title: 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Berg
authors.forename: Lili
authors.initials: L
authors.lastname: Wan
authors.forename: Ihab
authors.initials: I
authors.lastname: Younis
authors.forename: Michael D
authors.initials: MD
authors.lastname: Diem
authors.forename: Michael
authors.initials: M
authors.lastname: Soo
authors.forename: Congli
authors.initials: C
authors.lastname: Wang
authors.forename: Gideon
authors.initials: G
authors.lastname: Dreyfuss
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology*; D001710:Biotin / Q000737:chemistry; D057809:HEK293 Cells; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D009285:Naphthoquinones / Q000494:pharmacology*; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism*; D017381:Spliceosomes / Q000187:drug effects* / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology*
chemical_list: C506607:BN 82685; D016227:Benzoquinones; D009285:Naphthoquinones; D012322:RNA Precursors; D012333:RNA, Messenger; D013844:Thiazoles; D001710:Biotin; C035342:1,4-naphthoquinone