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The RNA helicase DDX46 inhibits innate immunity by entrapping m6A-demethylated antiviral transcripts in the nucleus Qingliang Zheng1,3, Jin Hou2,3, Ye Zhou2, Zhenyang Li2 & Xuetao Cao1,2 DEAD-box (DDX) helicases are vital for the recognition of RNA and metabolism and are critical for the initiation of antiviral innate immunity. Modification of RNA is involved in many biological processes; however, its role in antiviral innate immunity has remained unclear. Here we found that nuclear DDX member DDX46 inhibited the production of type I interferons after viral infection. DDX46 bound Mavs, Traf3 and Traf6 transcripts (which encode signaling molecules involved in antiviral responses) via their conserved CCGGUU element. After viral infection, DDX46 recruited ALKBH5, an ‘eraser’ of the RNA modification N 6-methyladenosine (m6A), via DDX46’s DEAD helicase domain to demethylate those m6A-modified antiviral transcripts. It consequently enforced their retention in the nucleus and therefore prevented their translation and inhibited interferon production. DDX46 also suppressed antiviral innate immunity in vivo. Thus, DDX46 inhibits antiviral innate responses by entrapping selected antiviral transcripts in the nucleus by erasing their m6A modification, a modification normally required for export from the nucleus and translation. Type I interferons have vital roles in the innate defense against viral infection. After viral infection, pattern-recognition receptors trigger activation of the kinase TBK1 and transcription factor IRF3 to induce the production of type I interferons1. Such interferon production during viral infection is tightly controlled to prevent harmful immunopathology after elimination of the invading virus2. Many positive or negative regulators in the cytoplasm have been identified that ensure that appropriate amounts of type I interferons are produced by activating or degrading key components of antiviral signaling3–5. Furthermore, factors in the nucleus are also important in regulating the production of type I interferons via various mechanisms, such as nuclear sumoylation mediated by the transmembrane adaptor TRIM5α, as well as acetylation of the DNA sensor IFI16, which promotes interferon production6,7, and inhibition of the activation of the transcription factor IRF7 via the transcription factor Myc8. However, molecular regulation of the production of type I interferons has focused mainly on cytosolic regulators of innate signaling; whether nuclear factors also have important roles in this process is largely unknown. Members of the DEAD-box (DDX) family of helicases with 12 conserved motifs are the largest family of helicases that are important in the antiviral innate immune response. Many members of the DDX family have been verified as sensing viral nucleic acids or regulating downstream signaling. DDX58 (RIG-I) recognizes viral RNA, DDX41 recognizes intracellular DNA and bacterial cyclic dinucleotides, and DHX9 senses viral double-stranded RNA in dendritic cells; this all

leads to the transcriptional induction of genes encoding type I interferons9–12. In addition, cytosolic DDX3 promotes antiviral signaling and interferon production by binding to the kinases TBK1 and IKKε13. However, there has been no report about negative regulation of the production of antiviral interferons by a member of the DDX family. Furthermore, most members of the DDX family are located in the nucleus and control nearly every aspect of RNA metabolism14,15; therefore, it is possible that these nuclear helicases regulate antiviral innate immune responses through RNA modification. In eukaryotic cells, control of mRNA metabolism is critical for managing the quantity of gene expression16. The post-transcriptional regulation of mRNA can help cells respond more rapidly to external signaling and stimuli than does regulation at the transcriptional level17. N6-methyladenosine (m6A) is the most prevalent internal modification on eukaryotic mRNA18. Dynamic m6A modification of mRNA is post-transcriptionally installed, erased and recognized by m6A methyltransferases, m6A demethylases and m6A-specific binding proteins, respectively19–22. The fundamental cellular functions of m6A have been connected directly to the splicing, translation and stability of mRNA and, consequently, have been associated with a broad set of biological functions23–25. ‘Erasure’ of the m6A modification substantially inhibits export of mRNA from the nucleus and induces retention in the nucleus of mRNA encoding molecules such as those involved in circadian rhythm26,27. However, whether the m6A modification participates in the regulation of antiviral innate immunity, especially in the regulating the production of type I interferons, is unknown.

1Department

of Immunology & Center for Immunotherapy, CAMS-Oxford University International Center for Translational Immunology, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China. 2National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China. 3These authors contributed equally to this work. Correspondence should be addressed to X.C. ([email protected]). Received 11 December 2016; accepted 7 August 2017; published online 28 August 2017; doi:10.1038/ni.3830

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Articles DDX46 has been shown to be required at the early step of pre-spliceosome assembly 28. It uses the energy released by the hydrolysis of ATP to rearrange local RNA–RNA or protein–RNA interactions29. However, the potential roles of DDX46 in RNA editing and whether DDX46 can regulate antiviral innate immunity by editing antiviral-molecule-encoding transcripts in the nucleus have remained elusive. Here we found that nuclear DDX46 acted as a negative regulator of the antiviral innate response by recruiting the m6A ‘eraser’ ALKBH5 to induce retention of antiviral innate mRNA in the nucleus. Our results might add insight into the action of the m6A RNA modification in regulating innate response and inflammation and might provide potential targets for controlling infection and inflammatory disease.

conventional dendritic cells or plasmacytoid dendritic cells or in human THP1 macrophages (Supplementary Fig. 1k–m). Together these data demonstrated that DDX46 was a negative regulator of the production of type I interferons in response to viral infection. To investigate the biological relevance of DDX46, we assessed the effect of its knockdown on VSV replication in macrophages. VSV replication was significantly inhibited by knockdown of DDX46, a result confirmed by measurement of intracellular VSV RNA replicates and the half-maximal tissue-culture infectious dose of VSV in cell supernatants (Fig. 1g,h). Moreover, overexpression of DDX46 promoted VSV replication (Fig. 1i). Thus, these data suggested that DDX46 was able to promote VSV replication in macrophages, probably by inhibiting the production of type I interferons.

