Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells.
Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells.
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DOI：
10.1371/journal.pone.0173246
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Heise T
中科院分区：
文献类型：
--
作者：
Sommer G;Fedarovich A;Kota V;Rodriguez R;Smith CD;Heise T
RNA结合蛋白La在许多肿瘤组织中过表达，并被认为通过结合并促进编码肿瘤促进因子和抗凋亡因子的mRNA的表达来支持肿瘤发生。因此，能够阻断La与特定RNA结合的小分子可以通过减少促肿瘤因子和抗凋亡因子的表达而具有治疗作用。对这种新的治疗策略，我们的目的是开发一种高通量的荧光偏振分析筛选小的化合物库的分子阻断La的结合到来自细胞周期蛋白D1 mRNA的RNA元件。在此，我们利用一个强大的荧光偏振测定和验证的主要命中电泳迁移率变动测定。我们最近表明，镧保护细胞对顺铂治疗刺激蛋白质合成的抗凋亡因子Bcl 2。在这里，我们通过RNA免疫沉淀实验表明，一个小的化合物特异性地损害与Bcl 2 mRNA在细胞中的La的协会和cipslatin诱导的细胞死亡的细胞敏感。总之，我们报告的应用程序的高通量荧光偏振分析，以确定小的化合物，损害的La在体外和细胞中的靶RNA的结合。
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.