Extended Data Figure 2. Broad rescue specificity of A59-PS(S_p) and U80-PS(S_p) spliceosomes.

a, Pre-incubation with EDTA reveals a branching defect for A59-PS(S_p). Extracts were pre-incubated at 4°C in the presence or absence of 2 mM EDTA and then incubated with the 3′O substrate. After affinity-purification, branching efficiency was quantified without further incubation (bottom). A representative gel is shown (top). EDTA pre-incubation caused an 8-fold reduction in branching efficiency, suggesting that splicing extracts may contain a thiophilic metal that supports branching by A59-PS(S_p) spliceosomes; note that pre-mRNA still immunoprecipitated efficiently, indicating catalytic activation of the spliceosome. In contrast EDTA pre-incubation has no effect on U6 WT spliceosomes (data not shown). b, The branching defects for A59-PS(S_p) spliceosomes are rescued by either Mn²⁺ or Cd²⁺. Assays were as in Fig. 2c. A representative gel (top) and quantification (bottom) are shown; ni, no incubation. Both Mn²⁺ and Cd²⁺ strongly rescue A59-PS(S_p) and U80-PS(S_p) spliceosomes (lanes 3,4,7,8), suggesting that even the weaker Mn²⁺-S interaction³⁸ at these positions can support branching. This broad specificity for branching may also explain why A59-PS(S_p) and U80-PS(S_p) spliceosomes also catalyzed exon ligation in the presence of thiophilic metals, whereas G60-PS(R_p), G78-PS(S_p), and U80-PS(R_p) spliceosomes, for which branching was only rescued in the presence of Cd²⁺, stalled after branching. Values are averages; error bars, s.d. (n=3).