RESULTS DDX46 negatively regulates the production of type I interferons Members of the DDX family bind to and metabolize RNA14. Published reports suggest that there are ten members of this family that are associated with pre-mRNA splicing30, and three members, DDX1, DDX3 and DDX41, have been shown to not only participate in the splicing of pre-mRNA in the nucleus but also serve important roles in the antiviral innate immune response in the cytoplasm as cytosolic sensors31. We sought to investigate whether the other members of the DDX family associated with pre-mRNA splicing, especially in the nucleus, also influence host antiviral immune responses. We screened the other seven members of the DDX family: DDX5, DDX17, DDX23, DDX39, DDX42, DDX46 and DDX48. By using small interfering RNA (siRNA) to knock down the expression of these DDX helicases in peritoneal macrophages, we found that knockdown of DDX46 led to the greatest increase in the production of interferon-β (IFN-β) after infection with vesicular stomatitis virus (VSV) (Supplementary Fig. 1a). DDX46 was present exclusively in the nucleus of macrophages with or without viral infection (Supplementary Fig. 1b). These results led us to focus on DDX46 and further investigate whether DDX46 negatively regulates antiviral responses. Because type I interferons have pivotal roles in antiviral immune responses, we further confirmed the screening data on the role of DDX46 in the negative regulation of interferon production through the use of two different siRNAs (Supplementary Fig. 1c,d). We found that knockdown of DDX46 significantly enhanced not only the production of type I interferons triggered by RNA viruses (VSV or Sendai virus) but also that triggered by a DNA virus (herpes simplex virus) in mouse macrophages and human THP1 macrophages (Fig. 1a–c and Supplementary Fig. 1e), while viral-infection-induced production of interferons was significantly inhibited in RAW264.7 mouse macrophages stably overexpressing DDX46 (Fig. 1d–f and Supplementary Fig. 1f). These data suggested that DDX46 was a negative regulator of the production of antiviral type I interferons. We also found that knockdown of DDX46 enhanced activation of the transcription factor NF-κB and promoted the production of inflammatory cytokines and IFN-β after infection of cells with VSV or stimulation of cells with lipopolysaccharide (LPS) (Supplementary Fig. 1g–i). Furthermore, overexpression of DDX46 markedly reduced the activation of an IFN-β luciferase reporter induced by RIG-I, the RNA helicase MDA5, the adaptor TRIF or the signaling adaptor MAVS (Supplementary Fig. 1j). However, DDX46 did not affect the activity of the IFN-β luciferase reporter induced by the adaptor MyD88, TBK1 or IRF3 (Supplementary Fig. 1j), which suggested that DDX46 functioned downstream of MAVS and upstream of TBK1. Additionally, DDX46 expression itself was not substantially influenced by viral infection in mouse macrophages, peripheral blood mononuclear cells, myeloid

Identification of DDX46-bound RNAs We then pursued the mechanisms responsible for the DDX46-mediated inhibition of the production of type I interferons. As DDX46 is located exclusively in the nucleus and functions mainly in the initial steps of pre-mRNA splicing29, we hypothesized that DDX46 might bind directly to a series of RNAs to inhibit the production of type I interferons. To identify DDX46-bound RNAs or the directly targeted RNAs, we performed iCLIP-seq (individual-nucleotide-resolution ultraviolet-irradiation crosslinking and immunoprecipitation coupled with high-throughput sequencing). Immunoblot analysis of protein–RNA complexes revealed extensive signals after immunoprecipitation with antibody to DDX46 (anti-DDX46) but not after immunoprecipitation with the control antibody to immunoglobulin G (anti-IgG) (Fig. 2a), which suggested enrichment for DDX46-bound RNAs after immunoprecipitation with anti-DDX46. cDNA libraries were then generated from the purified RNAs and submitted for deep sequencing with a high-throughput sequencer (Supplementary Fig. 2a,b). From two pooled iCLIP analyses of DDX46 (Supplementary Fig. 2c,d), we obtained 114 × 106 clean reads from the uninfected macrophages and 87 × 106 clean reads from the infected macrophages (Fig. 2b). After mapping the mm10 mouse genome and removing PCR duplicates, we achieved a yield of 169,745 peaks from the uninfected cells and 127,818 peaks from VSV-infected cells (Fig. 2b). Intergenic regions (~54%) and introns (~34%) were the main regions showing enrichment for DDX46 peaks, relative to the abundance of these peaks in mature RNA (including mature mRNA and noncoding RNA); coding sequences, 5′ untranslated regions, 3′ untranslated regions and noncoding RNA regions showed less enrichment for DDX46 peaks (~12%) (Fig. 2c). This suggested that DDX46 ‘preferentially’ bound to intergenic regions of RNA transcripts and pre-mRNA transcripts, consistent with the roles of DDX46 in splicing pre-mRNA29. In addition to unannotated RNA transcripts, most of these gene transcripts were mRNA, but 0.4% of the transcripts were noncoding RNAs; also, comparison of 16,713 gene transcripts from uninfected cells and 13,866 gene transcripts from VSV-infected cells showed that 12,129 of these overlapped (Supplementary Fig. 2e). Hexamer-enrichment analysis of iCLIPseq peaks within all annotated genes showed that DDX46 bound a CCGGUU element in mRNA transcripts (Fig. 2d). Together these data identified both the location and sequence ‘preference’ for DDX46bound mRNA. To understand the functional targets of DDX46, we used the DAVID bioinformatics database to identify terms from the Kyoto Encyclopedia of Genes and Genomes and gene-ontology terms for which proteincoding genes associated with DDX46 peaks identified by iCLIP-seq showed enrichment. We observed that, according to this analysis, DDX46 might take part in a set of biological processes and several cellular signaling pathways (Supplementary Fig. 2f–i). After viral infection,



