The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways

In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.


The EMBO Journal
Morgane Baldaccini et al

EV6
The EMBO Journal © The Author(s)

Figure
Figure EV3.PKR dimerization and/or catalytic activities do not change the infection outcome in NoDiceΔPKR FHA:CTRL cells.(A) Western blot analysis of DICER, p-PKR, PKR and CAPSID expression in SINV-GFP infected NoDiceΔPKR FHA:CTRL cells expressing MYC:EMPTY CTRL vector, MYC:PKR, MYC K296R or MYC:T451A at an MOI of 0.02 for 24 h.Alpha-Tubulin was used as loading control.Band intensity was quantified and normalized to Tubulin for three independent biological replicates, then represented as mean (+/− SD) under the corresponding lane; p-PKR = p-PKR/PKR quantification.(B) Mean (+/− SEM) of SINV-GFP viral titers in the same samples as in (A), infected at an MOI of 0.02 for 24 h (n = 3 biological replicates) from plaque assay quantification.Ordinary one-way ANOVA test with Dunnett's correction.ns: non-significant.Source data are available online for this figure.

Figure
Figure EV4.Dicer N1 cells express a different gene set from Dicer WT cells.(A) SINV-GFP genomic and subgenomic read distribution detected by RNA-sequencing analysis in NoDice FHA:DICER WT or N1 cells uninfected (Mock, gray) or infected at an MOI of 2 for 12 h (orange) or 0.02 for 24 h (purple).The viral reads number (mean +/− SEM) is normalized to the total mapped reads in each condition.TPM: transcripts per million.Ordinary two-way ANOVA test with Sidak's correction.****p < 0.0001; ns: non-significant.n = 3 biological replicates, error bars represent SEM.(B) Volcano plots showing for each gene the log2 fold change and adjusted p value (Wald test, DESeq2 package) between SINV-infected (MOI of 2 for 12 h) and mock NoDice FHA:DICER WT cells (left), or SINV-infected and mock NoDice FHA:DICER N1 cells (right).Each gene is marked as a dot (red: upregulated, blue: downregulated, gray: unchanged).The horizontal line denotes an adjusted p-value of 0.05 and the vertical ones the Log2 fold change cut-offs (−1 and 1) (n = 3 biological replicates).(C,D) GSEA enrichment plots for selected biological states and processes linked to inflammatory and antiviral pathways for mock NoDice FHA:DICER N1 vs mock NoDice FHA:DICER WT (C), or SINV-infected (MOI of 0.02 for 24 h) vs. mock NoDice FHA:DICER WT cells (D).Source data are available online for this figure.

Figure EV5 .
Figure EV5.Immune-related transcription factors activation is involved in the deregulation of Dicer N1 cells mRNAs.
(A-C) Histograms representing the cumulative probability of differentially expressed genes controlled by the transcription factors IRF2 (A), IRF3 (B) or STAT1 (C), plotted according to their Log2 fold change.The vertical lines stand for the Log2 fold change cut-offs (−1 and 1).The two-sample Kolmogorov-Smirnov test was used to assess whether each distribution was statistically different from the distribution of NoDice FHA:DICER WT cells infected with SINV vs. mock.p-values are indicated on each histogram.Black: WT SINV-002-24h vs WT MOCK; red: N1 SINV-002-24h vs N1 MOCK; blue: N1 MOCK vs WT MOCK.(D,E) Volcano plots for differentially expressed genes (DEGs) under the control of NF-kB/p65 (D) or STAT2 (E).Each gene is marked as a dot and plotted based on its log2 fold change and adjusted p values (Wald test, DESeq2 package) comparing SINV-infected (MOI of 0.02 for 24 h) vs mock NoDice FHA:DICER WT cells (left), or mock NoDice FHA:DICER WT vs mock NoDice FHA:DICER N1 cells (right).The horizontal line denotes an adjusted p-value of 0.05 and the vertical ones the Log2 fold change cut-offs (−1 and 1).n = 3 biological replicates.(F) Table of 15 representative upregulated NF-kB/p65 targets from the DEGs in the comparison mock NoDice FHA:DICER N1 vs mock NoDice FHA:DICER WT.Classification was made according to their Log2 fold change values.padj: adjusted p-value (Wald test, DESeq2 package).(G) RT-qPCR on selected DEGs controlled by either NF-kB/p65 or STAT2 in NoDice FHA:DICER WT and N1 cells infected or not with SINV-GFP at an MOI of 0.02 for 24 h.Mean (+/− SEM); n = 3 biological replicates.One-way ANOVA with Sidak's correction.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: non-significant.Source data are available online for this figure.