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 single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening 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 hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.
Wan
L
L
Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104-6148, USA.
Kim
J K
JK
Pollard
V W
VW
Dreyfuss
G
G
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
C439788:C1 HNRNP; C503635:HNRNPC protein, human; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008024:Ligands; D019151:Peptide Library; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D007930:Leucine