Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.
Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.
Cáceres
J F
JF
Cold Spring Harbor Laboratory, NY 11724-2208.
Krainer
A R
AR
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors
D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D004247:DNA