DETECTION AND ISOLATION OF RNA-BINDING PROTEINS BY RNA-LIGAND SCREENING OF A CDNA EXPRESSION LIBRARY

A screening assay for the detection of RNA-binding proteins was develo ped, it allows the rapid isolation of cDNA clones coding for proteins with sequence-specific binding affinity to a target RNA, For developin g the screening protocol, constituents of the human U1 snRNP were util ized as model system. The RNA partner consisted of the U1-RNA stem-loo p II and the corresponding protein consisted of the 102 amino acid N-t erminal recognition motif of the U1A protein, which was fused to beta- galactosidase and expressed by the recombinant lambda phage LU1A, Foll owing binding of the fusion protein to nitrocellulose membranes, hybri dization with a P-32-labeled U1-RNA ligand was carried out to detect s pecific RNA-protein interaction, Parameters influencing the specificit y and the detection limit of binding were systematically investigated with the aid of the model system. Processing the nitrocellulose membra nes in the presence of transition metals greatly increased the signal: background ratio, A simple screening protocol involving a single-buffe r system was developed, Specific RNA-protein interaction could be dete cted in the presence of a Targe excess of recombinant phages from a cD NA library Only moderate binding affinities (K-d = 10(-8) M) were requ ired. The suitability of the RNA-ligand screening protocol was demonst rated by the identification of new viroid-RNA binding proteins from to mato.