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Figure 1  DDX46 promotes VSV replication by suppressing VSV-triggered production of type I interferons in macrophages. (a–c) qRT-PCR analysis of Ifnb mRNA (a (left), b,c) or Ifna4 mRNA (a, right) and ELISA of IFN-β in the supernatants (a, middle) of macrophages transfected for 72 h with non-targeting control siRNA (NC) or DDX46-specific siRNA (siDDX46; one of two siRNA constructs (1 or 2 in a, left)) (key) and infected for 0, 8 or 12 h (horizontal axes) with VSV (a; multiplicity of infection (MOI) = 10), Sendai virus (SeV) (b; MOI = 100) or herpes simplex virus (HSV) (c; MOI = 10) (below plots); qRT-PCR results were normalized to those of the internal control gene Actb. (d–f) qRT-PCR analysis of Ifnb mRNA (d (left), e,f) and ELISA of IFN-β in the supernatants (d, right) of RAW264.7 cell clones with (DDX46) or without (Control) stable overexpression of DDX46 (key), infected for 0, 8 or 12 h (horizontal axes) with VSV (d), Sendai virus (e) or herpes simplex virus (f) as in c (below plots); qRT-PCR presented as in a–c. (g,h) qRT-PCR analysis of intracellular VSV RNA (g) and the halfmaximal tissue-culture infectious dose (TCID50) of VSV in supernatants (h) of macrophages transfected with siRNA as in a (key) and infected for 0, 8 or 12 h (horizontal axes) with VSV as in c. (i) qRT-PCR analysis of VSV RNA in RAW264.7 cell clones stably overexpressing DDX46 or not (key) and infected for 0, 8 or 12 h (horizontal axes) with VSV as in c. ND, not detected. *P < 0.01 (Student’s t-test). Data are representative of three independent experiments (mean + s.d. of technical triplicates).

DDX46-bound RNAs showed enrichment (relative to RNA encoding proteins in other pathways) for signaling pathway of antiviral innate immune responses, one of the pathways from the Kyoto Encyclopedia nature immunology  aDVANCE ONLINE PUBLICATION

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Figure 2  iCLIP-seq analysis of DDX46-bound RNA from VSV-infected macrophages. (a) Immunoblot analysis of complexes of DDX46 and biotinlabeled RNA immunopurified, by immunoprecipitation (IP) with IgG (control) or anti-DDX46 (above plots), from RAW 264.7 macrophages infected for 0 or 6 h with VSV (above lanes) and treated with RNase I (above lanes). Left margin, molecular size, in kilodaltons (kDa). Right margin, DDX46–RNA complexes (+RNA) or DDX46 alone (arrowhead). (b) iCLIP-seq alignment and processing pipeline that resulted in peaks. (c) Frequency of total nucleotides under substantial iCLIP-seq peaks, in various regions (horizontal axis; extracted from the reference sequence database (RefSeq) of the NCBI) of protein-coding genes (from RefSeq) in RAW 264.7 macrophages left uninfected or infected with VSV (MOI = 10) (key). UTR, untranslated region; CDS, coding sequence. (d) Composite motif ‘logos’ (as identified by P values) of the multiple-sequence alignment of the 1,000 hexamers with the greatest enrichment under significant iCLIP-seq peaks, in protein coding genes, in a comparison of hexamer frequency to 1,000 permutations of binding-site locations within bound transcripts for uninfected RAW 264.7 macrophages (top) or RAW 264.7 macrophages infected with VSV (bottom). Data are representative of three independent experiments with similar results (a) or two experiments (c,d).

of Genes and Genomes that showed over-representation was ‘RIG-I-like receptor signaling’ (Supplementary Fig. 2i). Thus, these results suggested that the iCLIP-seq peaks further reflected the biological activity of DDX46 in the regulation of antiviral innate immune responses. DDX46 increases retention of antiviral transcripts in the nucleus To further elucidate the mechanisms of DDX46-mediated inhibition of interferon production, we focused on the antiviral transcripts that showed considerable enrichment among DDX46-bound RNAs , relative to the abundance of transcripts in other pathways. In the iCLIPseq data, three transcripts of genes encoding antiviral molecules (Mavs, Traf3 and Traf6) were among the DDX46-bound RNAs (Fig. 3a). We found that transcripts encoding negative regulators of antiviral signaling, such as Tnfaip3, Dhx58, C1qbp, Cyld, Sike1 and Pin1, were not among the DDX46-bound transcripts, and further analysis indicated that the transcripts of these negative regulators lacked the conserved binding motif CCGGUU (Supplementary Table 1). The selective iCLIP interactions were further confirmed by RNA immunoprecipitation and qRT-PCR, which showed that DDX46 was able to bind Mavs, Traf3 and Traf6 transcripts, but not Ticam1 transcripts (encoding TRIF), and the DDX46-binding activity of these transcripts was significantly enhanced after VSV infection (Fig. 3b and Supplementary Fig. 3a). 

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Figure 3  DDX46 increases the retention of antiviral mRNA in the nucleus and decreases the expression of the protein encoded. (a) Sequencing read clusters (tracks from the IGV visualization tool for interactive exploration of genomic data sets) from iCLIP analysis of Mavs, Traf3 and Traf6 (left margin) in RAW 264.7 macrophages left uninfected or infected with VSV (MOI = 10) (left margin); above tracks, pre-mRNA genomic loci. (b) Abundance of Gapdh, Tbk1, Mavs, Traf3 and Traf6 transcripts in DDX46–RNA complexes obtained, by co-immunoprecipitation of DDX46 and RNA (RNA-binding protein immunoprecipitation (RIP)), from RAW264.7 cells infected for 0 or 6 h (key) with VSV (MOI = 10); results are presented relative to control immunoprecipitation of protein–RNA with IgG (RIP/IgG). (c,e) RT-PCR analysis of Mavs, Traf3 and Traf6 mRNA, as well as of Rnu6 and Gapdh mRNA (loading controls throughout), in the nucleus (Nu) and cytoplasm (Cyto) of RAW264.7 cells transfected for 72 h with control or DDX46-specific siRNA (top) (c) or for 24 h with control plasmid or plasmid encoding wild-type DDX46 or DDX46 with a mutant LAT domain (LET) or DEAD domain (HGAD) (top) (e), then infected for 6 h with VSV (MOI = 10). (d,f) qRT-PCR analysis of the distribution of Mavs, Traf3 and Traf6 RNA in the cytoplasm and nucleus of RAW264.7 cells treated as in c (d) or e (f); results are presented relative to those of Gapdh (cytoplasm) or Rnu6 (nucleus). (g,h) FISH analysis of Mavs transcripts in RAW264.7 cells treated as in c (g) or e (h). DAPI, DNA-binding dye. Scale bars, 5 µm. (i) Immunoblot analysis of total DDX46, MAVS, TRAF3, TRAF6 and TBK1, phosphorylated (p-) IRF3 and β-actin (loading control throughout) in macrophages transfected for 72 h with control or DDX46-specific siRNA (above blots), and infected for 0, 3 or 6 h (above lanes) with VSV (MOI = 10). (j) Immunoblot analysis of MAVS, TRAF3, TRAF6, TBK1 and Myc in RAW264.7 cells transfected with control plasmid (−) or increasing doses (wedge) of plasmid encoding Myc-tagged DDX46. NS, not significant (P > 0.05); *P < 0.01 (Student’s t-test). Data are representative of two experiments (a) or three independent experiments (b,d,f; mean + s.d. of technical triplicates) or are from three independent experiments with similar results (c,e,g–j).

