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.

A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore

“…22 Also, new details regarding the mechanism of action of SF3B1 targeted agents have been elucidated. 25 Additionally results of the genome-wide array analysis of sudemycin treated tumor cells, which shows that sudemycins cause a rapid wide-ranging change in alternative pre-mRNA splicing and that a biotin-labeled sudemycin probe directly interacts with the SF3B1 protein have been reported. 26 This collaborative project ultimately led to sudemycin D6 (SD6), 15 which is currently in preclinical development as an anticancer agent (see Figure 1).…”

Herboxidiene triggers splicing repression and abiotic stress responses in plants

“…Probing the functions of the splicing machinery and its regulation, and subsequently the interplay between these regulatory mechanism and stress signal inputs, requires the availability of small molecules capable of perturbing the splicing machinery in a targeted fashion [10, 17, 20, 21]. The use of such small molecule inhibitors in plant research would provide mechanistic insights into the splicing process and its intricate regulatory mechanism at various molecular levels and under a variety of growth and stress conditions.…”

“…The use of such small molecule inhibitors in plant research would provide mechanistic insights into the splicing process and its intricate regulatory mechanism at various molecular levels and under a variety of growth and stress conditions. Work in cultured mammalian cells has identified a group of splicing inhibitors, including PB, SSA and GEX1A [20, 22, 23]. Interestingly, such compounds have distinct and very different chemical structures, and target SF3B, a subcomplex of the U2 snRNP spliceosomal complex composed of SF3B1, SF3B2, S3B3, SF3B4, SF3B5, and SF3B6, subsequently disrupting the early stages of spliceosome assembly and impairing splicing functions.…”

“…cultures, exhibits antitumor activity by targeting the spliceosome U2 snRNP complex and inhibiting pre-mRNA splicing, this activity makes GEX1A a valuable starting point for the development of anticancer drugs [20, 24, 25]. Preliminary studies have shown that GEX1A functions as an herbicide, but the mode of action is currently unknown [18, 23].…”

Roles of Fe–S proteins: from cofactor synthesis to iron homeostasis to protein synthesis

“…SF3B1 encodes an essential component of the spliceosome [24]. The protein is involved in RNA splicing, mediating recognition of the branch point sequence and selection of the 3′ splice site.…”