RNase L regulates the antiviral proteome by accelerating mRNA decay, inhibiting nuclear mRNA export, and repressing transcription

Figures

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  Figure 1.. Flaviviruses trigger RNase L-mediated decay of cellular mRNA (A) smRNA-FISH for human GAPDH mRNA (gray), poly(A) RNA (green), and viral RNA (magenta) in parental (WT) or RNase L-KO (RL-KO) A549 cells 24 h post-infection (p.i.) with DENV-2, ZIKV, or WNV. As a control, cells were lipofected with poly(I:C) for 12 h. Arrows indicate cells that activate RNase L-dependent decay of cellular mRNA. Scale bar, 20 μm. (B) Quantification of GAPDH mRNA as represented in (A) in individual cells (dots). The percentage of cells with activated RNase L was determined by the percentage of cells with GAPDH mRNA copies below the lowest observed in RL-KO cells (dashed line). The bar represents the median. (C) Quantification of poly(A) RNA intensity as represented in (A). (D) Quantification of viral RNA copies as represented in (A). n = 29 cells for (B)–(D). Significance was determined by Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 2.. RNase L activation kinetics in response to poly(I:C) and ZIKV infection (A) Schematic of experimental design. (B and C) Live-cell imaging of mRUBY2-PABPC1 A549 cells during poly(I:C) transfection (B) or ZIKV infection (C). White arrows indicate nuclear PABPC1, and green arrows indicate stress granules. Scale bar, 20 μm. (D) Percentage of cells with RNase L active in a single field of view, as determined by initiation of PABPC1 translocation. (E) Intensity of nuclear PABPC1 after RNase L activation, normalized to the peak value in each individual cell. Error bars represent standard deviation for n = 10 cells.

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  Figure 3.. RNase L does not enhance cell death or apoptosis (A) Live-cell imaging of WT and RNase L-KO (RL-KO) cells expressing mRuby2-PABPC1 following the lipofection of poly(I:C). Cell death was monitored by imploded cell morphology, and apoptosis signaling was measured by caspase-3/7 detection dye (green). Dead/caspase-positive cells are indicated with blue arrows. Scale bar, 15 μm. (B) Similar to (A) but during ZIKV infection. Scale bar, 15 μm. (C) Quantification of data for viable cells (pink lines) and caspase-3/7-positive cells (green) from n = 5 biological replicates in WT and RL-KO cells at indicated times post-lipofection of poly(I:C) as represented in (A). (D) Similar to (C) but during ZIKV infection as represented in (B). (E) Representative images of WT and RL-KO cells expressing mRuby2-PABPC1 and treated with caspase-3/7 indicator dye at indicated times post-infection with ZIKV. In the top, the WT cell activates RNase L at 28 h p.i. based on PABPC1 translocation to the cell. The images were derived from Videos S1 and S2. Scale bar, 10 μm.

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  Figure 4.. RNase L alters the cellular proteome (A) Scatterplots of log2-transformed protein levels in mock conditions (x axis) and 16 h post-lipofection of poly(I:C) in WT or RL-KO cells. (B) Volcano plots showing proteins up- or downregulated in WT or RL-KO cells at 16 h post-lipofection of poly(I:C). Highlighted points have met significance based on two-sample t test with FDR < 0.05 (n = 6). (C) Venn diagrams of proteins that are significantly differentially regulated by dsRNA response. Significance was determined by two-sample t test with FDR < 0.05 (n = 6). (D) Boxplot of the log2FC of proteins within the indicated half-life cutoff. The line is the linear regression of mean log2FC for each half-life cutoff with R2 = 0.801 and p = 0.0065. (E) Heatmap of GO terms associated with the proteins that were significantly upregulated or downregulated in the indicated cell line 16 h post-lipofection of poly(I:C). (F) Heatmap of transcriptional regulatory relationships of differentially upregulated proteins in WT and RL-KO cells 16 h post-lipofection of poly(I:C).

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  Figure 5.. ISG mRNAs evade RNase L-mediated RNA decay, permitting their translation (A) Quantitative proteomic analyses in WT and RL-KO cells. (B) Heatmap showing the induction of immediate-early and ISGs in response to poly(I:C). (C) Heatmap showing the effect of RNase L from (B). (D) Western blot analysis of ISG proteins in WT and RL-KO at 0, 8, and 16 h post-lipofection of poly(I:C). (E) smRNA-FISH for the indicated ISG mRNAs and GAPDH mRNA under mock conditions or 8 h post-lipofection of poly(I:C). Scale bar, 10 μm. (F) Quantification of smRNA-FISH puncta as represented in (E). Each dot represents a single cell. n = 30 or more cells. (G) smRNA-FISH for OASL mRNA and GAPDH mRNAs and IFA for OASL protein. White arrows indicate cells with degraded GAPDH mRNA, yellow arrows indicate cells with GAPDH mRNA. Scale bar, 10 μm. (H) Quantification of smRNA-FISH puncta and mean fluorescence intensity (MFI) for OASL protein in individual cells (dots) as represented in (G). n = 36 cells. Significance was determined by Student’s t test (Excel). *p < 0.05, **p < 0.01, and ***p < 0.001.

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  Figure 6.. RNase L-dependent inhibition of ISG mRNA export dampens protein synthesis (A) smRNA-FISH for labeled mRNAs 8 h post-lipofection of poly(I:C) in WT and RL-KO cells. The dashed lines demarcate nuclei, as determined by DAPI staining, of cells displaying a retention of mRNAs. Quantification of total (W), cytosolic (C), or nuclear (N) ISG mRNA levels is shown. Significance was determined by Student’s t test (Excel). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar, 20 μm; n = 20 or more cells. (B) Co-IFA for OASL protein and smRNA-FISH for OASL mRNA. White arrows indicate cells displaying nuclear retention of OASL mRNA. Yellow arrow indicates a cell containing cytoplasmic OASL mRNA. Scale bar, 10 μm. (C) Mean fluorescence intensity (MFI) of OASL protein in WT and RL-KO cells as represented in (B). n = 18 cells. (D) Scatterplot of the MFI of OASL protein (x axis) and nuclear OASL mRNA (y axis) per cell. n = 18 cells.

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  Figure 7.. RNase L dampens ISG expression by repressing RNAPII-mediated transcription (A) Fluorescence microscopy of EU-click-488 in WT and RL-KO cells. Scale bar, 20 μm. (B and C) Quantification of EU mean fluorescence intensity (MFI) in cells (dots). The bars represent the average ± standard deviation. Significance was determined by Student’s t test (Excel). *p < 0.05, **p < 0.01, and ***p < 0.001. n = 35 cells. (D) smRNA-FISH for IFIT2 mRNA and poly(A)+ RNA and in WT cells containing EU-click-488 under mock treatment or 8 h post-lipofection of poly(I:C). The arrow in the insets indicate putative nascent mRNAs at the genomic locus. Scale bar, 10 μm. (E) Meta-analyses of RNA-seq data of RNA induced in WT and RL-KO A549 cells 6 h post-lipofection of poly(I:C).19 The mRNAs upregulated (green), unchanged (gray), or downregulated (magenta) by RNase L are indicated. (F) smRNA-FISH for ISG15 mRNA in WT and RL-KO cells at 0, 8, or 16 h post-lipofection of poly(I:C). The graph represents quantification of total ISG15 mRNA copies per cell. n = 20 or more cells. Scale bar, 10 μm. (G) Similar to (F) but displayed to show nuclear ISG15 transcription foci (arrows). The graph on the right is the…