Extended Data Fig. 7: Structural differences between pre-B^5’ss, pre-B^{5’ss+ATPγS} and B-like complexes.

a, BRR2 is anchored to its new position in pre-B^{5’ss+ATPγS} (top) and the B-like complex (bottom) via the PRP8^RH-PRP8^Jab1- linker whose ends are now stably bound to PRP8^En. The boxed regions are shown in expanded form at the right. Jab1 linker, the region of the RH-Jab1 linker proximal to the PRP8 Jab1 domain. RH linker, the region of the RH-Jab1 linker proximal to the PRP8 RH domain. b, Interaction of SNRNP27K with PRP8^En in the pre-B (top) and pre-B^5’ss (bottom) complexes. c, Rotation of PRP8^Jab1 around the RH-Jab1 linker is required to reach its B-like position. An overlay shows the position of the Jab1 domain in pre-B^{5’ss+ATPγS} (purple) and B-like (grey). PRP8^Jab1 appears to rotate around the amino acids of the RH-Jab1 liker directly upstream of the Jab1 domain, which may act as a hinge. d, Two different views of an overlay of BRR2 in pre-B^{5’ss+ATPγS} (multi-colored) and B-like (grey), showing that BRR2 in pre-B^{5’ss+ATPγS} must still rotate in order to reach its position in the B-like complex. e, Stepwise opening of the BBR2^NC. In pre-B and pre-B^5’ss (left panel), the opening of the RNA binding channel is blocked by the BRR2 PLUG domain and C-terminal (CT) tail of the PRP8 Jab1 domain. In pre-B^{5’ss+ATPγS}, pre-B^{5’ssLNG+ATPγS} and the B-like complex (middle and right panels), the BRR2^PLUG and PRP8 Jab1 CT tail are displaced. However, in pre-B^{5’ss+ATPγS}, pre-B^{5’ssLNG+ATPγS} (middle left panel), the separator loop (marked by a red arrowhead) of the RecA2 domain contacts an α-helix of the Sec63 domain (blue arrowhead), blocking the RNA binding channel. Thus, in the absence of the B-specific proteins, a new intermediate BRR2^NC state (i.e., with a free RNA channel entry site but a blocked RNA channel) is observed. Due to the presence of the U4 Sm domain, U4 snRNA cannot be threaded into the RecA domains. Instead, the latter must to be opened to allow U4 snRNA binding. Downward movement of the RecA1 and RecA2 domains of BRR2^NC and the concomitant movement of the separator loop, positions it further away from the BRR2^NC Sec63 domain, leading to an open RNA binding channel (middle right panel), allowing the binding of the single-stranded region of U4 snRNA (right panel) (See also Supplementary Video 6). f, PRP6 and PRP31 are phosphorylated in pre-B^ATP and pre-B^5’ss+ATP complexes. Proteins were isolated from the indicated complexes and the phosphorylation status of PRP6 and PRP31 determined by immunoblotting with antibodies specific for phosphorylated PRP6 or PRP31. The less intense signals in the B complex lane obtained with the anti-phosphorylated PRP6 and PRP31 antibodies is due to the fact that these proteins are thiophosphorylated in the B complexes, which were isolated in the presence of ATPγS, and thiophosphorylated proteins are poorly recognized by the anti-phospho antibodies. For blot source data, see Supplementary Fig. 1. Similar blot results where obtained with two independent experiments. g, Comparison of the position of BRR2 and the U4 Sm core in pre-B^{5’ss+ATPγS} (left) compared to the B-like complex (right). The structures are aligned via the PRP8^NTD. In pre-B^{5’ss+ATPγS}, the BRR2 helicase domain has not reached its final position. In addition, BRR2^NC exhibits a closed, inactive conformation and has not yet bound the U4 snRNA, and the U4 Sm core domain has also not reached its final, B-like position. Although BRR2’s PLUG and PWI domains are not visible, the N-terminal ca 50 amino acids of BRR2 still wrap around PRP8^Large at the same position as in the pre-B^5’ss complex. These data strongly support the idea that during the translocation of BRR2’s helicase domain across the large domain of PRP8, BRR2 does not dissociate and subsequently reassociate, but rather remains attached via its N-terminal region to the spliceosome. h, BRR2 is translocated, but the U4 Sm core is not docked to BRR2 in pre-B^{5’ss+ATPγS}. Right, zoomed-in view of the boxed region. U6-C37 is inserted into a protein pocket formed by SF3A1 aa 496-521 and PRP6, stabilizing the new position of U4/U6 stem III. Tethering of U6-C37 by SF3A1 and PRP6, may help to stabilize the short U6/5’ss helix after release of RBM42, which appears to contact U6 near C37, and after the repositioning of U4/U6 helix III, which prior to its movement likely constrains the position of U6 nucleotides in this region. In addition, capture of this U6 nucleotide fixes the path of the adjacent U6 snRNA region, which may facilitate the subsequent formation of the extended U6/5’ss helix upon disruption of U4/U6 stem III and release of U6-C37. i, The U4 Sm core contacts BRR2 in pre-B^{5’ssLNG+ATPγS}, and U4/U6 stem III is dissociated by binding of the long 5’ss oligo to U6 snRNA. Right, zoomed-in view of the boxed region. An extended (ext) U6/5’ss helix is formed, and aa 496-521 of SF3A1 are destabilized.