Structure of the core RNA–RNA network in yeast activated spliceosomes. (A) Secondary structure of the group II intron domain V (DV; adapted from Pyle 2010 with permission from Taylor and Francis © 2010). Essential RNA–RNA interactions with other intron elements are denoted with small Greek letters and nonstructural magnesium ions are indicated. The J2/3 linker is indicated and positions of intron domains III and IV are indicated schematically. Tertiary interactions are shown as gray lines, and stacking interactions as gray open rectangles. (B) Secondary structure of the yeast U6 ISL and U6/U2 helices Ia and Ib (adapted from Fica et al. 2014 with permission from Nature Publishing Group © 2014). Phosphates of nucleotides (colored in yellow with red outlines) that coordinate the two essential magnesium ions. Tertiary interactions of the AGC triad are shown as gray lines. Stacking of U6–G52 and U6–U80 is indicated (Fica et al. 2014). (C) Watson/Crick accessibilities of the nucleotides in the core of the spliceosomal RNA network in purified Bact (orange) and B* complexes (green). The size of the dots corresponds to the degree of accessibility to chemical modification. The asterisk denotes nucleotide U2–C41 that was hyperreactive toward DMS in the Bact complex. 5′ exon nucleotides are shown in lowercase letters. The last nucleotides of the 5′ exon are base-pairing to U5 loop 1 nucleotides. (D–F) Representative gels of the DMS (D) and CMCT (E) chemical modification of U6 snRNA, as well as DMS modification of U2 snRNA (F) in Bact and B* complexes (as indicated above the lanes), analyzed by primer extension. Key nucleotides are highlighted. See Supplemental Figures S1–S3 for the full data set of all RNAs and chemical probes. B* complexes were reconstituted by complementing purified Bact complexes with recombinant Prp2 and its cofactor Spp2 and ATP.
