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

ABSTRACT: The ATP analog ATP?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?S on pre-mRNA splicing in vitro. Addition of ATP?S to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATP?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?S-stalled B complexes (B(ATP?S)) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B(ATP?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?S) complexes exhibit a morphology highly similar to B complexes, indicating that the ATP?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?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?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.

SUBMITTER: Agafonov DE 

PROVIDER: S-EPMC4986889 | biostudies-other | 2016 Sep

REPOSITORIES: biostudies-other