Figure 2.

Spliceosome maturation is not affected by phosphorothioate substitution at U₈₀ of U6 but splicing only occurs in the S_P diastereomer with a thiophilic metal ion. a, U6/sU₈₀ forms mature spliceosome. Extracts were reconstituted with unmodified U6 (lanes 1 and 2) or U6/sU₈₀ (R_P, lanes 3–4; S_P, lanes 5–6), in the absence (lanes 1, 3 and 5) or presence (lanes 2, 4 and 6) of PRP2. The reaction mixture was sedimented through a glycerol gradient and the fraction containing the spliceosome was analysed on a native gel. The pre-catalytic and post-PRP2 spliceosomal markers (M; lanes 7 and 8) were generated as described¹⁶. b, The first transesterification reaction occurs in U6/sU₈₀(S_P)-containing post-PRP2 spliceosome in the presence of Mn²⁺. Spliceosomes isolated from glycerol gradients were incubated in buffer containing combinations of Mg²⁺, Mn²⁺, ATP, PRP2 and HP (a protein factor that facilitates splicing in purified spliceosomes¹⁶). c, Other thiophilic divalent metal ions can also rescue the transesterification reaction. The post-PRP2 U6/sU₈₀(S_P) spliceosome was incubated with metal ions without the addition of ATP, PRP2 or HP. Mg²⁺ was added as the sole divalent metal ion in lanes 1 (2.5 mM) and 2 (5 mM). The concentrations (mM) of the thiophilic metal (Mn²⁺, Ca²⁺, Cd²⁺ or Co²⁺) in each set were 0.02, 0.1, 0.5 and 2.5. Mg²⁺ was added to lanes 3–18 to make the total concentration of divalent metal ions 5 mM.