Separation of splicing factor SF3 into two components and purification of SF3a activity

Abstract

Components required for the splicing of nuclear messenger RNA precursors in vitro have been isolated from HeLa cells. Here we describe the separation of splicing factor SF3 into two components, SF3a and SF3b. Both activities are required together with several other protein factors and U1 and U2 small nuclear ribonucleoproteins for the assembly of a presplicing complex which represents the first ATP-dependent step in the assembly of the active spliceosome. SF3a has been purified to homogeneity by a combination of ion-exchange chromatography, gel filtration, and glycerol gradient sedimentation. It consists of a complex of three polypeptides of 60, 66, and 120 kDa. The association of SF3a activity with these polypeptides has been confirmed by immunoprecipitation and depletion experiments using a monoclonal antibody directed against the 66-kDa subunit.