Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes

Abstract

RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

Keywords: RNA-binding protein; small-molecule inhibitor; steroid hormones.

FIGURE 1.

Ro inhibits steroidogenesis. Dose-dependent effects of Ro on the percent of aldosterone (A), cell viability (PrestoBlue) (B), and DNA content (FluoReporter) (C) relative to DMSO in H295R cells stimulated with 10 nM Ang II. Error bars indicate SD from up to three independent rounds of experiments, each containing six independent replicates. (D) Heatmap representing selected enriched BP ontology terms in genes differentially expressed after Ro treatment during AngII stimulation time course (see Supplemental Fig. 1C,D for full results). (E) Fold change and P-values of mRNA levels compared with unstimulated controls for DMSO (black) or 5 µM Ro (red) treated H295R cells at multiple times post-AngII stimulation (10 nM). The likelihood ratio test was used to calculate P-values. (F) Model depicting the regulation of genes involved in key steps steroidogenesis by Ro. Solid lines show genes significantly down-regulated upon Ro treatment.

FIGURE 2.

Ro phenotypes are MSI2-independent. (A) Dose-dependent effects of Ro on the percent aldosterone relative to DMSO in H295R FLAG-MSI2-R100A cells with or without 5 µg/mL doxycycline. Error bars indicate the SD of two independent rounds of experiments, each with six independent replicates. (B) RIP-qPCR enrichment of MSI2 targets (CCSAP and SCD) and nontargets (ACTB and GAPDH) normalized to YARS in H295R cells. FLAG-MSI2-R100A RIPs (red, FLAG antibody) were from doxycycline-treated cells and endogenous MSI2 RIPs (gray, endogenous MSI2 antibody) were from untreated cells. Error bars indicate SE from three to six replicates. (C) Ratio of NanoLuc to Firefly luciferase normalized to uninduced cells (no dox, gray) upon expression of either FLAG-MSI2-R100A or FLAG-MSI2-WT protein (dox, red). P-values were calculated using Student's t-test (**) P < 0.001, (*) P < 0.05, (n.s.) ≥ 0.05. Error bars indicate standard error from two (HEK293) or one (H295R) independent rounds of experiments, each containing six (HEK293) or four (H295R) independent replicates, respectively. (D,E) RIP-qPCR enrichment of H295R cells with induced FLAG-MSI2-R100A (D) or endogenous MSI2 (E) treated with 5 µM Ro (red) or DMSO (gray). Error bars indicate SE from three to six replicates. (F) Ro dose-dependent effects on the percent cell viability (Cell-Titer-Glo) relative to DMSO in K562 cells transfected with FLAG-MSI2-R100A plasmid with or without 5 µg/mL doxycycline. Error bars indicate SD from six independent replicates.

FIGURE 3.

Ro interacts with multiple RRM containing RBPs. (A) Depiction of PISA assay. (B) Volcano plot of the change in relative temperature-dependent solubility of measured proteins with respect to Ro treatment compared with significance (−log₁₀ P-value). (C) Top 15 gene ontology molecular functions enriched in proteins identified as Ro interactors from PISA assay. (D) Bar plot showing the count of each RBD domain type for RBPs enriched as PISA interactors.

FIGURE 4.

Ro causes dose-dependent stress granule formation. (A) Correlation between gene expression changes in Ro-treated H295R cells and gene expression changes from RBP knockdown in K562 cells (ENCODE) for the subset of genes with Ro-dependent expression changes (see Materials and Methods). RBPs colored by the significance of the correlation coefficient. (Pink) Positive, (gray) not significan, (blue) negative. (B) CDF plot of the log₂ fold change in expression between Ro- and DMSO-treated cells for stress granule-localized transcripts (red) and all other transcripts (black). The Kolmogorov–Smirnov test was used to calculate statistical significance. (C) Overlap of Ro up-regulated transcripts and stress granule enriched transcripts. A hypergeometric test was used to calculate statistical significance. (D) Immunofluorescence of a stress granule marker, HuR/ELAVL1 (green), for DMSO- or Ro-treated H295R cells. Nuclei are shown in blue.

Kathryn Walters