title: Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells.
abstract: The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells.
authors.affiliation: Medical University of South Carolina, Department of Biochemistry & Molecular Biology, 173 Ashley Avenue, Charleston, SC, United States of America.
authors.forename: Gunhild
authors.initials: G
authors.lastname: Sommer
authors.affiliation: Medical University of South Carolina, Department of Biochemistry & Molecular Biology, 173 Ashley Avenue, Charleston, SC, United States of America.
authors.forename: Alena
authors.initials: A
authors.lastname: Fedarovich
authors.affiliation: Medical University of South Carolina, Department of Biochemistry & Molecular Biology, 173 Ashley Avenue, Charleston, SC, United States of America.
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Kota
authors.affiliation: Medical University of South Carolina, Department of Biochemistry & Molecular Biology, 173 Ashley Avenue, Charleston, SC, United States of America.
authors.forename: Reycel
authors.initials: R
authors.lastname: Rodriguez
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, 173 Ashley Avenue, Charleston, SC, United States of America.
authors.forename: Charles D
authors.initials: CD
authors.lastname: Smith
authors.affiliation: Medical University of South Carolina, Department of Biochemistry & Molecular Biology, 173 Ashley Avenue, Charleston, SC, United States of America.
authors.forename: Tilman
authors.identifier: http://orcid.org/0000-0002-3248-3129
authors.initials: T
authors.lastname: Heise
mesh_terms: D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D015335:Molecular Probes / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D012313:RNA / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D015335:Molecular Probes; D016601:RNA-Binding Proteins; D012313:RNA