Because DDX46 is known to function in pre-mRNA splicing28 and is located exclusively in the nucleus, we investigated whether the inhibitory effect of DDX46 on the production of type I interferons might be exerted through the splicing of Mavs, Traf3 and Traf6 mRNA. Our transcriptome-sequencing results showed that the splicing of Mavs, Traf3 and Traf6 mRNA was not influenced by DDX46 (Supplementary Table 2 and Supplementary Fig. 3b). We then investigated the effect of DDX46 on the intracellular distribution of the antiviral transcripts by assessing whether DDX46 affected the translocation of these innate mRNAs from the nucleus to the cytoplasm. The nuclear abundance of Mavs, Traf3 and Traf6 transcripts was lower, while cytoplasmic 

abundance of these transcripts was greater, in macrophages in which DDX46 was silenced than in those in which it was not silenced (Fig. 3c,d), which suggested that DDX46 increased the retention of these antiviral transcripts in the nucleus. Also, overexpression of wildtype DDX46 significantly increased the retention of Mavs, Traf3, and Traf6 transcripts in the nucleus, and overexpression of DDX46 with a mutant ATP-binding LAT (Leu-Ala-Thr) domain (LET (Leu-Glu-Thr)) still increased retention in the nucleus, while overexpression of DDX46 with a mutant DEAD (Asp-Glu-Ala-Asp) domain (HGAD (His-Gly-Ala-Asp)) failed to increase retention of these transcripts in the nucleus (Fig. 3e,f). Furthermore, we used a fluorescent in situ aDVANCE ONLINE PUBLICATION  nature immunology

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Articles hybridization (FISH) approach to monitor the intracellular distribution of Mavs transcripts and observed that Mavs mRNA was located mainly in the cytoplasm after knockdown of DDX46, while it was located mainly in the nucleus after overexpression of DDX46, but the intracellular distribution of Tbk1 transcripts was not influenced by DDX46 (Fig. 3g,h and Supplementary Fig. 3c). Moreover, DDX46 was found to be located in nuclear speckles (Supplementary Fig. 3d). Hence, DDX46 maintained retention of Mavs, Traf3 and Traf6 mRNA in the nucleus, and this was dependent on the helicase-activity DEAD domain of DDX46 but not its ATP-binding LAT domain. As DDX46 was found to maintain retention of Mavs, Traf3, and Traf6 mRNA in the nucleus, we next assessed the expression of the antiviral molecules encoded, after knockdown of DDX46. Knockdown of DDX46 significantly increased the expression of MAVS, TRAF3 and TRAF6 protein, while overexpression of DDX46 decreased the expression of these proteins, but knockdown of DDX46 had no effect on the expression of TRPM3, LRRC4C or RNF125 protein, which are encoded by mRNAs that also contain the CCGGUU motif (Fig. 3i,j and Supplementary Fig. 3e,f). Together with the data showing that DDX46 maintained retention of these antiviral transcripts in the nucleus, we concluded that DDX46 negatively regulated the production of type I interferons triggered by viral infection by inhibiting the protein expression of MAVS, TRAF3 and TRAF6, which are essential components of antiviral signaling.

Moreover, we replaced each of those sites specific to post-translational modification and found that mutant DDX46 with replacement of the lysine residue at position 479 with arginine (K470R) lost its binding to ALKBH5, but the substitutions in other DDX46 mutants had no effect (Fig. 4g and Supplementary Fig. 4d). Additionally, the inhibitory effect of DDX46 on MAVS-induced activation of the IFN-β luciferase reporter was significantly diminished when the DDX46 K470R mutant was used (Supplementary Fig. 4e). These data indicated that viral infection induced acetylation of DDX46 at Lys470, which was responsible for the interaction between DDX46 and ALKBH5 after viral infection. We further constructed plasmids expressing various truncated fragments of DDX46 and ALKBH5. Binding of DDX46 to ALKBH5 was found to be dependent on the interaction between the DEAD domain of DDX46 and the nucleotide-recognition NRL domain of ALKBH5 (Fig. 4h,i). DDX46 lacking its DEAD domain was no longer able to inhibit the MAVS-induced activation of the IFN-β luciferase reporter, while the DDX46 fragment containing only a DEAD domain retained this function, and the DEAD domain of DDX46 was also able to reduce the expression of MAVS (Supplementary Fig. 4f). Therefore, the DEAD domain of DDX46 and the NRL domain of ALKBH5 were responsible for their interaction and function, consistent with the observation that the DEAD domain of DDX46 induced the retention of RNA in the nucleus.

DDX46 recruits ALKBH5 after VSV infection To investigate the molecular mechanisms responsible for the DDX46mediated retention of Mavs, Traf3 and Traf6 transcripts in the nucleus, we immunoprecipitated DDX46 from lysates of uninfected or VSVinfected peritoneal macrophages, then used mass spectrometry to identify DDX46-associated proteins. Among the DDX46-interacting proteins detected in the immunoprecipitates, we selected the m6A eraser ALKBH5 as the candidate molecule to investigate because of its potential roles in regulating the export of mRNA from the nucleus and RNA metabolism26 (Supplementary Fig. 4a). We confirmed the interaction between DDX46 and ALKBH5 by immunoprecipitation (Fig. 4a), and we found that the endogenous interaction between DDX46 and ALKBH5 was significantly enhanced by infection of cells with VSV (Fig. 4b). We also found that interaction between DDX46 and ALKBH5 occurred after stimulation of cells with various pathogens (Supplementary Fig. 4b). We then investigated the potential role of ALKBH5 in VSV-infectiontriggered production of type I interferons. Knockdown of ALKBH5 significantly increased the VSV-infection-triggered production of type I interferons (Fig. 4c,d), while stable overexpression of ALKBH5 inhibited interferon production (Fig. 4e,f), which was a phenocopy of our results obtained for DDX46. ALKBH5 was also able to inhibit MAVS-induced activation of the IFN-β luciferase reporter and reduce the expression of MAVS, while another DDX46-bound serine-threonine kinase, SRPK2, failed to influence activation of the IFN-β luciferase reporter (Supplementary Fig. 4c). Thus, DDX46 and ALKBH5 might act cooperatively in negatively regulating the production of type I interferons. To elucidate the DDX46–ALKBH5 interaction further and to understand why their interaction increased after viral infection, we assessed post-translational modifications (including methylation and acetylation) of DDX46 that affected its activation after viral infection, by mass spectrometry. We found that after viral infection, five lysine residues of DDX46 (Lys316, Lys344, Lys394, Lys736 and Lys776) were methylated and seven lysine residues of DDX46 (Lys272, Lys344, Lys363, Lys394, Lys470, Lys716 and Lys779) were acetylated (Supplementary Table 3).

