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• W3111682023 abstract "•cGAS is essential for restricting RNA virus infection•Nuclear cGAS recruits Prmt5 upon virus infection•Prmt5 regulates histone arginine modification of IFN promoters•Prmt5 deficiency impairs the innate antiviral responses Cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS), upon sensing cytosolic DNA, catalyzes the production of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which activates STING-TBK1-IRF3 signaling. cGAS is also present in the nucleus, but the relevant nuclear function or mechanism remains largely unknown. Here, we report that nuclear cGAS is indispensable for inducing cytokines and chemokines triggered by RNA/DNA viruses. Unexpectedly, the DNA-binding/nucleotidyltransferase activity of cGAS is dispensable for RNA-virus-induced genes expression. cGAS deficiency does not affect the phosphorylation, dimerization, or nuclear translocation of IRF3 induced by double-stranded RNA (dsRNA). Mechanistically, nuclear-localized cGAS interacts with protein arginine methyltransferase 5 (Prmt5), which catalyzes the symmetric dimethylation of histone H3 arginine 2 at Ifnb and Ifna4 promoters, thus facilitating the access of IRF3. Deficiency of Prmt5 or disrupting its catalytic activity suppresses the production of type I interferons (IFNs), impairing the host defenses against RNA/DNA virus infections. Taken together, our study uncovers a non-canonical function of nuclear-localized cGAS in innate immunity via regulating histone arginine modification. Cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS), upon sensing cytosolic DNA, catalyzes the production of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which activates STING-TBK1-IRF3 signaling. cGAS is also present in the nucleus, but the relevant nuclear function or mechanism remains largely unknown. Here, we report that nuclear cGAS is indispensable for inducing cytokines and chemokines triggered by RNA/DNA viruses. Unexpectedly, the DNA-binding/nucleotidyltransferase activity of cGAS is dispensable for RNA-virus-induced genes expression. cGAS deficiency does not affect the phosphorylation, dimerization, or nuclear translocation of IRF3 induced by double-stranded RNA (dsRNA). Mechanistically, nuclear-localized cGAS interacts with protein arginine methyltransferase 5 (Prmt5), which catalyzes the symmetric dimethylation of histone H3 arginine 2 at Ifnb and Ifna4 promoters, thus facilitating the access of IRF3. Deficiency of Prmt5 or disrupting its catalytic activity suppresses the production of type I interferons (IFNs), impairing the host defenses against RNA/DNA virus infections. Taken together, our study uncovers a non-canonical function of nuclear-localized cGAS in innate immunity via regulating histone arginine modification. Sensing and responding to non-self-nucleic acids is a powerful host-defense strategy against microbial invasions in vertebrates. Toll-like receptors (TLR3, TLR7, and TLR8) recognize viral RNAs in the endosome (Alexopoulou et al., 2001Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4592) Google Scholar; Heil et al., 2004Heil F. Hemmi H. Hochrein H. Ampenberger F. Kirschning C. Akira S. Lipford G. Wagner H. Bauer S. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.Science. 2004; 303: 1526-1529Crossref PubMed Scopus (2787) Google Scholar), whereas RLR receptors (RIG-I and MDA5) detect viral RNAs in the cytosol (Yoneyama et al., 2004Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (2874) Google Scholar). All of these microbial RNA sensors converge to activate TBK1 kinase and, subsequently, phosphorylate the transcription factor IRF3, ultimately leading to the induction of type I interferons (IFNs) and pro-inflammatory cytokines. In parallel, several DNA sensors have been identified and characterized (Paludan, 2015Paludan S.R. Activation and regulation of DNA-driven immune responses.Microbiol. Mol. Biol. Rev. 2015; 79: 225-241Crossref PubMed Scopus (67) Google Scholar). cGAS is well established as a general cytosolic DNA sensor, whose catalytic pocket is structurally rearranged upon binding double-stranded DNA (dsDNA) and converts ATP and guanosine triphosphate (GTP) into cyclic guanosine monophosphate (GMP)-AMP (2′3′-cGAMP) (Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339: 786-791Crossref PubMed Scopus (1850) Google Scholar). The resulting secondary messenger binds to and activates the receptor stimulator of interferon genes (STINGs) on endoplasmic reticulum (ER) membranes (Wu et al., 2013Wu J. Sun L. Chen X. Du F. Shi H. Chen C. Chen Z.J. