Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn-RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a s ingle RNP motif RNA-binding domain. The RNA-binding domain (RBD) is compris ed of similar to 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RED in the binding to RNA. We have devised a phage display-based sc reening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnR NP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RED to be essentia l for C1 RNA binding, We also identified three mutations in the leucine-ric h C1-C1 interaction domain near the carboxyl terminus of the protein that b oth abolished C1 oligomerization and reduced RNA binding. These results dem onstrate that although the RED is the primary determinant of C1 RNA binding , residues in the C1-C1 interaction domain also influence the RNA binding a ctivity of the protein. The experimental approach we described should be ge nerally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.