Functional Analysis of pre-mRNA Splicing Factor SF2/ASF Structural Domains

EMBO J. 1993 Dec;12(12):4715-26.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alternative Splicing
  • Amino Acid Sequence
  • Base Sequence
  • DNA
  • Humans
  • Molecular Sequence Data
  • Mutagenesis
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Protein Conformation
  • RNA Precursors / metabolism*
  • RNA Splicing*
  • RNA-Binding Proteins
  • Serine-Arginine Splicing Factors

Substances

  • Nuclear Proteins
  • RNA Precursors
  • RNA-Binding Proteins
  • Serine-Arginine Splicing Factors
  • DNA