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA.Science. 2013; 339: 826-830Crossref PubMed Scopus (1001) Google Scholar), which in turn, translocates from ER to Golgi and recruits TBK1 and IRF3. Phosphorylated IRF3 dimerizes and translocates to the nucleus, driving the expression of type I IFNs and IFN-stimulated genes (ISGs) (Ishikawa and Barber, 2008Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (1523) Google Scholar). Recent reports are expanding the cGAS functions beyond its canonical roles in anti-DNA microbial immunity. cGAS-STING signaling triggers potent natural antitumor immunity, contributing to the effect of immune-checkpoint-blockade therapy (Deng et al., 2014Deng L. Liang H. Xu M. Yang X. Burnette B. Arina A. Li X.D. Mauceri H. Beckett M. Darga T. et al.STING-dependent cytosolic DNA sensing promotes radiation-induced type I interferon-dependent antitumor immunity in immunogenic tumors.Immunity. 2014; 41: 843-852Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar; Wang et al., 2017Wang H. Hu S. Chen X. Shi H. Chen C. Sun L. Chen Z.J. cGAS is essential for the antitumor effect of immune checkpoint blockade.Proc. Natl. Acad. Sci. USA. 2017; 114: 1637-1642Crossref PubMed Scopus (195) Google Scholar). cGAS can sense extranuclear self-DNA accumulated in aged cells, facilitating cellular senescence (Glück et al., 2017Glück S. Guey B. Gulen M.F. Wolter K. Kang T.W. Schmacke N.A. Bridgeman A. Rehwinkel J. Zender L. Ablasser A. Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence.Nat. Cell Biol. 2017; 19: 1061-1070Crossref PubMed Scopus (329) Google Scholar; Lan et al., 2019Lan Y.Y. Heather J.M. Eisenhaure T. Garris C.S. Lieb D. Raychowdhury R. Hacohen N. Extranuclear DNA accumulates in aged cells and contributes to senescence and inflammation.Aging Cell. 2019; 18: e12901Crossref PubMed Scopus (37) Google Scholar). During mitotic arrest, cGAS can promote apoptosis through mitochondrial outer-membrane permeabilization (Zierhut et al., 2019Zierhut C. Yamaguchi N. Paredes M. Luo J.D. Carroll T. Funabiki H. The cytoplasmic DNA sensor cGAS promotes mitotic cell death.Cell. 2019; 178: 302-315.e23Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Notably, the nuclear localization of cGAS and the underlying functions and consequences have captured a great deal of attention. Nuclear cGAS can detect DNA derived from HIV, which brings its nucleic acids to the nucleus (Lahaye et al., 2018Lahaye X. Gentili M. Silvin A. Conrad C. Picard L. Jouve M. Zueva E. Maurin M. Nadalin F. Knott G.J. et al.NONO detects the nuclear HIV capsid to promote cGAS-mediated innate immune activation.Cell. 2018; 175: 488-501.e22Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A circular RNA (cia-cGAS) blocks nuclear the ability of cGAS to sense self-DNA, thus maintaining host homeostasis (Xia et al., 2018Xia P. Wang S. Ye B. Du Y. Li C. Xiong Z. Qu Y. Fan Z. A circular RNA protects dormant hematopoietic stem cells from DNA sensor cGAS-mediated exhaustion.Immunity. 2018; 48: 688-701.e7Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Nuclear cGAS promotes tumor growth by suppressing DNA repair, which is independent of STING (Liu et al., 2018Liu H. Zhang H. Wu X. Ma D. Wu J. Wang L. Jiang Y. Fei Y. Zhu C. Tan R. et al.Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.Nature. 2018; 563: 131-136Crossref PubMed Scopus (175) Google Scholar). It remains intriguing to explore the DNA-sensor-independent functions of cGAS in the nucleus. Paradoxically, lentiviral-driven cGAS expression induces an antiviral program against several RNA viruses in vitro, and genetic ablation of murine Cgas indicates it is required to control RNA virus infection in vivo (Schoggins et al., 2014Schoggins J.W. MacDuff D.A. Imanaka N. Gainey M.D. Shrestha B. Eitson J.L. Mar K.B. Richardson R.B. Ratushny A.V. Litvak V. et al.Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity.Nature. 2014; 505: 691-695Crossref PubMed Scopus (501) Google Scholar). dsRNA was reported to bind to cGAS, but that interaction per se is not sufficient to stimulate the cGAS activity (Civril et al., 2013Civril F. Deimling T. de Oliveira Mann C.C. Ablasser A. Moldt M. Witte G. Hornung V. Hopfner K.P. Structural mechanism of cytosolic DNA sensing by cGAS.Nature. 2013; 498: 332-337Crossref PubMed Scopus (343) Google Scholar). Given the nuclear localization of cGAS, we wondered whether cGAS had any non-canonical antiviral functions, distinct from its role as a DNA sensor. In this study, we characterized nuclear cGAS to be indispensable for the host defense against RNA viruses, which was independent of its DNA-binding or nucleotidyltransferase activity. Specifically, nuclear cGAS recruits protein arginine methyltransferase 5 (Prmt5) and facilitates the binding of Prmt5 to the promoters and enhancers of Ifnb and Ifna4, thus enhancing type I IFN production by symmetric dimethylation of histone H3 arginine 2 (H3R2me2s) modification upon virus infection. Our study uncovers a non-canonical function of nuclear cGAS in innate immunity. Previous study has shown that induction of Ifnb and ISG by agonists of RIG-I-like receptors and RNA-activated Toll-like receptors was impaired in cGAS-knockout cells (Schoggins et al., 2014Schoggins J.W. MacDuff D.A. Imanaka N. Gainey M.D. Shrestha B. Eitson J.L. Mar K.B. Richardson R.B. Ratushny A.V. Litvak V. et al.Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity.Nature. 2014; 505: 691-695Crossref PubMed Scopus (501) Google Scholar). To explore the potential role of cGAS in preventing RNA virus infection, we employed two different pairs of small interfering RNA (siRNA) to knock down cGAS in mouse embryonic fibroblasts (MEFs) (Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339: 786-791Crossref PubMed Scopus (1850) Google Scholar) and then measured the induction of type-I IFNs and downstream genes via qPCR (quantitative PCR) upon various stimuli. As expected, both siRNA oligos significantly inhibited the IRF3-responsive genes (Ifnb, Ifna4, and Cxcl10), expression stimulated by exogenous DNA (immuno-stimulatory DNA [ISD], a cytosolic DNA mimic) (Figure 1A; Table S1), and the DNA virus HSV-1 (Herpes simplex virus 1) (Figure 1B). Surprisingly, knockdown of cGAS also drastically decreased the induction of the same set of genes triggered by either poly(I:C) (a ligand of the RNA sensor) (Figure 1C) or the RNA virus SeV (Sendai virus) (Figure 1D). Unexpectedly, the production of IRF3-responsive genes in cGAS-knockdown cells was markedly attenuated when stimulated with cGAMP (2′3′-cGAMP, a ligand that directly activates STING) (Figure 1E), which suggested that cGAS could function downstream of STING in addition to its pivotal role as a cytosolic DNA sensor. To substantiate that finding, silencing cGAS impaired the IFN-β protein secretion triggered by ISD, poly(I:C), or cGAMP, as measured by ELISA (enzyme-linked immunosorbent assay) (Figure 1F). These observations also held true in the murine fibrosarcoma cell line L929 and human HeLa cells (Figures S1A–S1H). To check the antiviral activity of cGAS, MEFs were, respectively, preincubated with culture supernatants from the ISD- or poly(I:C)-treated wild-type (WT) or cGAS-silenced MEFs, followed by virus infections. Cells pretreated with culture supernatants from either ISD- or poly(I:C)-treated, cGAS-silenced MEFs were more vulnerable to HSV-1 or VSV (vesicular stomatitis virus, an RNA virus) infections (Figure 1G). Consistently, knockdown of cGAS in L929 cells facilitated the replication of HSV-1-GFP and VSV-GFP, as evidenced by increased virus titers (Figure 1H), by stronger GFP-positive signals, and by increased number of GFP-positive cells (Figure 1I). Taken