DDX46 recruits ALKBH5 to erase the m6A modification We sought to determine how the DDX46–ALKBH5 interaction might regulate antiviral immune responses. We found that inhibition of the production of type I interferons mediated by overexpression of DDX46 was ‘rescued’ by knockdown of ALKBH5 (Supplementary Fig. 5a), which suggested that knockdown of ALKBH5 counteracted the effect of the overexpression of DDX46. Accordingly, overexpression of ALKBH5 reversed the increase in the production of type I interferons mediated by knockdown of DDX46 (Supplementary Fig. 5b). Moreover, in RAW264.7 macrophages in which was ALKBH5 knocked out, knockdown or overexpression of DDX46 failed to modulate the production of type I interferons induced by infection with VSV (Fig. 5a,b and Supplementary Fig. 5c). Thus, this showed that the role of DDX46 in the negative regulation of the production of type I interferons was dependent on ALKBH5. As ALKBH5 is an m6A eraser of RNA methylation, we investigated the m6A modification of RNA substrates after the recruitment of DDX46 and ALKBH5. We immunoprecipitated mRNA, with anti-m6A, from mRNA isolated from macrophages and found that the anti-m6A-bound fraction showed enrichment for not only the negative control (Gapdh mRNA) but also all mRNAs tested, especially the positive control (Tns3 mRNA)32, relative to the abundance of these mRNAs in fractions obtained with IgG (Fig. 5c); this confirmed the presence of m 6A sites on these transcripts. We also found that Rnf125, Trpm3 and Lrrc4c, which contain the CCGGUU motif (according to the iCLIP data) but whose encoded protein expression was not regulated by DDX46, did not have an m6A modification (Supplementary Fig. 5d). The anti-m6A-bound fraction showed greater enrichment for Mavs, Traf3 and Traf6, but not Gapdh, when DDX46 or ALKBH5 was knocked down, but less enrichment when the m 6A ‘writer’ METTL3 was knocked down (Fig. 5d). This indicated that the m6A sites on these potential DDX46-bound mRNAs were the direct substrates subject to ALKBH5-catalyzed demethylation. Furthermore, the enrichment for Mavs, Traf3 and Traf6 mRNA in fractions immunoprecipitated from macrophages with anti-m6A decreased after overexpression

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Articles Erasure of the m6A modification of mRNAs can induce retention of the mRNAs, such as Arntl and Per2, which encode molecules involved in the circadian rhythm27, in the nucleus. We found that

of DDX46 or ALKBH5 (Fig. 5e). These observations further supported the proposal that DDX46 was able to recruit ALKBH5 to erase the m6A modification of its substrates.

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Figure 4  DDX46 recruits ALKBH5 to inhibit VSV-triggered production of type I interferons. (a) Immunoprecipitation (with anti-Flag) and immunoblot analysis (IB) of HEK293T cells co-transfected to express various combinations (above lanes) of Flag-tagged DDX46 and Myc-tagged ALKBH5 (left) or Flag-tagged ALKBH5 and Myc-tagged DDX46 (right) and infected for 0 or 6 h (above lanes) with VSV (IP; top blots), and immunoblot analysis of whole-cell lysates without immunoprecipitation (WCL; bottom blots). (b) Immunoprecipitation (with anti-DDX46 (left two lanes) or IgG (far right lane)) and immunoblot analysis of lysates of macrophages infected for 0 or 6 h (above lanes) with VSV (MOI = 10). (c) qRT-PCR analysis of Alkbh5 mRNA (left) and immunoblot analysis of ALKBH5 (right) in macrophages transfected for 48 h with non-targeting control siRNA (NC) or ALKBH5-specific siRNA (siALKBH5 (one of two siRNA constructs (1, 2)); qRT-PCR results are presented relative to those of Actb. (d) qRT-PCR analysis of Ifnb (left) and ELISA of IFN-β in supernatants (right) of macrophages transfected as in c and infected for 0 or 12 h (horizontal axis) with VSV (MOI = 10); qPCR results resented as in c. (e,f) Immunoblot analysis of ALKBH5 (e), qRT-PCR analysis of Ifnb mRNA (f, left) and ELISA of IFN-β in the supernatants (f, right) of RAW264.7 cell clones with (ALKBH5) or without (Control) stable overexpression of Myc-tagged ALKBH5, left uninfected (e) or infected for 0, 8 or 12 h (horizontal axes) with VSV (MOI = 10) (f). (g–i) Immunoprecipitation and immunoblot analysis (left margin) of HEK293T cells transfected to express (above lanes) Myc-tagged wild-type DDX46 or various substitution mutants of DDX46, together with Flag-tagged ALKBH5 (g), Myc-tagged wild-type DDX46 or various truncation mutants of DDX46 (above blots), together with Flag-tagged ALKBH5 (h), or Myc-tagged wild-type ALKBH5 or various truncation mutants of ALKBH5 (above blots), together with Flagtagged DDX46 (i), and infected for 6 h with VSV. Numbers at top (h,i) indicate amino acid positions. HELICs, helicase superfamily carboxy-terminal domain; DSBH, double-stranded β-helix-fold. *P < 0.01 (Student’s t-test). Data are from three independent experiments with similar results (a,b,c (right), e,g–i) or are representative of three independent experiments (c (left), d,f; mean + s.d. of technical triplicates).



