Degradation of WTAP blocks antiviral responses by reducing the m 6 A levels of IRF3 and IFNAR1 mRNA
2021; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.15252/embr.202052101
ISSN1469-3178
AutoresYong Ge, Tao Ling, Yao Wang, Xin Jia, Xiongmei Xie, Rong Chen, Shangwu Chen, Shaochun Yuan, Anlong Xu,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle1 September 2021Open Access Source DataTransparent process Degradation of WTAP blocks antiviral responses by reducing the m6A levels of IRF3 and IFNAR1 mRNA Yong Ge Yong Ge Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Search for more papers by this author Tao Ling Tao Ling Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Search for more papers by this author Yao Wang Yao Wang Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China Search for more papers by this author Xin Jia Xin Jia Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China Search for more papers by this author Xiongmei Xie Xiongmei Xie Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Rong Chen Rong Chen Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Search for more papers by this author Shangwu Chen Shangwu Chen Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Shaochun Yuan Corresponding Author Shaochun Yuan [email protected] orcid.org/0000-0002-8755-7543 Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Anlong Xu Corresponding Author Anlong Xu [email protected] orcid.org/0000-0002-1419-3494 Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China Search for more papers by this author Yong Ge Yong Ge Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Search for more papers by this author Tao Ling Tao Ling Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Search for more papers by this author Yao Wang Yao Wang Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China Search for more papers by this author Xin Jia Xin Jia Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China Search for more papers by this author Xiongmei Xie Xiongmei Xie Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Rong Chen Rong Chen Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Search for more papers by this author Shangwu Chen Shangwu Chen Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Shaochun Yuan Corresponding Author Shaochun Yuan [email protected] orcid.org/0000-0002-8755-7543 Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China Search for more papers by this author Anlong Xu Corresponding Author Anlong Xu [email protected] orcid.org/0000-0002-1419-3494 Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China Search for more papers by this author Author Information Yong Ge1,2,†, Tao Ling1,2,†, Yao Wang1,3, Xin Jia1,4, Xiongmei Xie1, Rong Chen1,2, Shangwu Chen1, Shaochun Yuan *,1,2,5 and Anlong Xu *,1,3 1Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, China 3School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China 4School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China 5Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China † These authors contributed equally to the work *Corresponding author. Tel: +86 20 39332956; E-mail: [email protected] *Corresponding author. Tel: +86 20 39332990; E-mail: [email protected] EMBO Reports (2021)22:e52101https://doi.org/10.15252/embr.202052101 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract N6-methyladenosine (m6A) is a chemical modification present in multiple RNA species and is most abundant in mRNAs. Studies on m6A reveal its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. Although some recent discoveries indicate that m6A can affect the life cycles of numerous viruses as well as the cellular antiviral immune response, the roles of m6A modification in type I interferon (IFN-I) signaling are still largely unknown. Here, we reveal that WT1-associated protein (WTAP), one of the m6A "writers", is degraded via the ubiquitination-proteasome pathway upon activation of IFN-I signaling. With the degradation of WTAP, the m6A levels of IFN-regulatory factor 3 (IRF3) and interferon alpha/beta receptor subunit 1 (IFNAR1) mRNAs are reduced, leading to translational suppression of IRF3 and instability of IFNAR1 mRNA. Thus, the WTAP-IRF3/IFNAR1 axis may serve as negative feedback pathway to fine-tune the activation of IFN-I signaling, which highlights the roles of m6A in the antiviral response by dictating the fate of mRNAs associated with IFN-I signaling. Synopsis The m6A writer WTAP is degraded via the ubiquitination-proteasome pathway upon the activation of IFN-I