Extended Data Fig. 2: U2 snRNP is connected to the U4/U6.U5 tri-snRNP in CE pre-B by three major bridges, and localization of U1 and the MINX exon.

a, Fit of the pre-B model into the EM density of one pre-B protomer. On the left and right, enlargements of the major U2-tri-snRNP bridges are shown. Upper left, PRP4 kinase (PRP4K) bridges SF3B3^WD40B with the HAT repeats of the U5 protein PRP6. Lower left, interaction of an α-helix of SF3A1 (aa 461-473) with the U5 snRNP protein DIM1 and the PRP8 helical bundle (PRP8^HB). Right panels, two views of the U2/U6 helix II bridge with SF3B1 and SF3B6 at one end and the U6 Lsm ring at the other. In CI pre-B^18,19 the tri-snRNP and U2 snRNP are also connected via the U2/U6 helix II and the SF3A1-DIM1/PRP8 interaction. PRP4K is docked to the tri-snRNP via U4/U6 stem II and PRP6^HAT in both complexes, but its interaction with U2 SF3B3^WD40B appears to be more flexible in CI pre-B, as EM density connecting the two proteins is observed in only a subset of CI pre-B complexes¹⁸. Guided by crosslinks (see panel b) we can place the RRM domain of SF3B6 at the C-terminal HEAT repeats of SF3B1 directly adjacent to U2/U6 helix II, consistent with the idea that SF3B6 may help to facilitate the formation of U2/U6 helix II during the initial interaction of the tri-snRNP with the spliceosome, in both the cross-intron and cross-exon assembly pathways. b, Cross-links of SF3B6 with SF3B1. Crosslinked residues are depicted by circles connected by a reddish-brown line, where the numbers indicate the positions of the crosslinked amino acids. c, Crosslinks of SF3B3^WD40B with PRP4K. Labeling as in panel b. d, Left and middle, U2 snRNP is connected to the poorly-resolved, globular EM density by a thin density element. Right, the latter protrudes from SF3B1^HEAT at the position where the 3’ end of the intron was previously shown to exit the HEAT domain. Thus, this density is predicted to contain intron nucleotides between the BS and 3’ss, and the adjacent, poorly-defined globular domain highly likely contains the MINX exon and bound SR proteins, as well as U1 snRNP. e, Distance constraints exclude an alternative pre-B protomer organization. The distance between U1 and U2 SF3B1^HEAT in pre-B protomers organized as shown in Fig. 1 (left), and in an alternatively-organized protomer (right) are shown. For simplicity, only one of the U1 snRNPs is shown in each cartoon. Panel 2d shows that there is continuous density that links U2 SF3B1 of one promoter to the exon/U1 snRNP-containing globular density (GD) that is attached to PRP28 of the other protomer. Following this density, the distance between SF3B1 of one protomer and PRP28 of the other promoter would require ca 30 nts of RNA to cover. A similar RNA length would be required to reach the U1 bound to the 5’ss in the adjacent GD. Thus, our placement of the U1 snRNP is consistent with the 39 nt length of the MINX exon plus ca 10 nts of the PPT/3’ss that extend beyond SF3B1^HEAT. In the alternative organization, the same U2 snRNP, but instead the U1 from the other GD, would bind to the same MINX exon RNA substrate (and thus in this case U1 would interact in cis with PRP28). This would require that after exiting SF3B1^HEAT, the remaining PPT/3’ss nts plus the MINX exon would wrap back across SF3B1^HEAT and extend to PRP28 (and to the adjacent U1) within the same protomer. However, this would require more than 60 nts to cover this distance, without clashing with any tri-snRNP proteins, which is much longer than the length of our exon (39 nts) plus ca 10 nts of the PPT/3’ss that extend beyond SF3B1^HEAT. Alternative RNA paths that extend along the other side of the complex would be even longer. Therefore, an exon much longer than 60 nts would be required to form such an intra-protomer complex. Thus, in our CE pre-B complexes, PRP28 cannot interact within the same protomer with the U1 snRNP-bound 5’ss. f, Representative EM 2D class of the CE pre-B complex dimer. The “fuzzy” nature of the globular domains containing U1 and the exon binding proteins is likely due to the transient interaction of various SR proteins with the exon. g, Model of cross-exon, protein-protein interactions that indirectly bridge the U2 and U1 snRNPs in the CE pre-B complex based on protein crosslinking of the CE pre-B complex (see also Supplementary Table S2). Crosslinked residues are connected by black arrows. Although the cryo-EM structure of a cross-exon pre-A complex was recently reported³⁸, the nature of the bridge connecting the U2 and U1 snRNPs could not be discerned due to the poor resolution in this region of the complex. SR proteins are likely recruited to the defined exon by exonic splicing enhancers, as well as to the U1 snRNP³⁹, and have long been proposed to establish a network of protein-protein interactions across the exon that link the U2 and U1 snRNPs^40,41,42. The number and identity of the SR proteins interacting with the exon likely varies from one cross-exon complex to the next⁴³. Thus, there may be different combinations of SR proteins bound compared to those depicted in the model. The RRMs of SRSF1 crosslink with the U1-70K RRM, consistent with previous biochemical studies⁴⁴. Likewise, RBM39 crosslinks to U2AF, and the LUC7L paralogs LUC7L2 and LUC7L3 (labeled LUC7L) crosslink to several SR proteins. The U1-related protein PRPF40A crosslinks to U1-70K, U1-A and LUC7L3, consistent with previous studies revealing similar interactions of PRP40 in the yeast U1 snRNP in early splicing complexes^45,46. With the exception of a crosslink between SF3A1 and PRPF40A, crosslinks between U1 and U2 snRNP proteins are not observed, supporting the conclusion that U2 and U1 do not directly contact one another in cross-exon complexes, as previously proposed. The U1 snRNA stem-loop 4 was previously shown to interact with the U2 SF3A1 protein during the early stages of cross-intron spliceosome assembly⁴⁷. However, it is not clear whether this RNA-protein interaction, which would directly connect U1 and U2 snRNP, contributes to the molecular bridge linking U1 and U2 in cross-exon complexes.