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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

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Figure 5  DDX46 affects the m6A modification of transcripts via recruitment of ALKBH5 after viral infection. (a,b) qRT-PCR analysis of Ifnb mRNA in Alkbh5+/+ or Alkbh5−/− RAW264.7 cells (key) transfected with control or DDX46-expressing plasmid (below plot) (a) or with control or DDX46specific siRNA (below plot) (b) and infected for 12 h with VSV (MOI = 10). (c) Abundance of Gapdh, Mavs, Traf3, Traf6 and Tns3 transcripts among mRNA immunoprecipitated, with IgG or anti-m 6A (key), from macrophages; results are presented relative to those obtained with IgG. (d,e) qRT-PCR analysis of Gapdh, Mavs, Traf3 and Traf6 transcripts among mRNA immunoprecipitated, with anti-m 6A, from RAW264.7 after transfected for 72 h with control or DDX46-, ALKBH5- or METTL3-specific siRNA (key) (d) or transfected for 48 h with a control or DDX46- or ALKBH5-expressing plasmid (key) (e); results are presented relative to those obtained with IgG. (f,h) RT-PCR analysis of Mavs, Traf3 and Traf6 mRNA in the nucleus or cytoplasm (above lanes) of RAW264.7 cells transfected for 48 h with control or ALKBH5-specific siRNA (above gels) (f) or transfected for 24 h with control plasmid or plasmid expressing wild-type ALKBH5 or an ALKBH5 mutant lacking demethylation activity (H205A) (above gels) (h), then infected with for 6 h VSV (MOI = 10). (g,i) qRT-PCR analysis of the distribution of Mavs, Traf3 and Traf6 mRNA in the nucleus and cytoplasm of RAW264.7 cells treated as in f (g) or h (i); results presented relative to those of Gapdh (cytoplasm) or Rnu6 (nucleus). (j,k) FISH analysis of Mavs transcripts in RAW264.7 cells treated as in f (j) or h (k). Scale bars, 5 µm. (l) Immunoblot analysis of ALKBH5, MAVS, TRAF3 and TRAF6 in macrophages transfected for 72 h with control or ALKBH5-specific siRNA (above blots) and infected for 0, 3 or 6 h (above lanes) with VSV (MOI = 10). *P < 0.01 (Student’s t-test). Data are representative of three independent experiments (a–e,g,i; mean + s.d. of technical triplicates) or are from three independent experiments with similar results (f,h,j–l).

knockdown of ALKBH5 decreased the abundance of the antiviral Mavs, Traf3, and Traf6 transcripts in the nucleus but increased their abundance in the cytoplasm (Fig. 5f,g). Overexpression of ALKBH5 increased the abundance of these antiviral transcripts in the nucleus but decreased their abundance in the cytoplasm, while overexpression of an ALKBH5 mutant lacking demethylation activity failed to increase their retention in the nucleus (Fig. 5h,i). FISH analysis also confirmed that knockdown of ALKBH5 reduced the retention of Mavs mRNA in the nucleus, while overexpression of ALKBH5 increased its retention in the nucleus (Fig. 5j,k). Moreover, the ALKBH5 mutant that lacked demethylation activity had no effect on the intracellular localization of Mavs mRNA (Fig. 5k). We also found that the DDX46–MAVS complex overlapped nature immunology  aDVANCE ONLINE PUBLICATION

ALKBH5 in nuclear speckles (Supplementary Fig. 5e), and knockdown of ALKBH5 significantly increased the expression of MAVS, TRAF3 and TRAF6 protein (Fig. 5l). Hence, these data demonstrated that DDX46 recruited ALKBH5 to erase the m6A modification of those antiviral transcripts and thus induced their retention in the nucleus and decreased their translation into protein. These data also indicated that the regulatory effect of DDX46 on the expression of protein from its target genes depended on both a CCGGUU motif and m 6A modification of the target mRNAs. In vivo knockdown of DDX46 enhances antiviral responses To further investigate the function of DDX46 in vivo, we created targeted deletion of DDX46 in mice by removing exon 2 of the genomic 

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Figure 6  In vivo knockdown of DDX46 enhances the innate antiviral response. (a) Quantification of Ddx46+/+, Ddx46+/− or Ddx46−/− mice (horizontal axis) born or detected as embryos (above plot). (b) Immunoblot analysis of total DDX46, MAVS, TRAF3 and TRAF6 and phosphorylated TBK1 in lysates of Ddx46+/+ or Ddx46+/− macrophages (above blots) infected for 0, 3 or 6 h (above lanes) with VSV (MOI = 10). (c,d) ELISA of IFN-β in serum (c) and qRT-PCR analysis of Ifnb mRNA in the liver, spleen and lungs (below plots) (d) of Ddx46+/+ or Ddx46+/− mice (key) 12 h after intraperitoneal injection of PBS or VSV (1 × 108 plaque-forming units per gram of body weight) (horizontal axes); qRT-PCR results presented relative to those of Actb. (e,f) qRT-PCR analysis of intracellular VSV RNA (e) and the half-maximal tissue-culture infectious dose of VSV (f) in the liver, spleen and lungs of Ddx46+/+ or Ddx46+/− mice infected as in c,d. (g) Hematoxylinand-eosin staining of lung sections from Ddx46+/+ or Ddx46+/− mice treated as in c,d. Scale bars, 50 µm. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three experiments (a) or three independent experiments (c–f; mean + s.d. of technical triplicates) or are from three independent experiments with similar results (b,g).

