A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex

Abstract

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.

Keywords: S34F mutant; U2AF(35); U2AF(65); U2AF1; myelodysplastic syndrome; ribonucleoprotein targeting; spliceosome inhibition; splicing factor mutation; therapeutic strategy.

Figure 1.

A high-throughput screen for modulators of U2AF–RNA complexes. (A) Domains of U2AF1, U2AF2, and SF1 splicing factors. Gray, region excluded from the screened expression constructs. (B) Schematic diagram of the fluorescence polarization RNA binding assay. Fl, 5′ fluorescein. (C) Fluorescence polarization curve for U2AF1^S34F–U2AF2–SF1 binding into fluorescein DEK ^FLRNA. The nonlinear fit (solid curve) is overlaid on the mean data points ± SD of three replicates. A dashed line marks the protein concentration used for the enhancer screen. (D) Distribution of positive (blue) and negative (red) control signals used for the calculation of the Z′-factors of the assay. (E) Overview of the screen. Results are summarized to the right and detailed in Table S1.

Figure 2.

The hit enhancer of the U2AF2 – RNA complex stalls in vitro pre-mRNA splicing at the H/E-stage. (A) The fluorescence polarization dose-response of U2AF1^S34F–U2AF2–SF1–DEK ^FLRNA titrated with hit enhancer (NSC 194308, chemical structure inset). (B) qRT-PCR of spliced AdML products. The relative amounts of spliced product were normalized to a mock-treated negative control (1% v/v DMSO, gray). The positive control is an SF3B1 inhibitor (PB, 1 µM pladienolide-B). Two-tailed, unpaired t-tests with Welch’s correction compared the indicated NSC 194308 concentration with the mock-treated control: *, p<0.05; **, p<0.005; ***, p<0.0005. (C) Denaturing gel analysis of the radiolabeled pre-mRNA substrate and spliced products from reactions treated with the indicated concentrations of NSC 194308 or a mock-treated control. Spliced products are schematically diagrammed to the left. (D) Native gel analysis of spliceosome assembly at 30 minutes in the presence of the indicated concentrations of NSC 194308, positive control (SSA, 1 µM spliceostatin-A), or mock-treated control. The identities of spliceosome complexes are indicated (left), with assembly occurring in the following order: H/E → A → B → C. NCI DTP, NCI Developmental Therapeutics Program. Data represented in A and B are mean ± SD of three replicates. Data represented in C and D are representative of three replicates. See also Figure S1.

Figure 3.

The hit enhancer NSC 194308 binds U2AF2 RRM1/RRM2 (U2AF2^1,2L). (A) The favorable predicted binding site for NSC 194308 is located between RRM1 and RRM2 (blue) of the open U2AF2^1,2L conformation (PDB ID 5EV4). Candock scores are listed in Table S2. The bound oligonucleotide (magenta) of the structure is overlaid for reference. Structure-guided mutants are yellow, and for single-site mutations, shown as space-filling CPKs; NSC 194308 is a CPK representation colored by atom: carbon, cyan; oxygen, red; sulfur, orange; nitrogen, blue. (B) Locations of the mutated-regions (yellow) shown on the NMR model (blue) of closed U2AF2^1,2L (PDB ID 2YH0). (C) Representative sensorgram showing the aligned responses of NSC 194308 injected at the indicated concentrations over an immobilized GST-U2AF2^1,2L surface. (D) Plot of the average responses (two replicates) from the saturated regions of the sensorgrams fit to a nonlinear steady-state model. NSC 194308 binding to GST-U2AF2^1,2L, blue; separate GST-RRM1, dark gray; GST-RRM2, light gray. (E-H) Fluorescence polarization dose-responses of the hit enhancer titrated into DEK ^FLRNA bound to U2AF2^1,2L variants, including (E) wild-type, (F) inter-RRM linker deletion, (G) D215R/G319R, or (H) G154E. The mean ± SD of three replicates are shown in E - H. Schematic diagrams of the expected conformations of the U2AF2 variants are inset and colored as in A.

Figure 4.

Comparison of NSC 194308 variants shows that anti-splicing activity depends on the thiosulfate and is sensitive to variations of the hydrophobic group. (A) Chemical structures of hit compound variants. The (−)-cis-myrtanyl acetamidine was synthesized de novo (Compound 3, Star Methods and Figure S2) and other compounds were obtained from the NCI DTP. Me, Methyl. (B) Comparison of the spliced AdML product from HeLa nuclear extract treated with either 50 μM (left) or 25 μM (right) compound variants. The products were detected by qRT-PCR, normalized to a mock-treated negative control (1% v/v DMSO), and compared to a pladienolide B (PB) positive control. The mean ± SD of three reactions are graphed. Two-tailed, unpaired t-tests with Welch’s correction: *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005. Titrations are shown in Figure S2.

Figure 5.

NSC 194308 alters splicing of U2AF2-sensitive transcripts in HEK 293T cells. (A - C) qRT-PCR of gene transcripts with known responsiveness to U2AF2 family members. NSC 194308-treated samples (blue, left) are compared with samples treated with U2AF2-targeted Stealth™ siRNAs (or control siRNA) for either two days (magenta, center) or three days (purple, right). The products of the indicated gene transcripts were normalized to GAPDH controls. The qRT-PCR results are mean ± SD of three reactions. (D - F) RT-PCR of the indicated U2AF2-regulated gene transcripts following either treatment with NSC 194308 (left) or U2AF2 knockdown (right). A mocked-treated control (0.1% v/v DMSO) matches the NSC 194308 solvent. The mean ± SD exon-skipped:included ratios of background-corrected bands from three reactions (six for DMSO control) are plotted below representative ethidium-bromide-stained agarose gels. The expected PCR products are schematized (right). Two-tailed, unpaired t-tests with Welch’s correction: *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005. Immunoblots are shown in Figure S3.

Figure 6.

NSC 194308 preferentially kills K562 leukemia cells expressing MDS-associated S34F-mutant U2AF1 and alters splicing in these cells. (A) The fraction of viable cells (detected by trypan blue staining) normalized to a mock-treated sample (0.1% v/v DMSO, matching solvent) are plotted versus the log₁₀ concentration of NSC 194308. The mean ± SD of three replicates are shown. K562 cells with stably-integrated, doxycycline-induced WT or S34F-mutant U2AF1 or CRISPR-edited endogenous U2AF1 gene were treated with the indicated concentrations of NSC 194308 for five days. The inhibitory concentrations (IC₅₀) at half-maximal cell survival are inset above. (B - D) qRT-PCR (normalized to the GAPDH control of the WT sample) or (E - G) RT-PCR of the indicated gene transcripts from U2AF1-edited cells treated for seven hours with NSC 194308. The qRT-PCR results are mean ± SD of three reactions. The mean ± SD exon-skipped:included ratios of background-corrected bands from three reactions (six for DMSO control) are plotted below representative ethidium-bromide-stained agarose gels. Wild-type (WT), blue; S34F-mutant, red. Two-tailed, unpaired t-tests with Welch’s correction compare the indicated NSC 194308-treated sample with the matching WT or S34F mutant DMSO control: *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005. Immunoblots are shown in Figure S4.

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