Abstract
The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.
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Acknowledgements
We thank J. Lim (Icahn School of Medicine at Mount Sinai) for Kunjin virus; M. Evans (Icahn School of Medicine at Mount Sinai) for Gag-Pol; P. Palese (Icahn School of Medicine at Mount Sinai) for comments, anti-PB2 and unpublished observations; A. Telenti for comments; A. García-Sastre (Icahn School of Medicine at Mount Sinai) for comments, RIG-I deficient A549 cells and unpublished observations; R. Roeder (Rockefeller University) for TAF4 reagents; M.F. Lavin (The University of Queensland) for anti-SETX; and the Genomic facility at Icahn and the Proteomics facilities at Rockefeller University. Supported by the computational resources and staff of the Department of Scientific Computing at the Icahn School of Medicine at Mount Sinai; the Canadian Institutes of Health Research (M.S.M.); the US National Institutes of Health (U19AI106754 to I.M.); and the National Institute of Allergy and Infectious Diseases Center of Excellence for Influenza Research and Surveillance (HHSN272201400008C to I.M. and H.v.B.).
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M.S.M., L.M.-G., A.M.M. and A.F.-S. performed experiments with reporters and viruses; M.S.M., P.M., J.M., O.J.B. and M.F.L. performed experiments involving mice; A.R., M.T., N.P.M., Z.P., J.N., C.M. and I.M. performed next-generation sequencing and biochemical experiments; J.S.Y.H. and H.v.B. performed data analysis for next-generation sequencing experiments; C.F.B. and A.R.F. performed fractionation experiments; A.M.M., S.H., C.B., J.A.T.Y., A.F.-S., O.J.B. and M.F.L. provided materials and insights in experimental procedures; and H.v.B. and I.M. supervised the project and wrote the paper with feedback from coauthors.
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Supplementary Figure 1 Control experiments for Figure 1.
(a) Localization of SETX in untreated or PR8ΔNS1 virus infected A549 cells (4hours). Nuclear (DAPI) and Tubulin staining are shown. SETX antibody specificity was validated using human fibroblast cells proficient (WT) or deficient for SETX (SETX deficient). Shown are representative images from one of two independent experiments. Scale bars represent 50μm. (b) SETX co-elutes with high molecular weight complexes. Lysates from uninfected or PR8ΔNS1 infected A549 cells were subjected to glycerol gradient sedimentation and the presence of SETX, RNAPII or the Influenza A RNA polymerase PB2 subunit in sequential fractions revealed through immunoblotting. LARP7 is a control for elution in high molecular weight complexes. (c). Co-immunoprecipitation of RNAPII and SETX in the absence or presence of RNaseA. (d). Western blot analysis of SETX depletion in siSETX cells relative to siCtrl and untreated (No Si) condition prior to and after infection with PR8ΔNS1 virus. (e) The expression levels of 12 housekeeping genes were determined by microarray analysis of un-transfected (No Si), siSETX- or siCtrl- transfected cells under basal conditions (without infection). Results for individual probesets are shown for genes that are represented by multiple probesets on the microarray. A full list of genes and their expression in the experimental condition tested can also be found in Supplementary Table 1. (f) Western blot analysis of XRN2 depletion in siXRN2-treated cells relative to siCtrl and untreated (No Si) conditions prior to and after infection with PR8ΔNS1 virus. (g) Heatmap showing differentially expressed genes (Fold change relative to siCtrl >1.5, p < 0.01; ANOVA with post-hoc TUKEY test) in un-transfected cells (No si) or XRN2 siRNA (siXRN2) transfected cells as compared to control non-targeting siRNA transfected cells (siCtrl) at an early (4 hours) time point post-infection. (h). The expression levels of 12 housekeeping genes were determined by microarray analysis of un-transfected (No Si), siXRN2 or siCtrl transfected cells under basal conditions (without infection). Results for individual probesets are shown for genes that are represented by multiple probesets on the microarray. A full list of genes and their expression in the experimental condition tested can also be found in Supplementary Table 2. (i). Protein levels of SETX and phosphorylated IRF3 (pIRF3) were measured in siCtrl and siSETX cells left untreated or infected with PR8∆NS1 virus. Tubulin protein levels were used as a loading control. (j and k) Expression of virus-derived messenger RNA (j), and viral genomic RNA (k) from SETX siRNA transfected (siSETX, white bars), control siRNA transfected (siCtrl, Gray bars) and un-transfected (No si, black bars) cells infected for 4 hours with the PR8∆NS1 virus. Data shown are from three experiments (e-f, g-h and j-k; with the mean and s.d shown for j-k). A representative blot of two independent experiments (b and c) or blots of pooled extracts from two independent experiments are shown (d, f).