signaling. Degradation of WTAP leads to reduced m6A modifications of IRF3 and IFNAR1 transcripts at specific sites. Reduced m6A levels on both mRNAs lead to translational suppression of IRF3 and instability of IFNAR1 mRNA. The WTAP-IRF3/IFNAR1 axis may serve as a negative feedback loop to fine-tune the activation of IFN-I signaling. Proteasome mediated degradation of the m6A writer WTAP upon viral infection reduces m6A levels on IRF3 and IFNAR1 mRNAs and subsequently their protein output, thereby blocking IFN-I-mediated antiviral responses. Introduction Type I interferons (IFN-Is) have vital roles in antiviral innate immunity. After viral infection, germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs), can recognize nucleic acids derived from viruses, leading to activation of IRF3, IRF7, and nuclear factor-κB (NF-κB) to induce the production of IFN-I (Yan & Chen, 2012; Schlee & Hartmann, 2016). The secreted type I interferons (IFNα/β) then bind to the heterodimeric receptor complex composed of two chains (IFNAR1 and IFNAR2), which amplify and spread the antiviral response to surrounding uninfected cells via the Janus kinase/signal transducer and activator of transcription (Jak-STAT) signaling pathway (Villarino et al, 2017). Activation of the Jak-STAT pathway results in the transcription of hundreds of interferon-stimulated genes (ISGs), most of which encode products with profound antiviral effects, thus establishing an innate immune state against invading pathogens and maintaining homeostasis (Schneider et al, 2014; Barrat et al, 2019). Although IFN-I has critical roles in protection from pathogens, excessive IFN responses contribute to immune pathogenesis such as inflammatory autoimmune diseases and infectious diseases via aberrantly activating inflammation. Thus, to ensure the most favorable outcome, the host has developed sophisticated mechanisms at multiple levels to maintain appropriate IFN responses. For example, almost all ISGs are rich in ISRE (interferon-sensitive response element) motifs, and their transcription is initiated when the Jak-STAT signaling is activated (Levy et al, 1988). When the translational repressors 4E-BP1 and 4E-BP2 are lacking, the threshold for eliciting IFN-I production is lowered (Colina et al, 2008). In addition to transcriptional and translational regulation of many ISGs, the activation of IFN-I can be tightly regulated at the post-translational level. For instance, the activity and stability of many key components involved in IFN-I activation, such as the sensors RIG-I and cGAS, can be regulated by ubiquitination and deubiquitylation (Gack et al, 2007; Chen & Chen, 2019; Tao et al, 2020; Zhang et al, 2020). The activation of some key adaptors, such as MAVS, STING, and TRIF, is regulated by phosphorylation at their conserved pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site) motif (Liu et al, 2015). Recently, a new epigenetic modification, N6-methyladenosine (m6A), has aroused extensive attention. m6A is the most prominent mRNA modification in higher eukaryotes, governed by methyltransferase complex ("writers"), demethylases ("erasers"), and RNA-binding proteins ("readers") (Roignant & Soller, 2017; Frye et al, 2018). m6A is added to mRNA by a multisubunit "writer" complex composed of the METTL3/METTL14/WTAP heterotrimer and many additional adaptor proteins. METTL3 is the enzymatic component of the complex, and METTL14 is an allosteric activator that also binds to the target RNA. In this heterotrimer, WTAP is essential for m6A deposition and helps the METTL3-METTL14 heterodimer localize to transcription sites (Ping et al, 2014; Śledź & Jinek, 2016). m6A modification has been linked to various stages along the posttranscriptional trajectory of mRNA and, in particular, to promotion of mRNA splicing, export, decay, translation, and subcellular localization (Wang et al, 2014, 2015; Meyer & Jaffrey, 2017). Functionally, m6A has been shown to impact fundamental cellular processes in diverse organisms, including meiosis, the circadian clock, DNA damage repair, and the stress response (Xiang et al, 2017; Xu et al, 2017; Fustin et al, 2018; Zhou et al, 2018). Studies also show that different expression levels, post-translational modifications (PTMs), and cellular localization of "writer" proteins, depending on cell type and/or in response to environmental stimuli, can regulate the processing of m6A and provide unique potential to tune gene expression in different biological processes (Wang et al, 2016; Xiang et al, 2017; Du et al, 2018). In