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-specific 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 beta-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 detection 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 interaction 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.
Sägesser
R
R
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece. saegesser@nefeli.imbb.forth.gr
Martinez
E
E
Tsagris
M
M
Tabler
M
M
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008670:Metals; D011485:Protein Binding; D015347:RNA Probes; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014772:Viroids / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D008024:Ligands; D008670:Metals; D015347:RNA Probes; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D014764:Viral Proteins