Fig 1.
RNase L activation increases the concentration of RBPs in the nucleus.
(A) Immunofluorescence assay for PABPC1 in wild-type (WT) or RNase L knockout (RL-KO) A549 cells following four hours of lipofection with or without poly(I:C). (B) Quantification of the nuclear to cytoplasmic ratio of PABPC1 intensity as represented in (A). Between 56–118 cells were analyzed from three independent replicates. (B and C) Similar to (A and B) but for HuR. Between 15–22 cells were analyzed (E) Panels show indicated RBP immunofluorescence, with the nucleus outlined, in WT and RL-KO cells following transfection with poly(I:C). (F) Quantification of the nuclear to cytoplasmic ratio of immunofluorescence intensity of indicated RNA-binding proteins in WT and RL-KO cells transfected with or without poly(I:C), as represented in (E). Between 30–127 cells were counted from three independent replicates for G3BP1 an TIA-1, two replicates for FAM120A and Caprin-1, and one replicate for PABPC4. Note, only WT cells with RLBs or RL-KO cells with stress granules were analyzed in the poly(I:C)-treated cells since not all cells responds to poly(I:C). Images of PABPC4 and TIA during mock conditions can be observed in Fig 3. Images of FAM120A, Caprin-1, and G3BP1 are shown in S1A–S1C Fig. Statistical significance was determined using a one-way ANOVA with Tukey’s HSD. * is p < 0.05, ** is p < 0.01, *** is p < 0.001. Statistical analyses between the most relevant comparisons are shown the graphs. Comparisons between all groups are included in S1 Data.
Fig 2.
RNase L-mediated decay of RNAs specifically occurs in the cytoplasm.
(A) FISH for poly(A)+ RNA and immunofluorescence assay for G3BP1 in WT and R-KO A549 cells. WT cells with RNase L-dependent bodies (RLBs) or RL-KO cells containing stress granules (SGs) are indicated by white arrows. Cells without RLBs or SGs are non-responsive (n.r.) and are indicated by yellow arrows. (B) Quantification of poly(A)+ RNA signal in the nucleus (nuc.) or cytoplasm (cyto.) in WT and RL-KO cells with or without RLBs or SGs, respectively, as represented in (A). Between 18–35 cells from at least three fields of view were analyzed. (C) Immunoblot analysis of endogenous RNase L in whole cell lysate (w), nuclear fraction (n), or cytoplasmic fraction (c) from A549-WT cells. (D) Schematic showing RBP re-localization following either cytoplasmic RNase L activation (left) or activation of nuclear-localized RNase L (RL-NLS). (E) Immunoblot for RNase L in whole cell (w), nuclear (n), and cytoplasmic (c) crude fractions showing nuclear localization of the RNase L-NLS. (F) Immunofluorescence assay for G3BP1 and FISH for poly(A)+ RNA in parental A549 cells or A549 that express NLS-RNase L construct following mock or poly(I:C) lipofection. N.r. indicates cells not responding to poly(I:C) based on the lack of RLBs marked by G3BP1 and poly(A)+ RNA, and the absence of reduced cytoplasmic poly(A)+ RNA staining. (G) Quantification of mean poly(A)+ RNA signal intensity (arbitrary units, a.u.) in WT or WT-RL-NLS cells treated with poly(I:C) as represented in (F). Between 92–110 cells from at least ten fields of view were analyzed for each group. Statistical significance was determined using a one-way ANOVA with Tukey’s HSD. * is p < 0.05, ** is p < 0.01, *** is p < 0.001. Statistical analyses between the most relevant comparisons are shown the graphs. Comparison between all groups are included in S1 Data.
Fig 3.
RNase L-mediated re-localization of RBPs to the nucleus is dependent on intact nuclear RNA.
(A) Immunofluorescence for PABPC1 and FISH for poly(A)+ RNA in WT or WT cells expressing NLS-RNase L (NLS-RL) eight hours post-poly(I:C) transfection. (B) Quantification of the nuclear:cytoplasmic ratio of PABPC1 in mock-treated WT cells or WT or NLS-RL cells transfected with poly(I:C) as represented in (A). Between 48–70 cells from greater than six fields of view and two independent replicates were analyzed. (C-H) Similar analysis as (A) and (B) but for PABPC4 (C and D), HuR (E and F), or TIA1 (G and H). N.r. indicates cells that lack RLBs (G3BP1 foci) and did not reduce cytoplasmic poly(A)+ RNA levels, indicative of these cells not activating RNase L in response to poly(I:C), thus serving as internal controls. Statistical significance was determined using a one-way ANOVA with Tukey’s HSD. * is p < 0.05, ** is p < 0.01, *** is p < 0.001.
