A Quantitative High-Throughput In Vitro Splicing Assay Identifies Inhibitors of Spliceosome Catalysis

Abstract

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.

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.05788-11.

ACKNOWLEDGMENTS

We thank Hyunil Jo and the members of our laboratory, especially Byung Ran So, for stimulating discussions and comments on the manuscript. We give special thanks to Yoon Cho for technical assistance, Jeongsik Yong for technical advice, and Brian Carr for compounds (Cpd5 and PM-20). We thank Chao-Xing Yuan and staff at the University of Pennsylvania Proteomics Core facility for assistance with mass spectrometry and data analysis.

This work was supported by the Association Française Contre les Myopathies. G.D. is an Investigator of the Howard Hughes Medical Institute.