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 pr oteins that belong to the large RNP motif family of RNA-binding protei ns. This motif identifies an RNA-binding domain (RBD) that consists of a four-stranded antiparallel beta-sheet packed against two cy-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 R NA to determine the RNA-binding specificity of hnRNP C1. After 8 round s of selection/amplification nearly all RNAs contained contiguous stre tches of at least 5 U residues, and filter-binding assays demonstrated that this sequence constitutes a high-affinity (K-d = 170 nM) binding site for hnRNP C1. The highest affinity we measured for hnRNP C1 was for r(U)(14) (K-d = 14 nM). An RBD-containing peptide fragment of hnRN P C1 (amino acids 2-94) bound oligoribonucleotides containing an hnRNP C1 high-affinity binding site with nearly equal affinity to that of h nRNP C1. Unlike hnRNP C1, however, this peptide also bound oligoribonu cleotides that do not contain high-affinity hnRNP C1-binding sites. We identified a region of 10 amino acids, immediately COOH-terminal to t he 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 co nfers a general RNA binding activity to RNP motif RBDs and that the de terminants of RNA-binding specificity reside in the most variable regi ons, the loops connecting the beta-strands and/or the contiguous NH2 a nd COOH termini of the RBD.