Ddx46 locus using the genome-editing technology TALEN (‘transcription-activator-like effector nuclease’). We found that Ddx46−/− mice died in utero (Fig. 6a), but Ddx46+/− progeny reached adult and seemed to be smaller than their wild-type littermates (data not shown). Evaluation of DDX46 showed that its expression in macrophages from Ddx46+/− mice was significantly lower than its expression in macrophages from their wild-type littermates, and the expression of MAVS, TRAF3 and TRAF6 and VSV-infection-induced phosphorylation of TBK1 were significantly higher in macrophages from Ddx46+/− mice than in those from their wild-type littermates (Fig. 6b). We observed greater IFN-β production and a lower viral burden in Ddx46+/− mice than in their wild-type littermates (Fig. 6c–f) and, 

accordingly, found less viral-infection-mediated pathology, as well as less infiltration of polymorphonuclear cells and interstitial pneumonitis in the lungs, in Ddx46+/− mice than in their wild-type littermates, after VSV challenge (Fig. 6g). We also found a greater increase in inflammatory cytokines and Ifnb production in Ddx46+/− macrophages than in their wild-type counterparts after infection with VSV or stimulation with LPS (Supplementary Fig. 6a,b). These data convincingly demonstrated that the downregulation of DDX46 promoted interferon production by upregulating the expression of MAVS, TRAF3 and TRAF6 in vivo. On the basis of our findings, we propose the following working model to explain how nuclear DDX46 negatively regulates the production of type I interferons in the antiviral innate response (Supplementary Fig. 6c). Viral infection significantly enhances the binding of DDX46 to Mavs, Traf3 and Traf6 transcripts and recruits the m6A eraser ALKBH5 to demethylate the m6A modification of these antiviral transcripts. That demethylation in turn causes retention of these antiviral transcripts in the nucleus and leads to decreased expression of these antiviral molecules (as protein) in the cytoplasm, which consequently inhibits the production of type I interferons. DISCUSSION In this study, we demonstrated that nuclear DDX46 acted as a negative regulator of antiviral innate responses by binding the m6A eraser ALKBH5 and inducing retention of Mavs, Traf3 and Traf6 transcripts in the nucleus. DDX46 is a component of the 17S U2 small nuclear ribonucleic particle complex, which has an essential role in pre-mRNA splicing before or during prespliceosome assembly 28,33. DDX46 is reported to be required for the multi-lineage differentiation of hematopoietic stem cells and the development of the digestive organs and brain in zebrafish34,35. Here we found that DDX46 was a negative regulator of the production of type I interferons after viral infection and thus might help to prevent the overactivation of antiviral innate responses. Furthermore, we hypothesize that DDX46 operates in prespliceosome assembly to help with RNA splicing in uninfected conditions, but that after viral infection, DDX46 alters its binding ‘preferences’ to specific antiviral transcripts such as Mavs, Traf3 and Traf6 and then recruits ALKBH5. That protein–mRNA complex induces retention of those antiviral transcripts in the nucleus and inhibits the production of type I interferons. Therefore, we speculate that DDX46 might show considerable potential as a new target for the development of therapies directed against viral infection and inflammatory disease. We cannot at this time exclude the possibility that DDX46 might also target the mRNA from interferon-stimulated genes or from other host genes encoding molecules associated with viral replication. Our data have revealed the sequence selectivity for the recognition of diverse RNAs, including antiviral transcripts, by DDX46. We found that the sequences of DDX46-bound mRNA targets showed enrichment for CCGGUU elements. That binding site was different from those of other splicing factors and RNA-binding proteins previously described, such as UGGACC elements for the splicing factor SRSF1 in pre-mRNA processing and CA- or CT-repeat elements for DDX17 (refs. 25,36), which suggests that the CCGGUU element in diverse RNAs is indeed a specific binding site for DDX46. Notably, both DDX46 and RIG-I were induced to bind RNA transcripts after viral infection, but RIG-I, not DDX46, initiates the production of type I interferons37. This phenomenon might be due to their different recognition selectivity, as DDX46 detects CCGGUU elements of transcripts but RIG-I detects viral 5′-triphosphorylated double-stranded RNA. Additionally, DDX46 also binds other mRNA transcripts but does aDVANCE ONLINE PUBLICATION  nature immunology

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Articles not influence the expression of the proteins encoded when the transcripts lack m6A modification. We speculate that DDX46 might regulate the expression of those genes under some circumstances in which the mRNAs are m6A-methylated by their corresponding writers, which might represent another layer of the ‘fine tuning’ of gene expression. The retention of mRNA in the nucleus has an important role in the regulation of gene expression, which is mediated by a set of nuclear factors, such as the splicing factors MBNL1 and U2AF65 (ref. 38). It has been shown that RNAs retained in the nucleus include mainly long noncoding RNAs, hyper-edited mRNAs and incompletely spliced mRNAs, rather than the mature protein-coding mRNAs39,40. However, the abundance of many mature protein-coding mRNAs has been shown to be higher in the nucleus than in the cytoplasm41. We also found that DDX46 was able to induce the retention of antiviral transcripts in the nucleus after viral infection. Mature mRNAs, mRNAs retained in the nucleus and pre-mRNAs can be degraded in the nucleus42. However, whether the antiviral transcripts whose retention of in the nucleus was induced by DDX46 are degraded or not still needs to be investigated further. Given the wide range of mRNAs retained in the nucleus, it seems that retention of mRNA in the nucleus is a meaningful but previously underappreciated step in the life cycle of mRNA. Our study might prompt future work exploring RNA-modification-based regulation of gene expression in diverse physiological and pathological processes. We have demonstrated that erasure of the m6A methylation of antiviral transcripts by ALKBH5 led to their retention in the nucleus; future studies should investigate which specific m6A site within these antiviral transcripts are sufficient for retention in the nucleus. Additionally, we suggest that the ability of DDX46 to bind ALKBH5 might be important for the inhibitory effect of DDX46 on interferon production, because the DDX46 mutant lacking the ability to bind ALKBH5 had considerably diminished ability to mediate inhibition of the activation of IFN-β. We speculate that the viral-infectionenhanced interaction between DDX46 and ALKBH5 might be more important than their constitutive interaction in initiating ALKBH5mediated m6A-demethylation of the antiviral Mavs, Traf3 and Traf6 transcripts and consequent inhibition of the induction of IFN-β. This issue needs further investigation to reveal details of the mechanisms of the enhanced interaction between DDX46 and ALKBH5 in response to viral infection. Methods Methods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank T. Fang, M. Jin and Y. Li for technical assistance; X. Liu, C. Han, S. Xu, Y. Han and T. Chen for discussions; and the SIDANSAI Biotechnology Company (Shanghai, China) for helping to generate Ddx46+/− mice. Supported by the National Key Basic Research Program of China (2013CB530503 and 2012CB518900), the National Natural Science Foundation of China (31390431, 81422037 and 81671564) and the CAMS Innovation Fund for Medical Sciences (2016-12M-1-003). AUTHOR CONTRIBUTIONS Q.Z., J.H., Y.Z., and Z.L. performed the experiments; Q.Z., J.H. and X.C. analyzed data and wrote the paper; and X.C. designed and supervised the research. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

