Nucleic Acids Research, Vol 25, Issue 19 3816-3822, Copyright © 1997 by Oxford University Press
R Sagesser, E Martinez, M Tsagris and M Tabler
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.
ARTICLES
Detection and isolation of RNA-binding proteins by RNA-ligand screening of a cDNA expression library
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Crete, Greece. saegesser@nefeli.imbb.forth.gr
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