title: Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages.
abstract: The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1's role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.
authors.affiliation: Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, California 95064, USA Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
authors.forename: Kerstin A
authors.initials: KA
authors.lastname: Effenberger
authors.affiliation: Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, California 95064, USA Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
authors.forename: Veronica K
authors.initials: VK
authors.lastname: Urabe
authors.affiliation: Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, California 95064, USA Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
authors.forename: Beth E
authors.initials: BE
authors.lastname: Prichard
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, California 95064, USA Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Jurica
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D005091:Exons; D006801:Humans; D010750:Phosphoproteins / Q000037:antagonists & inhibitors*; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000235:genetics*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000037:antagonists & inhibitors*
chemical_list: D010750:Phosphoproteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human; D000255:Adenosine Triphosphate
keywords: SF3B1; inhibitor; pre-mRNA splicing; spliceosome