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 developed. It allows the rapid isolation of cDNA clones coding for proteins with sequence-spe-cific binding affinity to a target RNA. For developing the screening protocol, constituents of the human U1 snRNP were utilized as model system. The RNA partner consisted of the U1-RNA stem–loop II and the corresponding protein consisted of the 102 amino acid N-terminal recognition motif of the U1A protein, which was fused to β-galactosidase and expressed by the recombinant lambda phage LU1A. Following binding of the fusion protein to nitrocellulose membranes, hybridization with a 32P-labeled U1-RNA ligand was carried out to detect specific RNA–protein interaction. Parameters influencing the specificity and the detec-tion limit of binding were systematically investigated with the aid of the model system. Processing the nitrocellulose membranes in the presence of transition metals greatly increased the signal:background ratio. A simple screening protocol involving a single-buffer system was developed. Specific RNA–protein interac-tion could be detected in the presence of a large excess of recombinant phages from a cDNA library. Only moderate binding affinities (Kd = 10–8 M) were required. The suitability of the RNA-ligand screening protocol was demonstrated by the identification of new viroid-RNA binding proteins from tomato