Applying a high-Throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells

Gunhild Sommer, Alena Fedarovich, Venkatesh Kota, Reycel Rodriguez, Charles D. Smith, Tilman Heise

Research output: Contribution to journalArticle

3 Scopus citations

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 antiapoptotic 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 highthroughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells

Original languageEnglish (US)
Article numbere0173246
JournalPloS one
Volume12
Issue number3
DOIs
StatePublished - Mar 2017

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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