Abstract
We present a high throughput, versatile approach to identify RNA-protein interactions and to determine nucleotides important for specific protein binding. In this approach, oligonucleotides are coupled to microbeads and hybridized to RNA-protein complexes. The presence or absence of RNA and/or protein fluorescence indicates the formation of an oligo-RNA-protein complex on each bead. The observed fluorescence is specific for both the hybridization and the RNA-protein interaction. We find that the method can discriminate noncomplementary and mismatch sequences. The observed fluorescence reflects the affinity and specificity of the RNA-protein interaction. In addition, the fluorescence patterns footprint the protein recognition site to determine nucleotides important for protein binding. The system was developed with the human protein U1A binding to RNAs derived from U1 snRNA but can also detect RNA-protein interactions in total RNA backgrounds. We propose that this strategy, in combination with emerging coded bead systems, can identify RNAs and RNA sequences important for interacting with RNA-binding proteins on genomic scales.
Highlights
We present a high throughput, versatile approach to identify RNA-protein interactions and to determine nucleotides important for specific protein binding
To carry out the analysis, a protein-RNA complex is first formed and incubated with beads to which oligonucleotides complementary to the target RNA have been coupled
We demonstrate the versatility of the approach for 1) discriminating between mismatches in the oligonucleotides, 2) mapping protein recognition sites on RNA, 3) differentiating specific and nonspecific binding RNAs, and 4) detecting specific RNAs in complex mixtures
Summary
Plasmids—The U1A test transcript was constructed by annealing overlapping oligos and ligating the annealed product into pDP19 (Ambion) to create plasmid pPS2702. U1A-green fluorescent protein (GFP) was PCR-amplified from pPS2035 and ligated into prSETB (Invitrogen) to create pPS2699. Labeling with 32P verified a product of the expected size, and subsequent transcription reactions were purified by G-50 spin columns or phenol extractions followed by multiple ethanol precipitations. Total RNA from HeLa cells was prepared by the TRIzol method with high salt precipitations to reduce background GFP fluorescence. Bead Binding Assays—Binding was performed in 20 mM HEPES, pH 7.5, 300 mM KCl, 0.1% IGEPAL, 10 ng/l tRNA, 0.04 units/l superase-IN (Ambion), and 20 ng/l bovine serum albumin unless otherwise indicated. Pro Fit (Quantum Soft) was used to fit the fluorescence intensities to a Langmuir isotherm
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