short, the role of m6A in regulating mRNA fate and its functional importance in various cell types have been widely studied. Recently, some studies have suggested that m6A may also be involved in regulation of host immunity as well as viral replication (Zheng et al, 2017; Winkler et al, 2019). However, the specific mechanisms, especially whether and how m6A modification tightly regulates IFN-I signaling, are still unclear. In this study, we report that WTAP, one of the m6A "writer" proteins, is depleted via ubiquitination-proteasome-mediated degradation upon viral infection. Degradation of WTAP leads to reduced m6A methylation of IRF3 and IFNAR1 mRNAs, resulting in translational suppression of IRF3 and mRNA instability of IFNAR1. Thus, the precise control of IRF3 and IRNAR1 by m6A modification fine-tunes the antiviral responses to protect the host from immunopathology of over-reactive and harmful IFN-I production. In brief, our findings highlight WTAP as a regulator involved in antiviral immunity, providing a novel diagnostic marker and therapeutic target for related immune diseases. Results WTAP protein is decreased in virus-infected or nucleic acid analog-treated cells From previous studies, m6A is added to mRNA by a multisubunit writer complex, and the most important of which is the METTL3/METTL14/WTAP heterotrimer. The protein abundance of these writers can regulate m6A modification and control biological processes. To investigate the role of m6A modification in regulation of the IFN-I signaling pathway, we first analyzed the expression of the key m6A writer proteins-METTL3, METTL14, and WTAP in PBMCs (peripheral blood mononuclear cells) upon viral infection or nucleic acid analog stimulation. The results showed that, along with upregulation of RIG-I or MDA5, the protein level of WTAP was significantly decreased when PBMCs were challenged with VSV-eGFP (vesicular stomatitis virus with enhanced GFP), intracellular poly(I:C) LMW (low molecular weight), HSV-1 (herpes simplex virus 1), or intracellular HSV-60 (synthetic herpes simplex virus 1 DNA analog). However, as the key enzymatic components, the protein abundances of METTL3 and METTL14 were slightly changed only when PBMCs were challenged with poly(I:C) or HSV-60 (Fig 1A). Subsequently, the same treatments were performed in BMDMs (mouse bone marrow-derived macrophages) and similar results were obtained. The protein level of WTAP was significantly decreased with all indicated treatments in BMDMs, while METTL14 exhibited a downward trend only with poly(I:C) (LMW) or HSV-60 stimulation (Fig 1B). Then, we further validated the above phenotypes in human and mouse cell lines, such as A549, L929, and MEF (mouse embryonic fibroblast) cells. We also found that the protein abundance of WTAP, but not of METTL3 or METTL14, was reduced in A549 and L929 cells infected with VSV or SeV (Sendai virus) (Figs 1C and EV1A), or transfected with poly (I:C) (LMW) or poly (dA:dT) (Figs 1D and EV1B). In addition, WTAP was also significantly decreased in A549 cells (Fig EV1C) and MEFs (Fig EV1D) infected with HSV-1. However, the protein levels of METTL3 and METTL14 were not changed significantly upon the same treatments. Overall, the protein abundance of WTAP gradually decreased upon activation of IFN-I signaling after viral infection or nucleic acid analog treatment, while no uniform or broad trend in METTL3 and METTL14 expression was observed. These results indicate a potential relationship between m6A modification and the regulation of IFN-I signaling, in which WTAP may be an important regulator. Figure 1. WTAP protein is decreased in virus-infected or nucleic acid analog-treated cells A, B. Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in PBMCs (A) and BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1, or HSV-60 at the indicated time points. Data are representative of three independent biological experiments. C, D. Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in A549 cells upon challenge with VSV-eGFP or SeV (C) and poly (I:C) or poly (dA:dT) (D) at the indicated time points. Data are representative of three independent biological experiments. Source data are available online for this figure. Source Data for Figure 1 [embr202052101-sup-0002-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. WTAP protein is decreased in virus-infected or nucleic acid analog-treated cells A, B. Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in L929 cells upon challenge with VSV-eGFP or SeV (A) and poly (I:C) or poly (dA:dT) (B) at the indicated time points. Data are representative of three independent biological experiments. C, D. Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in A549 cells (C) and MEFs (D) upon challenge with HSV-1 at the indicated time points. Data are representative of three independent biological experiments. Source data are available online for this figure. Download figure Download PowerPoint WTAP is degraded through the K48-linked ubiquitination-proteasome pathway upon activation of IFN-I signaling It is well known that protein abundance is mainly regulated by transcriptional and post-translational modifications. To explore what affects the protein abundance of WTAP upon activation of IFN-I signaling, we first detected the transcriptional changes of WTAP. The results showed that the transcription of WTAP was slightly activated by both viral infections (VSV-eGFP or HSV-1) and nucleic acid analog treatments (poly (I:C), poly (dA:dT) or HSV-60) in PBMCs, BMDMs, and A549 cells (Fig 2A–C). However, stimulation with IFN-α or IFN-β in A549 cells (Fig 2D) and BMDMs (Fig EV2A) had no effect on the mRNA abundance of WTAP. These data indicate that the degradation of WTAP may be caused by post-translational modification instead of transcriptional regulation upon activation of IFN-I signaling. Figure 2. WTAP is degraded through the K48-linked ubiquitination-proteasome pathway upon the activation of IFN-I signaling A, B. Real-time PCR (qRT–PCR) analyses of WTAP mRNA expression in PBMCs (A) or BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1, or HSV-60 at indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. C. qRT–PCR analyses of WTAP mRNA expression in A549 cells upon challenge with VSV-eGFP, poly(I:C), HSV-1, or poly (dA:dT) at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. D. qRT–PCR analyses of WTAP mRNA expression in A549 cells stimulated with IFN-α or IFN-β at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. E. A549 cells were stimulated with poly (I:C) for 16 h and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 and 8 h. The cell lysates were analyzed by immunoblotting. Representative images from three independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F. A549 cells were stimulated with poly (I:C) for 16 h and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 and 8 h. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from three independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). G. Immunoassays of extracts of A549 cells infected with VSV-GFP for 16 h and then treated with PR-171 (10 μM) or DMSO for additional 4 and 8 h, followed by immunoprecipitation with anti-WTAP antibody and immunoblot analysis with anti-Ub antibody. Data are representative of three independent biological experiments. H. Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR-171 (10 μM). Data are representative of three independent biological experiments. Source data are available online for this figure. Source Data for Figure 2 [embr202052101-sup-0003-SDataFig2.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. WTAP is degraded through the K48-linked ubiquitination-proteasome pathway upon activation of IFN-I signaling A. qRT–PCR analyses of WTAP mRNA expression in BMDMs stimulated with IFN-α or IFN-β at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. B. A549 cells were stimulated with poly (dA:dT) or infected with VSV for 16 h and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 and 8 h. The cell lysates were analyzed by immunoblotting. Data are representative of three independent biological experiments. C. A549 cells were infected with VSV-eGFP for 16 h and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 and 8 h. The cell lysates were analyzed by immunoblotting. Data are representative of three independent biological experiments. D. Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin, and treated with DMSO or PR-171 (10 μM), followed by immunoprecipitation with anti-HA antibody and immunoblot analysis with anti-Flag antibody. Data are representative of three independent biological experiments. Source data are available online for this figure. Download figure Download PowerPoint It has been documented that WTAP can undergo degradation depending on the ubiquitin-proteasome pathway (Kuai et al, 2018). To explore whether WTAP is degraded when activating IFN-I signaling, the protein abundance of WTAP in the presence of the proteasome inhibitors carfilzomib (PR-171) and bortezomib (PS-341) or autophagy inhibitors chloroquine (CQ) and bafilomycin A1 (Baf