RNase L Reprograms Translation by Widespread mRNA Turnover Escaped by Antiviral mRNAs

Abstract

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-β. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.

Keywords: PABPC1; PKR; RNase L; dsRNA; eIF2a; innate immune response; interferon; mRNA degradation; mRNA metabolism; stress granule.

Figure 1.. RNase L catalytic activity alters SG assembly and reduces SG-associated RNAs.

(A) IF for SG-associated proteins G3BP1 and PABPC1 in WT and RL-KO U-2 OS cells. (B) G3BP1-positive foci from greater than thirty WT and RL-KO U-2 OS cells binned by volume. (C) IF for G3BP1 and PABPC1 in parental RL-KO A549 cells stably expressing either RNase L (RL) or RNase L-R667A (RL-CM) eight hours post-poly(I:C). Images for G3BP1 and PABPC1 staining are shown in Figure S1E. (D) G3BP1 and PABPC1 IF in PKR-KO and PKR and RNase L double KO (PKR/RL-KO) A549 cells rescued with RNase L (RL) or RNase L-R667A (RL-CM) six hours post-poly(I:C). (E) smFISH for AHNAK mRNA in WT and RL-KO U-2 OS cells +/− poly(I:C) with G3BP1 as a RLBs/SG marker. N.r. indicates non-responsive cells with respect to RLB/SG assembly. (F) Quantification of AHNAK mRNA smFISH from (E), with analysis of 17-30 RLB/SG cells in three fields of view. (G) RT-qPCR analysis of AHNAK mRNA in WT and RL-KO A549 cells at zero- and six-hours post-poly(I:C) transfection.

Figure 2.. RNase L promotes widespread turnover of mRNAs.

(A) smFISH for GAPDH mRNA (red) in WT and RL-KO A549 cells. (B) Quantification of GAPDH mRNA smFISH represented in (A). (C) smFISH/IF for GAPDH mRNA and G3BP1 in RL-KO cells rescued with RNase L (RL) or RNase L-R667A (RL-CM). (D) Quantification of GAPDH mRNA smFISH represented in (C). (E) qRT-PCR quantification in WT and RL-KO A549 cells transfected with or without (Mock) poly(I:C) for six hours. Bars represent the average C_t value differential +/− S.E.M. from at least five independent experiments. (F) Oligo(dT) (red) and G3BP1 (green) staining of WT and RL-KO A549 cells. (G) Quantification of mean oligo(dT) FISH signal in RLB/SG-positive cells as represented in (F). Greater than twenty cells were analyzed at the indicated times post-poly(I:C).

Figure 3.. IFN-β mRNA escapes RNase L-mediated mRNA turnover.

(A) smFISH/IF for IFN-β mRNA GAPDH mRNA and IF for G3BP1 in WT and RL-KO A549 cells. Cells that contain RLBs/SGs and IFN-β mRNA indicated by white arrows. Cells with RLBs/SGs without IFN-β mRNA are indicated by yellow arrows. Cells with IFN-β mRNA without RLBs/SGs are indicated by orange arrows. Cells lacking both RLBs/SGs and IFN-β mRNA indicated by blue arrows. (B) Quantification of IFN-β mRNA smFISH in WT and RL-KO A549 cells. (C) RT-qPCR analysis of IFN-β, IL-6, and GADD34 mRNA expression in WT and RL-KO A549 cells six-hours post-poly(I:C). Bars represent the average C_t value +/− the S.E.M from greater than 5 independent experiments.

Figure 4.. RNase L regulates global mRNA levels during dsRNA stress.

(A) Differential expression analysis without normalization from total RNA isolated from WT or RNase L-KO A549 cells before and six hours after poly(I:C) transfection. (Upregulated transcripts = Fold Change > 2, p-value < 0.05; downregulated transcripts = Fold Change < 0.5, p-value < 0.05). (B) Differential expression plot of mRNAs in WT cells treated with or without poly(I:C). The linear regression of the ERCC spike-in control RNAs is represented by dashed line. The color of the dots indicates their ERCC-normalized differential expression relative to RL-KO cells transfected +/− poly(I:C). Orange = RNase L-dependent decrease, blue = RNase L-dependent increase. (C) Scatterplot depicting half-life vs. expression levels (FPKM). Each transcript is color-coded by its ERCC-normalized differential expression between WT and RL-KO cells transfected +/− poly(I:C). (D) GO analysis of mRNAs that are induced in both WT and RL-KO cells post-poly(I:C) (greater than 4-fold, FPKM > 1 in either WT or RL-KO cells with poly(I:C) treatment) (Data S4).