Fig 4.
RNase L activation results in alterations to alternative splicing.
(A) Venn diagram comparing significant ΔPSI values in RL KO vs WT cells treated with poly(I:C) and WT control vs WT poly(I:C) treated cells. (B) List of genes significant in both comparisons from panel A. RBPs known to regulate splicing are shown in bold. (C) Validation of splicing events by low cycle radiolabeled RT-PCR. The red asterisk in the PTBP1 gel is a nonspecific product. Bar graphs show quantification of four biological replicates. * is p < 0.05, ** is p < 0.01, *** is p < 0.001, *** is p < 0.0001. Holm-Sidak was used to correct for multiple t-tests using an alpha of 0.05.
Fig 5.
RNase L activation results in alterations to host RNA processing.
(A) Distribution of intron/exon ratios of host RNAs in WT and RL-KO cells following mock or poly(I:C) lipofection. (B) IGV traces mapping to an example gene. Intron retention and DoG formation is highlighted in WT cells following poly(I:C) lipofection. (C) Distribution of DoG1-5000bp/exon ratios of host RNAs in WT and RL-KO cells following mock or poly(I:C) lipofection.
Fig 6.
RNase L promotes DoG transcriptional read-through and intron retention in type I and type III interferon RNAs.
(A) IGV traces mapping to IFNB1. Below shows the regions targeted by smFISH probes. (B) IGV traces mapping to IFNL1. Below shows the regions targeted by smFISH probes. (C) smFISH for IFNB1-DoG sixteen hours post-lipofection of poly(I:C) in WT and RL-KO cells. The cells that induced IFNB1, as determined by smFISH for the CDS of IFNB1 (S4A and S4B Fig), are demarcated by a white line. IFNB1 DoG smFISH foci are quantified in WT an RL-KO cells in the graph below. Greater than seventeen IFNB-positive cells were analyzed from at least four fields of view. (D) similar to (C) but for (D) IFNL1-DoG-1 RNA sixteen hours post-lipofection of poly(I:C). Greater than seventeen IFNB-positive cells were analyzed from at least five fields of view. (E) Similar to (D) but for IFNL1-intron RNA twelve hours post-lipofection of poly(I:C). Orange arrows indicate transcripts at the putative genomic loci of IFNL1. Greater than eleven IFNB1-positive cells were counted from at least 5 fields of view. Staining and quantification of IFNL1 CDS is shown in S4C–S4E Fig. Statistical significance was determined using student’s t-test. *< 0.05, ** is p < 0.01, *** is p < 0.001.
Fig 7.
DoG RNA included on interferon-encoding mRNAs correlates with their nuclear retention.
(A) Co-smFISH for the CDS and DoG-1 regions of IFNB1 sixteen hours post-lipofection of poly(I:C). White arrows indicate cells that lack both the mRNA export block and abundant DoG RNA outside the site of transcription. Red arrows indicate cells that display the mRNA export block and contain abundant and disseminated DoG RNA. Yellow arrow indicates cells that display the export block but do not contain abundant DoG RNA. (B) Box plots displaying of the number of IFNB1-DoG foci localized to the nucleus (n) or cytoplasm (c) in WT or RL-KO cells as represented in (A). Greater than seventeen IFNB-positive cells were analyzed from at least four fields of view. (C) Scatter plot of the ratio (nucleus/cytoplasm) of IFNB1-CDS foci (x-axis) and nuclear IFNB1-DoG foci (y-axis) show positive correlation between DoG RNA and nuclear retention. (D) Scatter plots of the quantity of nuclear IFNB1-DoG foci (y-axis) and the quantity of nuclear IFNB1-CDS foci shows IFNB1 DoG RNA increase as the absolute number of IFNB1 CDS smFISH in the nucleus increases. (E) Co-smFISH for the CDS and DoG-1 regions of IFNL1 sixteen hours post-lipofection of poly(I:C). (F and G) Quantification of (F) nuclear IFNL1-DoG-1 foci or (G) IFNL1-CDS foci as represented in (E). Greater than seventeen IFNB-positive cells were analyzed from at least five fields of view. (H) Scatterplot of the ratio (nucleus/cytoplasm) of IFNL1-CDS foci (x-axis) and nuclear IFNL1-DoG foci (y-axis). Statistical significance was determined using student’s t-test. *< 0.05, ** is p < 0.01, *** is p < 0.001.
