Analysis of RNA–protein interactions by a microplate-based fluorescence anisotropy assay

Abstract

Quantitative studies of RNA–protein interactions are quite cumbersome using traditional methods. We developed a rapid microplate-based fluorescence anisotropy (FA)/fluorescence polarization assay that works well, even with RNA probes >90 nucleotides long. We analyzed binding of RNA targets by vigilin/DDP1/SCP160p and by c-myc coding region instability determinant (CRD) binding protein, CRD-BP. Vigilin is essential for cell viability and functions in heterochromatin formation and mRNA decay. The CRD-BP stabilizes c-myc mRNA. Vigilin bound to a vitellogenin mRNA 3′-UTR probe with a two to three-fold lower affinity than to a Drosophila dodecasatellite ssDNA binding site and bound to the c-myc CRD with a two- to three-fold lower affinity than to the vitellogenin mRNA 3′-UTR. Competition between vigilin and CRD-BP for binding to the CRD may therefore play a role in regulating c-myc mRNA degradation. We analyzed suitability of the microplate-based FA assay for high-throughput screening for small-molecule regulators of RNA–protein interactions. The assay exhibits high reproducibility and precision and works well in 384-well plates and in 5 μl to 20 μl samples. To demonstrate the potential of this assay for screening libraries of small molecules to identify novel regulators of RNA–protein interactions, we identified neomycin and H33342 as inhibitors of binding of vigilin to the vitellogenin mRNA 3′-UTR.