DDX3X acts as a selective dual switch regulator of mRNA translation in acute ER stress

Figures

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  Figure 1.. DDX3X CLIP-Seq in MEFs, mRNA regulation and DDX3X CLIP density on the meta-mRNA in ER stress (A) Western blot of MEF lysates and IP beads eluates for DDX3X CLIP, and autoradiography of IP beads eluates to visualize crosslinked DDX3X-RNA complexes (highlighted in blue for −Tg and red for +Tg). (B) Western blot for indicated proteins in MEF lysates used for DDX3X CLIP. (C) DDX3X CLIP density across the meta mRNA, for all mRNAs in −Tg and +Tg treated cells. (D) Polysome profiling mRNA scatterplot for changes in abundance and polysome association in MEFs treated with Tg compared to no Tg treatment. The 7 highlighted territories demarcate mRNAs with changes in heavy polysome association and/or mRNA abundance, or no change thereof. Data from Guan et al, 2017. (E) Polysome profiling mRNA scatterplot as in (D) but plotted separately for the quintiles of DDX3X-bound mRNAs ranked by the log2 fold change in DDX3X CLIP abundance in +Tg treated cells vs −Tg cells. (F), (G) DDX3X CLIP density across the meta mRNA, for up-translated mRNAs (F), and down-translated mRNAs (G), in −Tg and +Tg treated cells, as the mRNAs are demarcated in (D).

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  Figure 2.. DDX3X CLIP-Seq in human cells, and the impact of Tg stress and DDX3X KD measured by polysome profiling. (A) DDX3X CLIP density across the meta mRNA in non-stressed 293T cells. (B) Western blot for indicated proteins in cell lysates used for polysome profiling, for indicated conditions. Lower panel: quantification of DDX3X signal from the same blot (vs tubulin). (C) 260 nm absorbance of polysome gradient collection, for indicated conditions. Vertical lines indicate individual fractions collected; blue colors indicate grouping of fractions for RT-qPCR analysis. (D) RT-qPCR analysis for ATF4 and GADD34 in grouped fractions as in (C), for indicated conditions. N=3, error bars: SD.

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  Figure 3.. mRNA abundance and polysome association changes upon DDX3X KD in the absence or presence of Tg induced stress. (A) Polysome profiling mRNA scatterplot for changes in abundance and polysome association in mock KD 293FT cells treated with Tg compared to no Tg treatment. Four individual mRNAs are highlighted. (B) Polysome profiling mRNA scatterplot for changes in abundance and polysome association for DDX3X KD compared to mock KD, no Tg treatment. mRNAs showing ≥1.5 fold increased (up-translated) and decreased (down-translated) polysome association are highlighted blue and red respectively. (C) mRNA scatterplot as in (A) with mRNAs highlighted according to polysome association change as in (B). (D) Violin plot for highlighted mRNAs in (C) and statistic test as indicated. (E) Polysome profiling mRNA scatterplot for changes in abundance and polysome association for DDX3X KD compared to mock KD, after Tg treatment. Up- and down-translated mRNAs are highlighted blue and red respectively. (F) mRNA scatterplot as in (A) with mRNAs highlighted according to polysome association change as in (E). (G) Violin plot for highlighted mRNAs in (E) and statistic test as indicated. (H) Cell viability…

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  Figure 4.. Association of DDX3X with eIF3 and DDX3X regulation of mRNAs in the context of 48S scanning process. (A) Silver stain of DDX3X IP from human cells. Major immunoprecipitated proteins are marked. Lane 1: negative control IP with normal rabbit serum (NRS IP). Lane 2: reducing elution of DDX3X IP beads. Lane 3: peptide elution of DDX3X IP, after treatment with RNase A. *: heavy and light Ab chain. (B) Number of unique and total peptides (pepts) of eIF3 subunits and eIF4G proteins, identified by mass spec of gel section containing the entire peptide-eluted DDX3X IP sample. The entire list of peptides can be found in Table S4. (C) DDX3X CLIP density on all mRNAs for eIF3D (magenta) and DDX3X CLIP (blue) in −Tg cells. (D) DDX3X CLIP density on up- (blue tones) and down-translated mRNAs (red tones) in DDX3X KD −Tg. (E) DDX3X CLIP abundance (log10 TPM) of 253 mRNAs with H/T ratio <0.5 in −Tg mock KD cells (see also Figure S7A) that are also up-translated in DDX3X KD −Tg. (F) Box plots of H/T ratios for the same 253 mRNAs in indicated conditions. Two-tailed t-test comparisons between −Tg mock and DDX3X KD (p-value: 6.1×10−22); mock KD −Tg and mock KD +Tg (p-value: 1.1×10−19), and three-way…

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  Figure 5.. Different mechanisms of translational regulation of two core subsets of DDX3X target mRNAs (A) Venn diagrams of DDX3X regulated mRNAs and statistic test (hypergeometric test) for the overlaps as indicated. The triple overlap is highlighted blue for 63 “DDU” mRNAs and red for 32 “UUD” mRNAs respectively. (B) STRING database analysis for “DDU” mRNAs. Statistically enriched terms (FDR< 0.05) are shown; mRNAs are colored by term enrichment. mRNAs selected for further analysis below are outlined in red. (C) STRING database analysis for “UUD” mRNAs. Statistically enriched terms (FDR< 0.05) are shown; mRNAs are colored by term enrichment. mRNAs selected for further analysis below are outlined in blue. (D) 260 nm absorbance of polysome gradients after harringtonine treatment for indicated conditions. RNA was extracted from the monosomal peak and from total input RNA. (E) Monosome-to-total (input) mRNA abundance ratios were measured for “DDU” mRNAs by RT-qPCR following polysome profiling with harringtonine treatment, which captures newly initiated ribosomes. Each line connects the mean values of the ratio for an individual mRNA in mock KD and DDX3X KD conditions. Significance was…

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  Figure 6.. mRNA co-dependence on DDX3X and ac4C modification for translational regulation. (A) ac4C density across the meta mRNA in DDX3X KD −Tg cells, for down-translated mRNAs (left, red), up-translated mRNAs (middle, blue) and all mRNAs (right, grey). Data from Yan et al., 2023, showing two separate ac4C-Seq methods that the authors employed, one using an Ab against ac4C, and a second one using a chemical probe. (B) Western blot for indicated proteins in in mock and NAT10 KD cell lysates, as indicated. Lower panel: quantification of NAT10 signal from the same blot (vs GAPDH). (C) Immuno-dot blot for ac4C using mock and NAT10 KD total RNA, top panel. Methylene blue stain, bottom panel, was used as a loading reference. Ac4C signal quantification is shown to the right of the top panel, one representative experiment shown. (D) 260 nm absorbance of polysome gradient collection, for indicated conditions. Vertical lines indicate individual fractions collected; blue colors indicate grouping of fractions for RNA-Seq library preparation. (E) Polysome profiling mRNA scatterplot for changes in abundance and polysome association in NAT10 KD compared to mock KD cells, −Tg. Four individual mRNAs are…

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  Figure 7.. A model of DDX3X function as a dual switch regulator in translation initiation in non-stressed cells and cells during acute ER stress. “Rest” mRNAs denote mRNAs that are translated in non-stressed cells which show a DDX3X-dependent switch in translation in cells during acute ER stress; “Stress” mRNAs show opposite DDX3X-dependent regulation in the same conditions. The positioning of DDX3X relative to the start codon represents the peak CLIP density for the mRNAs in these categories.