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. 2015 Jan 1;29(1):81–93. doi: 10.1101/gad.253708.114

Figure 3.

Figure 3.

Prp5 interacts directly with U2 snRNA on the U257G spliceosome. (A) Splicing reactions were carried out without (lanes 2–7) or with wild-type (lanes 8–13) or U257G (lanes 14–19) pre-mRNA in Prp9-V5 extracts, and the reaction mixtures were separated into three aliquots. One aliquot was not treated further (lanes 2,3,8,9,14,15), another was subjected to denaturation (lanes 4,5,10,11,16,17), and the third was irradiated with UV254nm followed by denaturation (lanes 6,7,12,13,18,19). The reaction mixtures were then precipitated with anti-V5 or anti-Prp5 antibody. Precipitated materials were analyzed by Northern blotting probed with five snRNAs. (B) Prp5-U2 cross-linked product was isolated as in A using Prp5-V5 extracts for precipitation of Prp5 cross-linked products with anti-V5 antibody and analyzed by primer extension to map cross-linking sites using primer U2-B. (C) The proposed structure of the U2–BSL form with the Prp5 cross-linked residues highlighted; branch site-interacting residues are indicated in red.