Fig 4.

Binding affinities of Yju2 domains to the spliceosome. (A) (Top) Splicing reactions were carried out in Yju2-depleted extracts for 20 min, and ATP was then depleted by addition of 10 mM glucose and incubation for 5 min. Following the addition of recombinant HA-tagged Yju2, V5-tagged Yju2-N, or HA-tagged Yju2-C at final concentrations of 0, 10, 50 100, 200, and 400 nM, the reaction mixtures were incubated for 10 min and precipitated with anti-HA (lanes 3 to 8 and 15 to 20) or anti-V5 (lanes 9 to 14) antibody. (Bottom) Binding affinities for the spliceosome of Yju2, Yju2-N, and Yju2-C. The fractions of Yju2-containing activated spliceosome, calculated as the ratio of the total amount of Yju2-bound spliceosome to that of the Ntc20-associated spliceosome, are plotted against the concentration of Yju2 added. (B) Splicing reactions were carried out in Yju2- and Prp16-depleted extracts supplemented with 0, 1, 2, 3, and 4 μM V5-tagged Yju2-N (lanes 1 to 5), and each reaction mixture was precipitated with anti-V5 antibody. (C) Splicing reactions were carried out in Yju2- and Prp16-depleted extracts supplemented with 0.3 μM HA-tagged full-length Yju2 (lanes 1 to 3), 1.5 μM V5-tagged Yju2-N (lanes 4 to 6), 0.4 μM HA-tagged Yju2-C (lanes 7 to 9), or 1.5 μM V5-tagged Yju2-N and 0.4 μM HA-tagged Yju2-C (lanes 10 to 13), followed by immunoprecipitation with anti-Ntc20 (lanes 2, 5, 8, and 11), anti-HA (lanes 3, 9, and 13) or anti-V5 (lanes 6 and 12) antibody. RXN, 1/10 of the reaction mixture used for immunoprecipitation.