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.

Keywords: RT-qPCR; high-throughput assay; inhibitor; pre-mRNA splicing; spliceosome.

Figure 1

High-throughput approach to identify splicing inhibitors. a) Two-step splicing reaction showing pre-mRNA, first step intermediates (5′ exon and lariat intron intermediates) and second step products (mRNA and intron lariat). b) Schematic of TaqMan® based RT-qPCR assay. mRNA is reverse transcribed (RT) and quantified via a dual-labeled oligo probe to the splice junction by release of a fluorophore reporter (R) from a quencher (Q). c) Schematic of automated splicing assay. d) Data used to calculate Z′ value for the assay using no-splicing control (black diamonds) and normal splicing (gray diamonds) reactions. For each reaction, C_T is plotted vs. well number. e) Screening results of NCI library collection. The histogram plots the number of compounds screened vs. C_T value, and the position of candidate inhibitors is indicated. f) The chemical structures of three verified hits (C1, C2 and C3).

Figure 2

Three compounds inhibit splicing chemistry in a dose-dependent manner. Denaturing gel analysis of RNA from splicing reactions with indicated concentration of C1, C2, and C3. Quantification of splicing efficiency vs. inhibitor concentration is plotted below each gel along with estimated IC₅₀ values. a) Inhibition in HeLa nuclear extract. Identities of bands are schematized to the left as (from top to bottom) lariat intermediate, pre-mRNA, mRNA, 5′ exon intermediate. b) Inhibition in yeast extract. Identities of bands are schematized to the left as (from top to bottom) lariat intermediate, intron lariat, pre-mRNA, mRNA, 5′ exon intermediate.

Figure 3

C1 stalls human and yeast spliceosomes at an A-like complex. Native gel analysis of spliceosome assembly. a) Thirty minute time points of splicing reactions in HeLa nuclear extract supplemented with 2% DMSO or indicated concentration of C1. The identity of complexes is denoted with assembly occurring in the following order: H/E → A → B → C. The arrow indicates the A-like complex. b) Time course analysis of splicing reactions in HeLa nuclear extract in 2% DMSO or 1 mM C1. Time points are indicated in minutes. c) Time course analysis of splicing reactions in HeLa nuclear extract in the presence or absence of ATP and C1 as indicated using a pre-mRNA with wild type or mutant branch point. d) Twenty minute time points of splicing reactions in yeast extract supplemented with 1% DMSO or indicated concentration of C1. The identity of complexes is denoted with assembly occurring in the following order: CC1 → CC2 → PS/SP. e) Time course analysis of splicing reactions in yeast extract in 1% DMSO or 500 μM C1.

Figure 4

A B-like complex accumulates in the presence of the C3 compound. a) Native gel analysis of spliceosome assembly. Left panel shows thirty minute time points of splicing reaction in HeLa nuclear extract supplemented with 2% DMSO or indicated concentration of C3. Right panel shows time course analysis of splicing reactions in HeLa nuclear extract in 2% DMSO or 1 mM C3. Complexes are labeled as in Figure 3. b) Left panel shows twenty minute time points of splicing reactions in yeast extract supplemented with 1% DMSO or indicated concentration of C3. Right panel shows time course analysis of splicing reactions in yeast extract in 1% DMSO or 1 mM C3. Complexes are labeled as in Figure 3. c) Chemical structure of C3 and related compounds. d) Denaturing gel analysis of in vitro splicing reactions with increasing concentrations of C3 or indicated compounds in HeLa nuclear extract. Bands are schematized as in Figure 2. e) Denaturing gel analysis of in vitro splicing reactions with increasing concentrations of C3 or NSC224124 in yeast extract. f) Quantification of the splicing efficiency relative to compound concentration of the splicing reactions shown in (d) (top panel) and (e) (bottom panel). Estimated IC₅₀ values are indicated.

Figure 5

Inhibition by naphthazarins is partially rescued by excess DTT. a) Denaturing gel analysis of in vitro splicing reactions in HeLa nuclear extract inhibited by C3 or related compounds supplemented with increasing concentrations of DTT. Bands are schematized as in Figure 2. b) Quantification of the splicing efficiency relative to compound concentration of the splicing reactions shown in (a). c) Same analysis as in (a) but with yeast splicing. d) Quantification of the splicing efficiency relative to compound concentration of the splicing reactions shown in (c). Estimated IC₅₀ values are indicated.

Figure 6

Presence and orientation of a nitrophenyl ring are important for splicing inhibition by C2. a) Thirty minute time points of splicing reactions in HeLa nuclear extract supplemented with 2% DMSO or indicated concentration of C2. b) Time course analysis of splicing reactions in HeLa nuclear extract in 2% DMSO or 1 mM C2. c) Denaturing gel analysis of in vitro splicing reactions with increasing concentrations of C2 or indicated compounds in HeLa nuclear extract. d) Chemical structure of C2 and related compounds. e) Quantification of the splicing efficiency relative to compound concentration of the splicing reactions shown in (c). Estimated IC₅₀ values are indicated.