Supplementary Figure 2 GRO-seq controls.
(a) Meta-analysis of GRO-seq signals across all RefSeq genes >1kb in A549 cells upon infection with influenza PR8ΔNS1, before (dashed lines) and 4 hours after infection (solid lines) in siCtrl (blue) and siSETX (red) treated cells. Y axis indicates the geometric mean coverage per 5 million reads. (b) Travelling ratios (TR) of all active genes for the conditions indicated in (a). TRs were calculated as the average number of tags per bp in the promoter-proximal region (30 to +300 bp around TSS) divided by that on the gene body (+300 bp to the end of the gene). The number of active genes identified in each condition is indicated between brackets. (c) GRO-seq read coverage for the RPL10 gene, showing no changes in siCtrl over siSETX conditions. Y-axis indicates base coverage per 5 million reads. (d) Overview of the fraction of GRO-seq reads mapping to genic (exons, introns and antisense) and intergenic regions. (e) Expression of IFNB1 and IFIT1 mRNA levels in siSETX and siCtrl A549 cells in the presence of flavopiridol (blue bars), DMSO vehicle control (red bars), or untreated control cells (gray bars). Values represent the mean and standard deviation of three independent experiments; Asterisks (*and **) indicate t-test p-values < 0.05 and < 0.005, respectively.
Supplementary Figure 3 SETX has ATP-dependent helicase activity and affects the induction of transcription start site-associated short RNA at gene promoters.
(a) Agilent bioanalyzer gel-like image of a helicase assay, showing a decrease in relative intensity of the DNA/RNA hybrid band (top) vs. the dissociated simplex band (bottom), as a function of the concentration of SETX. Samples were analyzed using the RNA 6000 Pico Kit. Hybrid-only and RNase treated lanes are shown as controls. (b) Log2 ratio of quantified simplex/hybrid signals (time-corrected peak area) as shown in (a). (c). Same as in a., with NS3 Helicase from hepatitis C virus, wild type SETX (wt), SETX ATPase mutant (ATP MUT) and RNA-binding Mutant (RNA MUT). (d) Log2 ratios of quantified simplex/hybrid signals (time-corrected peak area) as shown in (c). (e) Colloidal Coomassie staining of 3-8% Tris-Acetate gel of wildtype SETX (WT), ATPase (ATP MUT) RNA binding mutant (RNA MUT) purified from HEK 293 cells and used in (a) and (c), as indicated. Co-precipitation of exosome components with SETX (Supplementary. Fig.4 and Supplementary Table 4) does not exclude the possibility that wildtype SETX activity could be modulated by residual exosome activity. (f) Overview of whole-genome short-RNA-Seq data (30-70nt fragments) generated for cells overexpressing wildtype (WT) or mutant SETX (ATPase mutant: MUT1 and RNA binding mutant: MUT2) induced with daIRF3. Raw reads were first mapped against a reference set of sn(o)RNA, tRNA and rRNA transcripts that are typically found in the same size range, and the remainder were mapped against the hg19 human reference genome using Bowtie (v0.12.9). Reads mapping to more than one location in the genome were discarded from analysis. TSS-proximal reads were defined as reads mapping within 300 bp of a transcription start site (GENCODE v18). The table lists the read counts obtained at each stage of data processing. (g) Scatterplots comparing normalized TSS-proximal short RNA-Seq read counts (sense strand only) in cells over-expressing either WT or mutant SETX, after induction with daIRF3. Correlation coefficients (r) are indicated for each comparison. (h) Short RNA-Seq profiles (blue) of antiviral induced genes IFIT1, IFIT2 and IFIT3 (top) and GAPDH and ACTG1 controls (bottom). Short-read track scales correspond to the normalized read coverage per 25,000 uniquely mapped genomic reads in each dataset. Promoter-proximal TSS areas are highlighted in grey. No mapped reads were found at the IFNB1 locus in the SETX mutant or WT profiles (data not shown), presumably due to limitations in read depth. Top tracks (black) indicate the frequency of initiation site usage across each gene, derived from 5’-end RNA-sequencing tags of capped RNAs >300 nt obtained from the same cells. Short-read tags align exactly to the preferred transcription initiation site of each gene.
Supplementary Figure 4 Interaction between SETX and TAF4.
