Extended Data Fig. 9: Cryo-EM and image-processing of the pre-B^AMPPNP complex, and localization of the U1 snRNP.

a, Cartoon showing organization of the protomers in the pre-B^ATP dimers (left). Right, Schematic of the RNA-RNA network in the pre-B^ATP dimer. The protomers in the pre-B (Fig. 1a) and pre-B^ATP dimers, are organized in an anti-parallel manner, in contrast to the parallel organization of the protomers in the pre-B^{5’ss+ATPγS} dimers (Extended Data Fig. 5p). Addition of ATP and the 5’ss oligo, thus not only triggers rearrangements within the protomers, foremost the large-scale translocation of BRR2, but also a reorganization of the protomers relative to each other from an anti-parallel orientation to a parallel one. Although the mechanism for this reorganization is not clear, in particular whether it involves partial or complete detachment of the protomers comprising the dimer, it would in both cases likely require the dissociation of protein-protein contacts involving the PRP28 region of the tri-snRNP of one protomer and components of the globular domain encompassing the exon of the adjacent protomer, such as SR proteins and U1 snRNP. During formation of pre-B^{5’ss+ATPγS}, the 5’ss oligo disrupts the U1 base pairing interaction with the 5’ss of the MINX exon RNA and at the same time prevents U6 from interacting with the latter, freeing up the MINX exon RNA. In contrast, in pre-B^ATP, formation of the base pairing interaction of U6 with the 5’ss of the MINX exon RNA would stabilize the anti-parallel orientation and thus hinder the change in polarity of the dimer during the BRR2 translocation events in the two pre-B protomers. b, RNA composition of pre-B^AMPPNP complexes. Purified pre-B complexes were incubated with the non-hydrolyzable ATP analog AMPPNP, and then subjected to glycerol gradient centrifugation. RNA from the fastest-sedimenting gradient peak was isolated, separated on a NuPAGE gel, and visualized by staining with SyBr gold. For gel source data, see Supplementary Fig. 1. The nucleotide (nts) lengths of the snRNAs and MINX exon RNA are indicated on the right. The RNA composition was analysed from two independent pre-B^AMPPNP purifications with similar results. c, Representative cryo-EM 2D class averages of the pre-B^AMPPNP dimers. d, Cryo-EM computation sorting scheme of the pre-B^AMPPNP. e, Local resolution estimation of the tri-snRNP core region of the pre-B^AMPPNP complex. f, Orientation distribution plot for the particles contributing to the reconstruction of the tri-snRNP region in of the pre-B^AMPPNP. g, Fourier shell correlation (FSC) values indicate a resolution of 4.1 Å for the tri-snRNP core and 6.1 Å for the tri-snRNP plus adjacent U1 snRNP. h, Map versus model FSC curves generated for the tri-snRNP core and tri-snRNP plus adjacent U1 snRNP regions of pre-B^AMPPNP. i, Fit into the EM density (low-pass filtered to ~20 Å resolution) of the molecular model of a pre-B^AMPPNP complex monomer interacting in trans with a U1 snRNP bound to a second MINX exon RNA. On the right, the molecular architecture of the U1 snRNP and adjacent BRR2 and PRP28 proteins without EM density, as well as a cartoon of the unassigned EM density adjacent to U1, is shown. In pre-B^AMPPNP, U1 snRNA stem-loop III is located close to PRP8^En of the adjacent protomer, and the U1 Sm domain contacts the PRP28 RecA1 domain. j, Fit into the EM density (low-pass filtered to ~20 Å resolution) of the molecular model of a pre-B complex monomer interacting in trans with PRP28 from the adjacent pre-B monomer. On the right, the molecular architecture of BRR2 and PRP28 without EM density, as well as a cartoon of the unassigned EM density that likely contains the U1 snRNP, is shown. k, Close up of PRP28 docked to U1 of the adjacent monomer in the pre-B^AMPPNP dimer. A molecular model of PRP28 with a closed conformation and bound with a single-stranded RNA (based on the crystal structure of Mss116p) can be fit into the pre-B^AMPPNP EM density. In pre-B^AMPPNP, there is density between the two PRP28 RecA domains that appears to accommodate a single-stranded (ssRNA) but not double stranded RNA. Given this is indeed the case, our data would thus suggest that in the presence of AMPPNP, PRP28 has disrupted the U1/5’ss helix of the adjacent protomer, but due to the lack of ATP hydrolysis, its RecA domains are trapped in a closed conformation. l, EM density fit of PRP28 with an open conformation in the corresponding region of the cross-exon pre-B complex. In the CE pre-B complex, PRP28 adopts an open conformation, and there is no stable docking of U1. m-o, The PRP28 RecA domains undergo a major conformational change. Spatial organization of the PRP28 RecA domains in the closed (m) and open (n) conformations, with an overlay shown in panel o. The open conformation of a DEAD-box helicase such as PRP28 is its default state when it is not bound to a substrate⁵⁰. When the helicase encounters a double-helical RNA in the presence of ATP, it transitions to the closed conformation, where the subsequent closure of the two RecA domains physically separates the two strands⁵⁰, resulting in closed RecA domains bound to a single-stranded RNA (ssRNA). p, Fit of the U6/5’ss helix into the EM density of pre-B^ATP. q, A U6/5’ss helix is not formed in pre-B^AMPPNP. EM density that accommodates a U6/5’ss helix (as seen in panel p) is absent in pre-B^AMPPNP, confirming that this helix does not form.