Extended Data Fig. 1: Cryo-EM and image-processing of the cross-exon pre-B complex.

a, RNA composition of purified CE pre-B complex dimers. For gel source data, see Supplementary Fig. 1. Pre-B complexes formed on the MINX exon RNA (Fig. 1a) were affinity-purified and RNA from fractions of the fastest sedimenting peak (typically fractions 14-18) was isolated, separated on a NuPAGE gel, and visualized by staining with SyBr gold. The nucleotide (nts) lengths of the snRNAs and MINX exon RNA are indicated on the right. The RNA composition was analysed from three independent pre-B complex purifications with similar results. The MINX exon RNA⁷ was generated from the MINX pre-mRNA³⁷, which is a derivative of the Adenovirus Major Late (ADML) pre-mRNA. In the MINX exon RNA, the 5’ exon and adjacent downstream intron nucleotides of the MINX pre-mRNA have been deleted, leaving the 3’ exon (a truncated version of the ADML exon 2) and 64 nts of the 3’ end of the upstream, adjacent ADML intron, which contains an anchoring site followed by the branch site, polypyrimidine tract and 3’ss AG. A 5’ss was introduced at the 5’ end of the truncated exon, by adding the last 6 nts of the wildtype ADML exon 2 plus 22 nts of the adjacent downstream ADML intron, which are followed by a short linker and three RNA stem-loops that bind the MS2 protein (see Fig. 1a). Previous studies in our lab showed that cross-exon A-like complex formation on the MINX exon RNA is enhanced by the presence of the downstream 5’ss⁷. Furthermore, depletion of either U1 or U2 from the nuclear extract also led in each case to substantial reduction in the formation of the cross-exon A-like complex⁷. Thus, the complexes formed on the exon-containing substrate used in this study are exon-defined. b, Close up of the interfaces of the pre-B dimer. An expanded view of the boxed regions at the interfaces of the pre-B dimer is shown at the right. The two protomers contact each other via BRR2 and the RecA domains of PRP28 of one protomer, and the globular density of the other protomer, which contains U1 snRNP bound to the 5’ss and exon-binding proteins (see Extended Data Fig. 2). The functional relevance of these interfaces, as well as dimer formation in general, is currently not known. c, Representative cryo-EM 2D class averages of the pre-B dimers, where the top two represent class 1 dimers and the bottom two, class 2 dimers (see below). d, Cryo-EM computation sorting scheme. All major image-processing steps are depicted. For a more detailed explanation, see the EM data processing section in the Methods. Two major classes of the pre-B dimers are detected. In the class 2 dimer, the structure of only one pre-B complex is well-defined, whereas in class 1 both protomers are well-defined. The poorly resolved protomer in class 2 could potentially be a CE A-like complex. The tri-snRNP core is comprised of all tri-snRNP components excluding the U4 Sm core, U5 Sm core and BRR2. e, Local resolution estimation of the tri-snRNP region of the pre-B complex. f, Orientation distribution plot for the particles contributing to the reconstruction of the tri-snRNP region. g, Fourier shell correlation (FSC) values for the listed parts of the CE pre-B complex indicate a resolution of 3.5 Å for the tri-snRNP core, 4.2 Å for BRR2, 6.1 Å for the U4 core and 12 Å for the U2 snRNP. h, Map versus model FSC curves generated for the tri-snRNP core, BRR2 and U4 core regions of CE pre-B using PHENIX mtriage. i, Schematic of the RNA-RNA interaction network in the human CE pre-B protomer. The U1/5’ss base pairing interaction is inferred from previous biochemical characterization of CE pre-B complexes (previously denoted 37 S exon complexes)⁷. A dot between two nucleotides indicates that they do not base pair, but that a helix involving these nucleotides is formed (e.g., the extended U2/BS helix). j-k, U4/U6 stem III and the quasi pseudoknot are present in CE pre-B. Panel j, fit of the U4/U6 stem III and quasi-pseudoknot, as well as RBM42, to the CE pre-B EM density. Panel k, 3D molecular model corresponding to the right view in panel j.