title: A high-throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes.
abstract: The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing, myriad subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high-throughput assay for in vitro splicing using a reverse transcription followed by quantitative PCR readout. In a pilot screen of 3080 compounds, we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly affect spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.
authors.affiliation: 1Department of Molecular Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA, USA.
authors.forename: Kerstin A
authors.initials: KA
authors.lastname: Effenberger
authors.forename: Rhonda J
authors.initials: RJ
authors.lastname: Perriman
authors.forename: Walter M
authors.initials: WM
authors.lastname: Bray
authors.forename: R Scott
authors.initials: RS
authors.lastname: Lokey
authors.forename: Manuel
authors.initials: M
authors.lastname: Ares
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Jurica
mesh_terms: D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects*; D048348:Reverse Transcription; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D017381:Spliceosomes / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D012322:RNA Precursors; D054852:Small Molecule Libraries
keywords: RT-qPCR; high-throughput assay; inhibitor; pre-mRNA splicing; spliceosome