A. Krämer

Mammalian splicing factor SF1 consists of a single polypeptide of 75 kDa and is required for the formation of the first ATP-dependent spliceosomal complex. Three cDNAs encoding variant forms of SF1 have been isolated and four highly related cDNAs have been found in current databases. Comparison of the cDNA sequences suggests that different SF1 mRNAs are generated by alternative splicing of a common pre-mRNA. In agreement with this idea, at least three mRNAs that are differentially expressed in different cell types have been detected by northern blot analysis. All SF1 cDNAs identified encode proteins with a common N-terminal half that contains two structural motifs implicated in RNA binding (an hnRNP K homology [KH] domain and a zinc knuckle), but the proteins differ in the length of a proline-rich region and have distinct C-termini. Three SF1 isoforms expressed in insect cells via baculovirus transfer vectors show comparable activities in the assembly of a pre-splicing complex. Consistent with the presence of a KH domain and a zinc knuckle, we show that SF1 binds directly to RNA. This interaction appears to be largely sequence-independent with a preference for guanosine- and uridine-rich sequences. The KH domain of SF1 is embedded in a 160-amino acid sequence that is shared with human Sam68, a target of Src during mitosis, as well as Caenorhabditis elegans GLD-1 and mouse Qkl, both of which play roles during cellular differentiation. The presence of this shared region in SF1 suggests functions in addition to its role in pre-spliceosome assembly.

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.

Six fractions derived from a HeLa cell nuclear extract are necessary for the generation of spliced mRNA in vitro. To establish a function for the protein factors present in these fractions, their role in the formation of splicing complexes was analyzed by electrophoresis in native polyacrylamide gels. Two of the fractions are sufficient to assemble the adenovirus major late mRNA precursor into a presplicing complex with characteristics similar to the presplicing complex assembled in nuclear extract. One fraction supplies splicing factor (SF) 1 and at least one small nuclear ribonucleoprotein particle, U2 snRNP. The other fraction contains SF3. Extensive fractionation of this protein has revealed that it is essential for presplicing complex assembly and the splicing reaction.