The determinants of RNA-binding specificity of the heterogeneous nuclear ribonucleoprotein C proteins.
The hnRNP C proteins (C1/C2) are tenacious nuclear pre-mRNA-binding proteins that belong to the large RNP motif family of RNA-binding proteins. This motif identifies an RNA-binding domain (RBD) that consists of a four-stranded antiparallel beta-sheet packed against two alpha-helices. Despite considerable information on the structure of the hnRNP C RBD, little is known about its RNA-binding properties. To address this we used in vitro selection/amplification from pools of random sequence RNA to determine the RNA-binding specificity of hnRNP C1. After 8 rounds of selection/amplification nearly all RNAs contained contiguous stretches of at least 5 U residues, and filter-binding assays demonstrated that this sequence constitutes a high-affinity (Kd = 170 nM) binding site for hnRNP C1. The highest affinity we measured for hnRNP C1 was for r(U)14 (Kd = 14 nM). An RBD-containing peptide fragment of hnRNP C1 (amino acids 2-94) bound oligoribonucleotides containing an hnRNP C1 high-affinity binding site with nearly equal affinity to that of hnRNP C1. Unlike hnRNP C1, however, this peptide also bound oligoribonucleotides that do not contain high-affinity hnRNP C1-binding sites. We identified a region of 10 amino acids, immediately COOH-terminal to the RNP motif (amino acids 95-104), that prevents the minimal RBD from binding nonspecific RNA ligands. We propose that the highly conserved beta alpha beta beta alpha beta core structure of the RNP motif RBD confers a general RNA binding activity to RNP motif RBDs and that the determinants of RNA-binding specificity reside in the most variable regions, the loops connecting the beta-strands and/or the contiguous NH2 and COOH termini of the RBD.
Görlach
M
M
Howard Hughes Medical Institute, Philadelphia, Pennsylvania.
Burd
C G
CG
Dreyfuss
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008969:Molecular Sequence Data; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
C439788:C1 HNRNP; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012332:RNA, Heterogeneous Nuclear; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins