a, The minimal length of RNA needed to connect the upstream branch point (BP) and downstream 5′ SS in the A complex is modelled using the Rosetta RNP-denovo method. The A complex (PDB ID 6G90) is shown in grey. The pre-mRNA is shown in green. The upstream branch point and downstream 5′ SS are shown as purple space-filling models. Twenty-eight nucleotides are sufficient to connect the upstream branch point and downstream 5′ SS (not including the branch point and 5′ SS themselves) without any chain break or clashes. b, Schematics of wild-type and mutant DYN2 pre-mRNA (mutated nucleotides shown in red), IEI, and untagged IEI used for the EDC assembly and in vivo exon definition experiments. Stem-loops represent the MS2 binding sites, and the red line represents the DNA oligonucleotide used for RNase H cleavage. c, SDS–PAGE shows protein components of complexes assembled on wild-type and IEI substrates (lanes 1, 2), on wild-type in the presence of competing untagged IEI (lane 3), and on IEI after RNase H treatment in the absence and presence of the DNA oligo (lanes 4, 5). This experiment was repeated one additional time with similar results. d, RNA components of the same complexes as in lanes 4, 5 of c, confirming that RNase H treatment in the presence of the oligonucleotide cleaves the pre-mRNA. The smaller cleaved fragment (61 nucleotides) is difficult to see because EtBr stains short single-stranded RNA with low efficiency. This experiment was repeated two additional times with similar results. e, Mass spectrometry analyses of spliceosome assembled on the IEI and wild-type DYN2 pre-mRNA indicate that the two complexes have the same components in similar quantities with the exception of NCBP1 and 2, which are absent from the IEI complex. f, 2D classification of negative-stain TEM images of the E complex assembled on DYN2 IEI pre-mRNA. This experiment was repeated one additional time with similar results.