Fig. 8. PtdIns(4,5)P₂-associated factors are required for splicing. (A) 2C11 but not IgM depleted HeLa nuclear extracts are impaired in their ability to support the splicing of -crystallin mRNA. The inhibition is blocked by pre-incubation of the 2C11 beads with GroPIns(4,5)P₂ but not GroPIns. The inhibition of splicing seen is comparable to that observed using an anti-snRNP antibody (Y12). A schematic representation of the start, intermediates and product of the splicing reaction is shown on the right. (B) Immunoprecipitated material can be eluted from 2C11 beads by incubation with an excess of PtdIns(4,5)P₂ or GroPIns(4,5)P₂, as seen by western blotting using anti-RNA Pol IIo (H5; lower panel; P, pellet; S, supernatant). Re-addition of these eluates to the depleted splicing reaction partially restores the splicing activity, whereas re-addition of control eluates or the lipids alone have no effect.