together, these data strongly indicate that cGAS has a non-canonical role in antiviral responses. We further obtained the cGAS knockout L929 and HeLa cell lines by CRISPR-Cas9-mediated targeting. The absence of cGAS suppressed poly(I:C) or HT-DNA induced IRF3-responsive genes expression in both L929 and HeLa cells, which was confirmed in multiple knockout clones (Figures S2A–S2C). Similarly, Cgas-deficient L929s showed defects in IRF3-responsive gene induction in response to RNA viruses (SeV, VSV, NDV, and Zika) and HSV-1 infection (Figures 2A and 2B ). Moreover, re-expression of WT cGAS restored the deficiency of poly(I:C)-induced Ifnb mRNA in Cgas-deficient L929s. It is reported that the cGAS K3A (K335/372/382A) triple-point mutant is deprived of its DNA binding affinity (Cui et al., 2017Cui Y. Yu H. Zheng X. Peng R. Wang Q. Zhou Y. Wang R. Wang J. Qu B. Shen N. et al.SENP7 Potentiates cGAS Activation by Relieving SUMO-Mediated Inhibition of Cytosolic DNA Sensing.PLoS Pathog. 2017; 13: e1006156Crossref PubMed Scopus (41) Google Scholar), whereas the cGAS D307A, E211A, or D213A single-point mutants lack its catalytic activity (Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cyclic [G(2’,5’)pA(3’,5’)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar; Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339: 786-791Crossref PubMed Scopus (1850) Google Scholar). Intriguingly, all of the above cGAS mutants showed rescue effects as WT cGAS (Figures 2C and S2D). Similarly, reconstitution with cGAS K384Q (a human plasmid deprived of its catalytic activity) (Dai et al., 2019Dai J. Huang Y.J. He X. Zhao M. Wang X. Liu Z.S. Xue W. Cai H. Zhan X.Y. Huang S.Y. et al.Acetylation blocks cGAS activity and inhibits self-DNA-induced autoimmunity.Cell. 2019; 176: 1447-1460.e14Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) in cGAS-deficient HeLa cells also rescued the poly(I:C)-triggered expression of IFNB comparable to that of a WT cGAS plasmid (Figure S2E). Recent studies highlighted that cGAS is present in both the cytoplasm and nucleus. We confirmed those observations in different cell lines, including L929, MEF, and HeLa cells, which led us to speculate whether nuclear-localized cGAS might mediate this non-canonical function. It was revealed that the second nuclear localization sequence (NLS2) had a possible role in regulating the nuclear translocation of cGAS, whereas the DNA sensing function of cGAS was dispensable for its nuclear localization (Liu et al., 2018Liu H. Zhang H. Wu X. Ma D. Wu J. Wang L. Jiang Y. Fei Y. Zhu C. Tan R. et al.Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.Nature. 2018; 563: 131-136Crossref PubMed Scopus (175) Google Scholar). In contrast to WT cGAS, ectopically expressed cGAS δNLS2 mutants failed to activate the IFN-β-luciferase reporter (Figure 2D), suggesting a potential function of nuclear cGAS in antiviral immunity. Subcellular fractionation analysis confirmed that deletion of the second nuclear localization sequence of cGAS decreased the nuclear localization of cGAS (Figures 2E and 2F). Moreover, reconstitution with cGAS δLS2 in cGAS-deficient HeLa cells failed to rescue poly(I:C)-induced Ifnb expression as compared with that of WT cGAS (Figure 2G). Taken together, these results indicate that nuclear cGAS functions non-canonically in innate immune response to microbial RNAs, which is independent of its DNA sensing function and catalytic activity. It is reported that some RNA viruses infection could lead to mitochondrial stress and subsequent Bax/Bak macropores mediated-mitochondrial DNA (mtDNA) release, which triggers cGAS-STING pathway (McArthur et al., 2018McArthur K. Whitehead L.W. Heddleston J.M. Li L. Padman B.S. Oorschot V. Geoghegan N.D. Chappaz S. Davidson S. San Chin H. et al.BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.Science. 2018; 359: eaao6057Crossref Scopus (220) Google Scholar; Riley et al., 2018Riley J.S. Quarato G. Cloix C. Lopez J. O’Prey J. Pearson M. Chapman J. Sesaki H. Carlin L.M. Passos J.F. et al.Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis.EMBO J. 2018; 37: e99238Crossref PubMed Scopus (95) Google Scholar; Tumilasci et al., 2008Tumilasci V.F. Olière S. Nguyên T.L. Shamy A. Bell J. Hiscott J. Targeting the apoptotic pathway with BCL-2 inhibitors sensitizes primary chronic lymphocytic leukemia cells to vesicular stomatitis virus-induced oncolysis.J. Virol. 2008; 82: 8487-8499Crossref PubMed Scopus (40) Google Scholar). To test whether mtDNA release contributes to the effect of cGAS on the host response to microbial RNAs, we challenged cGAS-deficient Bax−/−Bak−/− MEFs with RNA viruses (SeV and VSV) or the DNA virus HSV-1. Silence of cGAS greatly suppressed the Ifnb induction in Bax−/−Bak−/− MEFs (Figure 2H), suggesting cGAS-mediated innate immune defense against RNA viruses was independent of mtDNA-cGAS-STING signaling. Moreover, knockdown of cGAS in Sting-deficient MEFs also impaired poly(I:C) or SeV-triggered IRF3-responsive genes expression (Figure 2I). Similarly, crippled cGAS expression in STING-deficient HeLa cells decreased the same gene expression when transfected with poly(I:C) (Figure 2J). These data collectively demonstrate cGAS-mediated innate defense against RNAs is independent of mtDNA-cGAS-STING signaling. As expected, the phosphorylation of TBK1 and IRF3, as well as IRF3 dimerization, were comparable in WT and Cgas-deficient L929s when stimulated with poly(I:C). By contrast, cGAS deficiency led to a marked decrease in the phosphorylation of TBK1 and IRF3, as well as IRF3 dimerization in response to poly(dA:dT) stimulation (Figure 2K). Similar phenomena were also observed in primary bone-marrow-derived macrophages (BMDMs) from WT and Cgas−/− mice (Figure S2F). In addition, cGAS depletion had no effect on the nuclear translocation of IRF3 in response to poly(I:C) treatment (Figures 2L and 2M). These data clearly reveal that cGAS functions downstream of IRF3 in response to RNA virus infection. We obtained cGAS knockout (Cgas−/−) mice by CRISPR-Cas9 technology (Figures S3A and S3B) (see methods part for details). Western blot analysis of proteins from the spleen, kidney, liver, lung, and heart tissues of wild-type and knockout mice confirmed the specific and efficient depletion of cGAS in mice (Figure S3C). The Cgas−/− mice bred in normal Mendelian ratios and displayed no aberrant fertility or developmental defects (Figure S3D). Primary bone marrow-derived macrophages (BMDMs) from WT or Cgas−/− mice were stimulated respectively with poly(I:C), ISD, poly(dA:dT) or cGAMP. As expected, the IRF3-responsive genes were drastically downregulated in Cgas−/− primary BMDMs after stimulation with poly(I:C), dsDNA mimics, or cGAMP (Figure 3A). Consistently, the IFN-β secretion was much lower in Cgas−/− than in WT BMDMs upon exposure to these stimuli (Figure 3B). Moreover, cGAS depletion also led to the defects in type I IFNs induction triggered by different doses of RNA viruses (SeV and VSV) for different times (Figures 3C, S3E, and S3F). In contrast, knocking out cGAS did not affect the nuclear factor κB (NF-κB) signaling triggered by either lipopolysaccharide (LPS) or tumor-necrosis factor α (TNF-α) (Figures 3D and S3G). WT or Cgas−/− mice were injected intravenously with VSV. The expression of Ifnb mRNA was significantly lower in the spleens and lungs of Cgas−/− mice than in those of WT mice (Figure 3E). In contrast, the levels of VSV-specific mRNAs and VSV-G proteins were much higher in the spleens and lungs of Cgas−/− mice compared with those of WT mice (Figures 3F and 3G). Moreover, the lungs showed more-severe injury in Cgas−/− mice than they did these in WT mice after VSV infection, as illustrated by hematoxylin and eosin (H&E) staining (Figure 3H). The concentrations of serum IFN-β were markedly lower in Cgas−/− mice than they were in WT mice (Figure 3I). Consequently, the Cgas−/− mice were much more vulnerable to VSV infection compared with WT mice because all Cgas−/− mice died within 3 days, whereas 80% of WT mice remained alive until 5 days after VSV infection (Figure 3J). To further elucidate the function of nuclear cGAS, we performed proteomic analysis by ectopically