Extended Data Fig. 4: Spatial organization of B-specific proteins in B-like complexes, and structural comparisons of pre-B, B-like and CI B.

a, Schematic of the domain organization of SMU1, RED, FBP21, SNU23, MFAP1 and PRP38A. Domains localized in the B-like cryo-EM structure are colored. NTR, N-terminal region; LisH, Lissencephaly type 1-like homology; GAC, globular α-helical core; WD40, β-propeller-like domain comprised of ca 40 amino acids that often contains a C-terminal tryptophan-aspartic acid (W-D) dipeptide; ZnF, Zinc Finger; WW; ca 40 amino acid-long domain containing two tryptophan residues; CTR, C-terminal region. b, U2 snRNP, whose molecular architecture is similar in B-like and B complexes, is stably-attached to the remodeled tri-snRNP, not only via U2/U6 helix II and interactions involving SF3A1, but also by the B-specific proteins SMU1 and RED. The latter form a the hetero-tetrameric SMU1-RED complex, whose domains are located at the same positions in the B-like and CI B complexes²³. That is, the SF3B3-WD40B domain interacts with the WD40 domain of one of the SMU1 subunits (SMU1-B^WD40) and also the SMU1 NTRs, while SMU1-A^WD40 is also docked to BRR2 at the interface between both helicase domains. As in the CI human B complex, SMU1 forms a homodimer that forms primarily via the interaction of the LisH domain in the NTR of each SMU1 molecule, and each SMU1 additionally interacts with one copy of the RED protein²³. The identity of the individual SMU1 NTR domains (A or B) cannot be determined unambiguously. c, In addition to SMU1, several other B-specific proteins, including FBP21, SNU23 and MFAP1, interact with BRR2 at its new position in B-like, as they do in the CI B complex²³. Aside from a potential role in helping to tether BRR2 to its new activation position, several of these proteins may thus additionally, or instead, regulate BRR2 activity in both B-like and B complexes. Our data do not directly implicate the B-specific proteins in regulating alternative splicing events. However, as our studies indicate that splice site pairing occurs at the B complex stage, proteins that play a role in B complex formation (which include the B-specific proteins) are clearly regulatory candidates. Indeed, RNA-mediated knockdowns of several B-specific proteins have revealed that they modulate multiple alternative splicing events in the cell. For example, Papasaikas et al showed that knockdown of SMU1 and RED leads to alternative splice site usage and exon skipping⁴⁸. In addition, the C. elegans homologue of MFAP1 was shown to affect alternative splicing⁴⁹. d, Comparison of the position of PRP31 and SAD1 in CE pre-B (left), B-like (middle) and CI B (PDB 6AHD)¹⁹ (right) complexes. The structures are aligned via PRP8^NTD. Surprisingly, SAD1, which is displaced in the CI B complex concomitant with the translocation of the BRR2 helicase domain from PRP8^RT to PRP8^{EN 19,22}, is still stably bound to SNU114 and PRP8^RT in the B-like complex. This appears to be due to structural differences in B versus B-like, and might arise due to the slightly different conformation of PRP8^En because a 5’ exon binding channel is not formed by UBL5 and MFAP1^CTR in B-like complexes due to the absence of a 5’ exon. e, Conformational change in the PRP6 HAT repeats during the conversion of the CE pre-B complex (left) into a B-like complex (middle left). Overlays of the PRP6 HAT repeats in CE pre-B versus B-like (middle right) and B-like versus CI B (right) (PDB 6AHD)¹⁹. The structures were aligned via the C-terminal HAT repeats.