(a) Validation of the IP-MS/MS via immunoprecipitation of SETX and immunoblotting for the indicated endogenous proteins in cells expressing Flag-tagged SETX. Input (Inp) and the IgG control IP are shown. (b) Association of TAF4 with SETX. FLAG tagged wildtype SETX and the SETX ATPase (M1) and RNA binding mutants (M2) were immobilized on FLAG-beads and probed with cell extracts (prepared via Dignam-Roeder method). After 4h binding in BC100 (20 mM Tris–HCl pH 7.9, 20% glycerol, 0.1 mM EDTA, 150 mM KCl), 4 washes in BC150 + 0.1% NP40 in the presence of absence of RNAse, the immunoprecipitated (IP) material was subjected to western blotting with the anti-Flag antibody (to control for equal levels of SETX protein bound to beads) and the TAF4 antibody (detecting endogenous TAF4). UT: untreated control; EV: empty vector control (c) Association of SETX with TAF4. As in (b) but with immobilized wild type TAF4 (WT) and TAF4 ΔTAFH mutant (Δ). After 4h of binding in BC100 followed by 4 washes in a BC150 + 0.1% NP40, the IP material was subjected to western blotting with the anti-Flag antibody (to control for equal level of TAF4 proteins on beads) and SETX antibody (detecting endogenous SETX). For a-c, representative blots of pooled extracts from biological replicates or two independent experiments are shown.
Supplementary Figure 5 SETX deficiency inhibits viral biogenesis.
(a and b). Growth of influenza PR8 (a) and PR8ΔNS1 (b) viruses in A549 cells untreated or treated with control non-targeting siRNA (siCtrl) or SETX-specific siRNA (siSETX). Cells that were not siRNA treated (No si) were also included as controls. Viral titers were determined by plaque assays on MDCK cells (for PR8) or MDCK cells stably expressing NS1 (for PR8ΔNS1). (c) Growth of influenza PR8 virus in siSETX-, siCtrl-treated or untreated (No si) A549 cells that stably express a shRNA targeting DDX58 (RIGI). (d) Western blot showing reconstitution of SETX-deficient cells with FLAG-tagged wildtype SETX and the SETX ATPase mutant (Mut). (e) Quantification of secreted cytokine levels in SETX-deficient and control human fibroblasts 4 hours post infection with PR8ΔNS1. Note that although amounts of IP-10 levels are easily detectable via ELISA, IFNA2 levels were much lower and could result from an IFNB1-dependent induction of IFNA2. (f) Growth of influenza PR8 virus in SETX-deficient and WT human fibroblasts. Viral titers were determined by plaque assay on MDCK cells. Note that final viral titers are similar to A549 cells but the kinetics of viral growth differs between the two cell types. (g) Expression of IFNB1, IFIT1, CXCL9 and SETX in SETX-deficient and wild-type control fibroblasts 4 hours post-infection with West Nile Virus (Kunjin strain; WNV KUNV). **P < 0.005 (t-test). (h) KUNV growth in wildtype and SETX-deficient primary human fibroblast cells. Viral titers were determined by focus-forming assay on Vero cells. (i). Western blot of SETX, RIG-I, IFIT1 and Tubulin proteins isolated from human glial cells after 0, 4, 12 and 24 hours post-infection with PR8∆NS1. Data are from three independent experiments (mean and s.e.m in a-b and f; s.d in c, e, g and h). Representative western blots are shown in d and i. (j) Model of RNAPII- and activator-dependent recruitment of SETX at antiviral loci during infection. Promoter proximal termination is elicited via SETX translocation over nascent transcript as a result of sequence-specific RNA recognition (upper panel) or via helicase activity and R-loop resolution as a result of structure-specific recognition of RNA/DNA hybrids at promoters (lower panel).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 (PDF 3219 kb)
Supplementary Table 1
List of probesets that are affected by siSETX treatment in uninfected cells. (XLS 238 kb)
Supplementary Table 2
List of probesets that are affected by siXRN2 treatment in A549 cells in uninfected cells (XLSX 18 kb)
Supplementary Table 3
List of genes with significantly changed levels of engaged RNAPII in human A549 cells after 4 hours of infection with influenza PR8ΔNS1. (XLS 293 kb)
Supplementary Table 4
Shared Proteins (XLSX 32 kb)
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Miller, M., Rialdi, A., Ho, J. et al. Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis. Nat Immunol 16, 485–494 (2015). https://doi.org/10.1038/ni.3132
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DOI: https://doi.org/10.1038/ni.3132
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