A1) was tested. PR-171 is an irreversible proteasome inhibitor, while PS-341 is a reversible inhibitor of the proteasome (Fricker, 2020), and both are commonly used in clinical treatment and scientific research. The results showed that PR-171 and PS-341 (Figs 2E and EV2B), but not CQ and Baf A1 (Figs 2F and EV2C), blocked the VSV-eGFP- or nucleic acid analog-mediated degradation of WTAP in A549 cells. In addition, higher levels of ubiquitinated WTAP were observed in the presence of PR-171 than in the presence of DMSO (Fig 2G), revealing that WTAP undergoes ubiquitination upon viral infection. To determine what type of ubiquitin chain is added to WTAP, we transfected 293T cells with Flag-tagged WTAP together with HA-tagged K48-linked ubiquitin (K48-Ub), K63-linked ubiquitin (K63-Ub), or wild-type ubiquitin (HA-Ub), and found that WTAP undergoes both K48-linked and K63-linked ubiquitination. However, only K48-linked, but not K63-linked ubiquitination of WTAP, was markedly increased in the presence of the proteasome inhibitor PR-171 (Figs 2H and EV2D). All these results suggest that K48-linked ubiquitination-proteasomal degradation is the main reason for the decrease of WTAP protein upon activation of IFN-I signaling. WTAP positively regulates the antiviral immune responses To determine the relationship between WTAP and the activation of IFN-I signaling, we first knocked out WTAP using CRISPR-Cas9 systems (Ran et al, 2013) in A549, L929, and 293T cells. However, only one WTAP−/− 293T subclone was obtained after a series of efforts (Fig 3A). We then used this subclone to investigate the role of WTAP in IFN-β production via dual-luciferase assays. The results showed that the activities of IFN-β promoter-based luciferase reporter (IFN-β-luc) and interferon-stimulated response element (ISRE)-based luciferase reporter were upregulated upon viral infection in 293T cells. However, when endogenous WTAP was knocked out, the increased IFN-β-luc and ISRE-luc activities mediated by VSV-eGFP infection or poly(I:C) and poly (dA:dT) stimulation were significantly reduced (Fig 3B). Such defects could be rescued in a dose-dependent manner when exogenous WTAP was overexpressed in WTAP−/− 293T cells infected with VSV-eGFP or treated with poly(I:C) or poly (dA:dT) (Fig EV3-EV5). Similarly, decreased mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 were observed in WTAP−/− 293T cells upon VSV-eGFP, poly(I:C), or poly (dA:dT) treatments (Fig 3C). These data indicate a positive regulation mediated by WTAP in the IFN-I signaling pathway. Figure 3. WTAP positively regulates the antiviral immune responses A. WTAP knockout efficiency in 293T cells detected by immunoblotting. Data are representative of three independent biological experiments. B. Luciferase activity analyses in wild-type (WT), or WTAP−/− 293T cells, transfected with a luciferase reporter for IFN-β (IFN-β-luc) or ISRE (ISRE-luc) followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP. The results are expressed relative to Renilla luciferase activity. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. C. qRT–PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP−/− 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP. Data are representative of three independent biological experiments. D. Immunoblot analyses to detect the WTAP knockdown efficiency in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 h. Data are representative of three independent biological experiments. E, F. qRT–PCR analyses to detect the mRNA abundance of IFNB1 (E), RANTES, and ISG15 (F) in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by treatment with poly(I:C)-HMW at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. G, H. ELISA analyses to detect IFN-β (G) and CCL5 (H) secretion in supernatants from E at 16 h. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. I, J. Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies. Data are representative of three independent biological experiments. K, L. qRT–PCR analyses to detect the mRNA abundances of IFNB1 (K) and RANTES (L) in A549 cells transfected with empty vector (EV) or WTAP-HA plasmid, followed by treatment with poly(I:C)-HMW at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. M, N. ELISA analyses to detect IFN-β (M) and CCL5 (N) secretion in supernatants from K. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test. Source data are available online for this figure. Source Data for Figure 3 [embr202052101-sup-0004-SDataFig3.zip] Dow
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