A Quantitative High-Throughput In Vitro Splicing Assay Identifies Inhibitors of Spliceosome Catalysis
A Quantitative High-Throughput In Vitro Splicing Assay Identifies Inhibitors of Spliceosome Catalysis
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DOI：
10.1128/mcb.05788-11
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发表时间：
2012-04-01
影响因子：
5.3
通讯作者：
Dreyfuss, Gideon
中科院分区：
文献类型：
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作者：
Berg, Michael G.;Wan, Lili;Dreyfuss, Gideon
尽管进行了深入的研究，但用于调节和剖析mRNA剪接途径的试剂非常少。在这里，我们描述了一种新的方法来识别这样的工具，基于检测外显子连接复合物（EJC），一个独特的分子签名，剪接mRNA上的叶子。我们开发了一种高通量，剪接依赖性EJC免疫沉淀（EJIPT）测定定量mRNA剪接从生物素标记的前mRNA在细胞提取物中，使用抗体EJC组件Y14和真核生物翻译起始因子4aIII（eIF 4AIII）。部署EJIPT，我们进行了高通量筛选（HTS）结合二级检测，以确定剪接抑制剂。我们描述了1，4-萘醌类和1，4-杂环醌类作为有效的选择性剪接抑制剂的已知抗癌活性的鉴定。有趣的是，与先前描述的小分子不同，其中大多数靶向早期步骤，我们的抑制剂以苯并噻唑-4，7-二酮BN 82685为代表，阻断剪接中两个酯交换反应中的第二个，防止内含子lactone的释放和外显子的连接。我们发现，BN 82685抑制激活的剪接体的精细结构重排，这是第二步催化所需的，允许定义剪接体停滞在催化中间。EJIPT为剪接和EJC调节剂的表征和发现提供了一个平台。
Despite intensive research, there are very few reagents with which to modulate and dissect the mRNA splicing pathway. Here, we describe a novel approach to identify such tools, based on detection of the exon junction complex (EJC), a unique molecular signature that splicing leaves on mRNAs. We developed a high-throughput, splicing-dependent EJC immunoprecipitation (EJIPT) assay to quantitate mRNAs spliced from biotin-tagged pre-mRNAs in cell extracts, using antibodies to EJC components Y14 and eukaryotic translation initiation factor 4aIII (eIF4AIII). Deploying EJIPT we performed high-throughput screening (HTS) in conjunction with secondary assays to identify splicing inhibitors. We describe the identification of 1,4-naphthoquinones and 1,4-heterocyclic quinones with known anticancer activity as potent and selective splicing inhibitors. Interestingly, and unlike previously described small molecules, most of which target early steps, our inhibitors represented by the benzothiazole-4,7-dione, BN82685, block the second of two trans-esterification reactions in splicing, preventing the release of intron lariat and ligation of exons. We show that BN82685 inhibits activated spliceosomes' elaborate structural rearrangements that are required for second-step catalysis, allowing definition of spliceosomes stalled in midcatalysis. EJIPT provides a platform for characterization and discovery of splicing and EJC modulators.