ATP gamma S stalls splicing after B complex formation but prior to spliceosome activation

The ATP analog ATP gamma S inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATP gamma S on pre-mRNA splicing in vitro. Addition of ATP gamma S to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATP gamma S concentrations >= 0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATP gamma S-stalled B complexes (B-ATP gamma S) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B-ATP gamma S complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B-ATP gamma S complexes exhibit a morphology highly similar to B complexes, indicating that the ATP gamma S-induced block in the transformation of the B to B-act complex is not due to a major structural defect. Likely mechanisms whereby ATP gamma S blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATP gamma S may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B-act spliceosomal complex.