Fig 8.
RNase L promotes nuclear RBP influx and DoG transcriptional read-through of IFNB1 during SARS-CoV-2 infection.
(A) Immunofluorescence assay for PABP in WTACE2 and RL-KOACE2 A549 cells forty-eight hours post-infection with SARS-CoV-2 (MOI = 5) or mock-infected WT cells. To identify infected cells, smFISH for SARS-CoV-2 ORF1b mRNA was performed. (B) Scatter plot of mean intensity values for PABP staining in the nucleus (y-axis) or cytoplasm (x-axis) in mock or SARS-CoV-2-infected WTACE2 or RL-KOACE2 A549 cells as represented in (A). Dots represent individual cells. Between 20–44 cells were analyzed for group from at least three fields of view. (C) Box plot of the ratio (nucleus/cytoplasm) of the mean intensity of PABP as represented in (A). (D) smFISH for IFNB1-CDS and IFNB1-DoG forty-eight hours post-infection with SARS-CoV-2 in WTACE2 or RL-KOACE2 A549 cells (MOI = 5). (E) Quantification of IFNB1 DoG RNA in WT and RL-KO cells. Statistical significance was determined using student’s t-test.
Fig 9.
RNase L promotes nuclear RBP influx and DoG transcriptional read-through and intron retention during dengue virus infection.
(A) Immunofluorescence assay for PABP in WT and RL-KO A549 cells infected with dengue virus serotype 2 (DENV2) forty-eight hours post-infection (MOI = 1). smFISH for DENV2 mRNA was performed to identify infected cells. (B) Scatter plot of mean intensity values for PABP staining in WT or RL-KO cells that did or did not activate the dsRNA response based on RLB assembly (WT cells) or SG assembly (RL-KO) cells as represented in (A). (C) Box plot of the ratio (nucleus/cytoplasm) of the mean intensity of PABP (B). (D) smFISH for IFNL1-CDS, IFNL1-intron, and DENV mRNA in WT or RL-KO cells forty-eight hours post-infection with DENV2 (MOI = 1). (E) smFISH for IFNB1-CDS and IFNB1-DoG in WT or RL-KO cells forty-eight hours post-infection with DENV2 (MOI = 1). (F) Box plots quantifying the smFISH from individual cells (D and E). Twenty-five WT cells and eight RL-KO cells were analyzed from greater than five fields of view for IFNB-DoG-1. Fifteen WT cells and nine RL-KO cells were analyzed from greater than five fields of view. Statistical significance was determined using student’s t-test. *< 0.05, ** is p < 0.01, *** is p < 0.001.
Fig 10.
PABP accumulation in the nucleus is not sufficient for mRNA export block or DoG transcriptional read-through of IFNB1.
(A) Immunofluorescence for PABPC1 and smFISH for IFNB1 mRNA in A549 six hours after poly(I:C) transfection. Left panel shows a non-responsive cell that did not activate RNase L or induce IFNB1. The numbers in the top left corner are the number of nuclear IFNB smFISH foci and the percentage that those represent out of the total smFISH foci in the cell. Below the panels are intensity plots of PABPC1 signal as indicated by the white line. (B) Scatter plot of nuclear PABP intensity (y-axis) and the percent of IFNB1 mRNA in the nucleus (x-axis). (C) smFISH for IFNB1 mRNA in A549-RL-KO cells that express either mRuby2-PABPC1 or mRuby2-PABPC1 containing a nuclear retention signal (NRS). Quantification the percent of IFNB1 mRNA in the nucleus is shown in S12A Fig. (D) Western blot of XRN1 from A549 transfected with XRN1-targeting siRNAs (siXRN1) or negative control siRNAs (siNC). (E) IF for PABP and smFISH for GAPDH mRNA and IFNB1 mRNA in A549-WT cells transfected with either siNC or siXRN1. (F) IF for PABP and smFISH for IFNB1 CDS and DoG-1 in A549-WT cells transfected with either siNC or siXRN1. Quantification of both (E) and (F) are shown in S12B and S12C Fig.
Fig 11.
Model for RNase L-mediated regulation of nuclear RNA processing.
Activation of RNase L in response to viral double-stranded RNA (dsRNA) results decay of cytoplasmic RNAs. RNA-binding proteins (RBPs) disassociate from degraded cytoplasmic RNA and shuttle to the nucleus where they bind to nuclear RNA. The binding of RBPs to nuclear RNA alters RNA processing events. In addition, degraded cytoplasmic RNA may also antagonize nuclear RNA processing.