expressing Flag-tagged cGAS in L929s (Figure S4A). The Prmt5 was unequivocally identified (Figure 4A). We further confirmed the interaction between cGAS and Prmt5 by coimmunoprecipitation (coIP; Figure S4B). cGAS specifically interacted with Prmt5, but not with other members of that family (Figure 4B). We mapped the region of cGAS (amino acids 241–380) and the region of Prmt5 (amino acids 241–360) to mediate this interaction (Figures 4C and 4D). Confocal microscope imaging confirmed cGAS co-localized with Prmt5 endogenously in nucleus upon poly(I:C) stimulation (Figure 4E). Furthermore, coIP of subcellular fractionation confirmed cGAS interacted robustly with Prmt5 endogenously in the nucleus but not in the cytoplasm when stimulated with either poly(I:C) (Figure 4F) or VSV (Figure S4C). Similar endogenous association was also observed when stimulated with herring testis DNA (HT-DNA; Figure S4D). Such association disappeared in Cgas-deficient cells (Figures S4E and S4F), demonstrating the specificity of the endogenous interaction between cGAS and Prmt5. Previous study showed that Prmt5 catalyzes the reaction of transferring methyl groups from S-adenosylmethionine (SAM) to the guanidine nitrogen of its substrate protein arginine (Blanc and Richard, 2017Blanc R.S. Richard S. Arginine methylation: the coming of age.Mol. Cell. 2017; 65: 8-24Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Prmt5 predominantly localizes in the cytoplasm and its translocation into nucleus plays an important role in controlling early embryos development and tumor progression by reprogramming genes expression (Ancelin et al., 2006Ancelin K. Lange U.C. Hajkova P. Schneider R. Bannister A.J. Kouzarides T. Surani M.A. Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells.Nat. Cell Biol. 2006; 8: 623-630Crossref PubMed Scopus (352) Google Scholar; Hou et al., 2008Hou Z. Peng H. Ayyanathan K. Yan K.P. Langer E.M. Longmore G.D. Rauscher 3rd, F.J. The LIM protein AJUBA recruits protein arginine methyltransferase 5 to mediate SNAIL-dependent transcriptional repression.Mol. Cell. Biol. 2008; 28: 3198-3207Crossref PubMed Scopus (157) Google Scholar). Interestingly, we observed a marked nuclear translocation of Prmt5 upon stimulation with either poly(I:C) (Figure 4G) or HT-DNA (Figure S4G). Importantly, this nuclear translocation was abolished when depleting endogenous cGAS (Figure 4G). Together, these results indicate that nuclear cGAS recruits Prmt5 and facilitates its cytoplasm-to-nucleus translocation in response to foreign RNA or DNA challenges. To analyze the potential role of Prmt5 in antiviral innate immunity, we generated Prmt5-knockout L929 cells via CRISPR-Cas9 technology. We obtained three Prmt5-knockout-cell clones with different genomic RNAs (gRNAs) and validated the ablation of Prmt5 protein in those cells (Figure S5A). The absence of Prmt5 decreased the expression of type I IFNs in response to poly(I:C) or HT-DNA treatment, which was confirmed in multiple knockout clones (Figures S5B and S5C). As expected, Prmt5 deficiency drastically impaired the expression of IRF3 responsive genes (Ifnb, Ifna4, and Cxcl10) upon poly(I:C) transfection or RNA virus infection (VSV or Zika) (Figures 5A–5C). Consistently, knocking out Prmt5 markedly attenuated the same set of antiviral genes expression when stimulated with HT-DNA or HSV-1 (Figures S5D–S5F). In accordance, IFN-β secretion was remarkably decreased in Prmt5-deficient compared with WT L929s upon exposure to poly(I:C) and VSV (Figure 5D) or HT-DNA and HSV-1 (Figure S5G). We then explored whether Prmt5 regulated virus replication by challenging cells with GFP-expressing virus. As expected, Prmt5 silencing facilitated the replication of VSV-GFP (Figures 5E and 5F) as well as HSV-1-GFP (Figures S5H and S5I). Because Prmt5 catalyzes the symmetric dimethylation of arginine, we impaired its catalytic activity by using a specific and potent inhibitor, EPZ015666 (EPZ) (Chan-Penebre et al., 2015Chan-Penebre E. Kuplast K.G. Majer C.R. Boriack-Sjodin P.A. Wigle T.J. Johnston L.D. Rioux N. Munchhof M.J. Jin L. Jacques