Figure 5.. RNase L drives translational shut-off.

(A) S-35 metabolic labeling of newly synthesized proteins in WT, RL-KO, and PKR-KO A549 cells post-poly(I:C). (B) Quantification of experiments represented in (A). Bars represent the average signal from at least two independent replicates +/− S.D. normalized to time zero. (C) Representative images from SUnSET puromycin-labeling (green) analysis of indicated cells lines four hours post-poly(I:C) transfection. Dapi-stained nuclei (blue). Scale bar is 25 μm. (D) Similar to (C) but enlarged. (E) Quantification of the mean intensity normalized to mock-treated cells of puromycin staining in cells (between 51-176 cells analyzed for each cell line) that contain RLBs/SGs four hours post-poly(I:C). (F) Quantification of the percentage of cells translating (puromycin signal greater than 80% of average signal from untreated cells) four hours post-poly(I:C). Bars represent the average +/− S.D. normalized to untreated cells from at least three independent replicates in which cells from at least five fields of view were analyzed. (G) Representative image of simultaneous SUnSET puromycin-labeling (green), PABP IF (red), and GAPDH smFISH (white) in A549 cells post-poly(I:C). Cells that do not contain RLBs or do not display PABP translocation are labeled non-responsive cells (n.r.).

Figure 6.. RNase L-driven translational repression is independent of rRNA degradation and p-eIF2α.

(A) TapeStation analysis of the 28S and 18S rRNAs. (B) Immunoblot analysis of eIF2α, eIF2α-P51S (p-eIF2α), GADD34, and GAPDH in indicated A549 cell lines. (C) Quantification of the p-eIF2α:eIF2α ratio as represented in (B). Bars represent the average ratio +/− SEM from independent replicates (n=5-9). (D) S-35 metabolic in A549 cells transfected with poly(I:C) or treated with 250 uM sodium arsenite then treated with or without ISRIB (50 nM). (E) Quantification of IFN-β secretion from WT and RL-KO A549 via ELISA. Limit of quantification was 50 pg/ml. Bars represent Average +/− S.D. from three independent experiments. *** indicates p-value <0.001 as determined by student’s t-test. (F) Diagram of the eGFP expression vector in which eGFP ORF containing the IFN-β UTRs is driven by the IFN-β promoter. (G) smFISH for GAPDH and eGFP mRNAs in A549-WT cells with the IFN-β promoter-eGFP vector stably incorporated. GFP fluorescence is shown. (H) Similar to (G) but in A549- RL-KO cells. (I) Quantification of mean eGFP intensity from at least fifteen WT and RL-KO cells as represented in (G) and (H).

Figure 7.. IFN-β mRNA escapes RNase L-mediated decay via resistance to RNase L and RNase L-promoted transcriptional induction.

(A) Diagrams of endogenous IFN-β gene and eGFP constructs driven by the IFN-β promoter. (B) smFISH for IFN-β (top images) and eGFP (lower images) mRNAs from expression constructs depicted directly to the left in (A) eighth hours post-poly(I:C). IFN-β smFISH was performed in WT and RL-KO A549 cells with the IFN-β_pI₅,G_ORFI₃ construct stably incorporated. (C) Quantification of smFISH foci per cell as represented in the images in (B) directly to the left. (D) Diagram of CMV promoter-driven eGFP. (E) smFISH for eGFP mRNA driven by the CMV promoter in WT cells with or without poly(I:C) transfection. smFISH for GAPDH mRNA and GFP fluorescence in WT and RL-KO cells is shown in Figure S7A,B. (F) Quantification of eGFP smFISH represented in (E). (G) Immunoblot analysis of p-IRF3 (rep 1). Normalized p-IRF3 band intensity from two experiments are shown below. (H) IFN-β smFISH from non-deconvolved images. Arrows mark high-intensity RNase A-and Actinomycin D-sensitive foci consistent with nascent transcripts at IFN-β loci (Figure S7D,E,F). (I) Quantification of the relative intensity of IFN-β TSS in WT and RL-KO cells represented in (H). Between 111-138 foci were analyzed from three independent experiments. (J) Model of RNase L-mediated regulation of translation via mRNA degradation escaped by antiviral mRNAs.