Figure 2. Impact of WIN site inhibitors (WINi) on the translatome of MLLr cancer cells.

(A) Volcano plots depicting alterations in translation efficiency (TE) induced by 48 hr treatment of MV4;11 cells with either 2 µM C6 (left) or 100 nM C16 (right) compared to DMSO (n = 2; red indicates false discovery rate [FDR] < 0.05 and Log2 FC > 0.25), as determined by Ribo-seq. (B) Number of mRNAs with significantly (FDR < 0.05 and Log2 FC > 0.25) altered TE levels following treatment of MV4;11 cells with C6 (2 µM) or C16 (100 nM) for 48 hr. See Figure 2—source data 1 for complete output of Ribo-seq analysis. (C) Overlap of mRNAs with significantly decreased TE in response to C6 or C16 treatment. (D) TE of mRNAs in DMSO-treated MV4;11 cells plotted against translation efficiencies of mRNAs in cells treated with either C6 (left) or C16 (right). Red indicates mRNAs with significantly altered translation efficiencies following inhibitor treatment (FDR < 0.05 and Log2 FC > 0.25). (E) Numbers of differentially translated mRNAs (∆TE) in each quartile of genes (stratified by TE in DMSO) in cells treated with C6 (left) or C16 (right). (F) Enrichment analysis of common mRNAs suppressed by C6/C16 at the mRNA (blue) and translational (red; TE) level in MV4;11 cells. Hallmark.MSigDB pathways are shown. The x-axis indicates the number of suppressed genes in each category; the italic numbers are the corresponding FDR. See Figure 2—source data 2 for the full Hallmark.MSigDB analysis, as well as for Reactome and KEGG pathways. (G) Enrichment analysis of mRNAs suppressed translationally by C6/C16 but with no significant changes in mRNA levels. Gene Ontology (GO) Biological Process (BP) and Molecular Function (MF) categories are shown, as well as KEGG pathways. The x-axis displays -Log10 FDR; the number of mRNAs is shown in italics in each bar. See Figure 2—source data 3 for extended enrichment analyses, broken down by TE and mRNA direction changes. (H) TE changes in WDR5-bound (left) and non-bound (right) RPGs elicited by C6 (top) or C16 (bottom).

Figure 2—figure supplement 1. WIN site inhibitors (WINi) suppress bulk protein synthesis.

(A) Representative histograms from protein synthesis assays in MV4;11 cells treated 24, 48, or 96 hr with either 0.1% DMSO (blue), 2 µM C6 (red), or 100 nM C16 (orange). Cells were pulsed with O-propargyl-puromycin (OPP) to label nascent proteins, Alexa Fluor 647 linked to incorporated OPP in Click chemistry reactions, and fluorescence measured by flow cytometry analysis. MV4;11 cells treated 30 min with 100 µg/mL cycloheximide (‘CHX’; green) serve as a positive control for inhibited protein synthesis. MV4;11 cells pulsed with DMSO (‘No OPP’; black) serve as a control for background fluorescence. (B) Quantification of protein synthesis assays. Fluorescence from CHX-treated cells was set as the baseline, and fluorescence presented relative to DMSO-treated (DM) cells at each time point (n = 3; normalized geometric mean ± SEM). p-Values calculated by Student’s t-tests are represented by asterisks: *<0.05, **<0.01, ***<0.001.

Figure 2—figure supplement 2. WIN site inhibitors (WINi) suppress translation.

(A) Distribution of ribosome-protected fragment (RPF) lengths in each Ribo-seq sample/replicate. The length distribution of RPFs in mammalian Ribo-seq experiments typically peaks at 30–31 nucleotides. (B) Proportion of RPFs mapping to the coding sequence (CDS) or 5′ or 3′ untranslated regions (UTR) of transcripts. Color of dots is the same as in (A). (C) Proportion of RPFs mapping to each reading frame in the 5′ UTR (left), the CDS (middle), and the 3′ UTR (right). Color of dots is the same as in (A). (D) Magnitudes of significant translation efficiency (TE) alterations of mRNAs in each quartile (stratified by TE in DMSO) in cells treated with C6 (left) or C16 (right). Color dots represent individual genes. Bottom, middle, and top horizontal lines of each box represent first, second, and third quartiles, respectively. Vertical lines extend to data points within 1.5-fold of the interquartile range. Black dots represent values beyond 1.5-fold of the interquartile range. (E) Changes in TE induced by C6 (left) or C16 (right) in MV4;11 cells, binned according to mRNA TOPscores (Philippe et al., 2020). Dashed lines represent the median; dotted lines indicate quartiles. Significance by t-test is indicated compared to group with TOPscore 0–1 (*≤0.05, **≤0.0001). (F) UpSet plot, showing the breakdown of genes encoding PRMT5 substrates Radzisheuskaya et al., 2019 whose transcript levels and/or translation efficiencies decrease following WIN site inhibition (p-value calculated by hypergeometric test for over-representation of genes encoding PRMT5 substrates in genes with decreased translation efficiencies). (G) Overlap of C6/C16 common mRNAs with decreased abundance (RNA; blue) and those with decreased TE (salmon), grouped according to the indicated Hallmark.MSigDB categories. (H) Overlap of all C6/C16 common mRNAs with altered abundance and decreased TE. (I) The top row of the heatmap displays the codon stability coefficient (CSC) for each codon (Wu et al., 2019) ranked from lowest ('non-optimal') to highest ('optimal'). The middle row displays enrichment of each codon in mRNAs that are decreased at both the TE and mRNA levels (RNA + TE) versus those that show a decrease in TE without an accompanying decrease in mRNA abundance (TE only). Bottom row is -Log10 false discovery rate (FDR).

Figure 2—figure supplement 3. WIN site inhibitors (WINi) impair translation of mitochondrial ribosomal proteins.

(A) Top: transcript level changes in mitochondrial ribosomal protein genes elicited by C6 or C16, as indicated. Bottom: translational efficiency (TE) changes in mitochondrial ribosomal protein genes elicited by C6 or C16. All of the mitochondrial RPGs are nuclear-encoded; none have detectable binding of WDR5.