Analysis of RNA-protein interactions by a microplate-based fluorescence anisotropy assay.
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 microl to 20 microl 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.
Mao
Chengjian
C
Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801-3602, USA.
Flavin
Kathryn Goolsby
KG
Wang
Stanley
S
Dodson
Robin
R
Ross
Jeffrey
J
Shapiro
David J
DJ
D020413:3' Untranslated Regions / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D015342:DNA Probes / Q000138:chemical synthesis; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D008832:Microchemistry; D012313:RNA / Q000378:metabolism*; D015347:RNA Probes / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D014819:Vitellogenins / Q000235:genetics
D020413:3' Untranslated Regions; D002352:Carrier Proteins; D015342:DNA Probes; C406144:IGF2BP1 protein, human; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014819:Vitellogenins; C075197:high density lipoprotein binding protein; D012313:RNA