNA network near the branch helix and contacts U2 snRNA, intron-lariat RNA, U5 snRNA, and U6 snRNA.