S.L. et al.A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.Nat. Chem. Biol. 2015; 11: 432-437Crossref PubMed Scopus (271) Google Scholar). Consistent with the observations in Prmt5 knockout cells, EPZ treatment significantly decreased the expression of IRF3-responsive genes (Ifnb, Ifna4, and Cxcl10) in a dose-dependent manner when stimulating cells with either poly(I:C) (Figure 5G) or poly(dA:dT) (Figure S5J). Similar phenomena were also observed in HeLa cells when treated with EPZ (Figure S5K). Collectively, these data establish that Prmt5 is crucial for promoting innate antiviral response. To further address the function of Prmt5 in cGAS-mediated innate antiviral response, we overexpressed cGAS in WT and Prmt5-deficient L929s, respectively. It was observed that ectopically expressed cGAS synergized poly(I:C)-triggered expression of IFNs in a dose-dependent manner in WT L929s, whereas such synergistic effects of cGAS diminished in Prmt5-deficient L929s (Figure 6A). Analogously, ectopically expressed Prmt5 synergized the poly(I:C)-triggered expression of IFNs in a dose-dependent manner in WT but not in Cgas-deficient L929s (Figure 6B). Consistently, reconstituted with cGAS WT, Cgas-deficient L929s could more robustly enhance the poly(I:C)-triggered expression of IFNs than could cGAS δ241–380, which could not interact with Prmt5 (Figure 6C). Likewise, the induction of type I IFNs by poly(I:C) was obviously increased in the Prmt5-deficient L929s when reconstituted with Prmt5 WT, but not with Prmt5 δ241–360 (Figure 6D). Additionally, it is notable that Prmt5 was expressed normally in cells lacking cGAS and vice versa (Figure S6A). Taken together, these data indicate that cGAS and Prmt5 act synergistically in the nucleus to promote antiviral genes expression. Next, we performed chromatin immunoprecipitation (ChIP) assays to monitor the presence of Prmt5 on the promoters of type I IFN genes and the potential effect of cGAS on that. Upon poly(I:C) stimulation, Prmt5 was observed at the enhancers and promoters of Ifnb and Ifna4 in WT L929s. Prmt5 was enriched at the Ifnb enhancer spanning the −466 to −590 bp upstream of the transcription start site (TSS) (Figure 6E) and at the Ifna4 enhancer spanning the −336 to −449 bp upstream of the TSS (Figure 6F). Notably, endogenous cGAS knockout dramatically decreased the presence of Prmt5 on the Ifnb enhancer (Figure 6E) and decreased the occupancy of Prmt5 on −336 to −449 bp of the Ifna4 enhancer (Figure 6F). These data demonstrate that Prmt5 could be recruited to the Ifnb enhancer in a cGAS-dependent manner and to the Ifna4 enhancer in a partially cGAS-dependent manner in response to microbial RNAs. Prmt5 catalyzes the symmetric dimethylation of H3R2 (H3R2me2s), which promotes transcriptional activation by excluding co-repressor complexes and attracting coactivator complexes (Migliori et al., 2012Migliori V. Müller J. Phalke S. Low D. Bezzi M. Mok W.C. Sahu S.K. Gunaratne J. Capasso P. Bassi C. et al.Symmetric dimethylation of H3R2 is a newly identified histone mark that supports euchromatin maintenance.Nat. Struct. Mol. Biol. 2012; 19: 136-144Crossref PubMed Scopus (201) Google Scholar). Poly(I:C) treatment markedly increased the level of H3R2me2s modification in WT L929s, whereas Prmt5 depletion resulted in the loss of H3R2me2s modification (Figure 6G). Consistently, we observed more H3R2me2s modification in WT than in Prmt5-deficient L929s in response to HT-DNA (Figure S6B) but not LPS (Figure S6C). A ChIP-qPCR assay detected enriched H3R2me2s modification at the Ifnb and Ifna4 enhancers and promoters in WT L929s (Figures 6H and 6I). Deficiency of Prmt5 and cGAS marginally affected the H3R2me2s modification on parts of the IFN promoters in resting cells. By contrast, the levels of H3R2me2s at those promoters and enhancers were robustly increased upon stimulation with poly(I:C), and ablation of Prmt5 eliminated such histone arginine modific" @default.
• W3111682023 created "2020-12-21" @default.
• W3111682023 creator A5013038112 @default.
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