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Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1. Schlee, M. & Hartmann, G. Discriminating self from non-self in nucleic acid sensing. Nat. Rev. Immunol. 16, 566–580 (2016). 2. Ivashkiv, L.B. & Donlin, L.T. Regulation of type I interferon responses. Nat. Rev. Immunol. 14, 36–49 (2014). 3. Gack, M.U. et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-Imediated antiviral activity. Nature 446, 916–920 (2007). 4. Cao, X. Self-regulation and cross-regulation of pattern-recognition receptor signalling in health and disease. Nat. Rev. Immunol. 16, 35–50 (2016). 5. Zheng, Q. et al. Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27. Cell Res. 25, 1121–1136 (2015). 6. Portilho, D.M. et al. Endogenous TRIM5α function is regulated by SUMOylation and nuclear sequestration for efficient innate sensing in dendritic cells. Cell Rep. 14, 355–369 (2016). 7. Ansari, M.A. et al. Herpesvirus genome recognition induced acetylation of nuclear IFI16 is essential for its cytoplasmic translocation, inflammasome and IFN-b responses. PLoS Pathog. 11, e1005019 (2015). 8. Kim, T.W. et al. Transcriptional repression of IFN regulatory factor 7 by MYC is critical for type I IFN production in human plasmacytoid dendritic cells. J. Immunol. 197, 3348–3359 (2016). 9. Loo, Y.M. & Gale, M. Jr. Immune signaling by RIG-I-like receptors. Immunity 34, 680–692 (2011). 10. Zhang, Z. et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat. Immunol. 12, 959–965 (2011). 11. Parvatiyar, K. et al. The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response. Nat. 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35. Hozumi, S. et al. DEAD-box protein Ddx46 is required for the development of the digestive organs and brain in zebrafish. PLoS One 7, e33675 (2012). 36. Zarnack, K. et al. Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell 152, 453–466 (2013). 37. Weber, M. et al. Incoming RNA virus nucleocapsids containing a 5′-triphosphorylated genome activate RIG-I and antiviral signaling. Cell Host Microbe 13, 336–346 (2013). 38. Sun, X. et al. Nuclear retention of full-length HTT RNA is mediated by splicing factors MBNL1 and U2AF65. Sci. Rep. 5, 12521 (2015).

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aDVANCE ONLINE PUBLICATION  nature immunology

ONLINE METHODS

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Mice. C57BL6/J mice (6–8 weeks old) were obtained from the Joint Ventures Sipper BK Experimental Animal Company (Shanghai, China). Ddx46+/− mice were generated by the TALEN approach. Mice were housed and bred in specific pathogen-free conditions. All animal experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Second Military Medical University (Shanghai, China). Cell culture and transfection. HEK293T, THP-1 and RAW264.7 cell lines were obtained from American Type Culture Collection (ATCC) and were cultured as described previously43. Mouse peripheral blood mononuclear cells, myeloid conventional dendritic cells and plasmacytoid dendritic cells were obtained and cultured as described previously44. Jet-PEI or Jet-PRIME transfection reagents (Polyplus-transfection, Illkirch, France) were used for co-transfection of plasmids or plasmids and RNAs according to the manufacturer’s instructions. Thioglycollate-elicited mouse peritoneal macrophages were transfected with siRNA using INTERFERin (Polyplus-transfection) according to the manufacturer’s instructions. To establish stably transfected RAW264.7 cells, G418 was added (1,000 µg/ml) 48 h after transfection and was maintained at 800 µg/ml for 3 weeks for positive selection. For stably transfected cells, expression of DDX46 and ALKBH5 was confirmed by immunoblot analysis. Stably transfected RAW264.7 cells were subsequently cultured in complete medium with 500 µg/ml G418. All cells tested negative for mycoplasma contamination. Reagents and pathogens. Antibodies to the following were used for immunoprecipitation (IP), immunoblot analysis (IB) and immunofluorescence (IF): β-actin (sc-8432, 1:1,000 for IB), TRAF6 (sc-7721, 1:500 for IB), TRAF3 (sc949, 1:500 for IB) and HRP-coupled secondary antibody (sc-2749, 1:1,000 for IB), all from Santa Cruz Biotechnology; DDX46 (ab72083, 1:1,000 for IB and 1:200 for IP or IF), TRPM3 (ab56171, 1:1,000 for IB), LRRC4C (ab111572, 1:1,000 for IB), RNF125 (ab74373, 1:1,000 for IB) and ALKBH5 (ab174124, 1:1,000 for IB and 1:200 for IF), all from Abcam; and MAVS (4983, 1:1,000 for IB), TBK1 (3013, 1:1,000 for IB), phosphorylated TBK1 (5483, 1:1,000 for IB), phosphorylated p65 (3033, 1:1,000 for IB), phosphorylated IκBα (9246, 1:1,000 for IB), IκBα (9247, 1:1,000 for IB), phosphorylated IRF3 (4947, 1:1,000 for IB) and Myc-tag (2278, 1:1,000 for IB), all from Cell Signaling Technology. The Flag tags (F7425, 1:1,000 for IB) as well as the agarose used for IP were from Sigma-Aldrich. DAPI (62247, 1:1,000 for IF), Alexa Fluor 488 (A-21202, 1:500 for IF) and Alexa Fluor 546 (A-11071, 1:500 for IF) were from Thermo Fisher Scientific. Antibody specific to m6A (202003, 1:200 for IP) was from Synaptic Systems. VSV was propagated and amplified by infection of a monolayer of Vero cells. The supernatant was harvested 24 h later and clarified by centrifugation. Viral titers were determined by evaluation of the half-maximal tissue-culture infectious dose (TCID50) in Vero cells5. IFN-β was measured with an ELISA kit (PBL Biomedical Laboratories). RNA interference. Macrophages or RAW264.7 cells were transfected with siRNA (final concentration, 20 nM) using the INTERFERin reagent (Polyplus-transfection). All siRNAs were obtained from GenePharma (Supplementary Table 4). RNA fractionation, RNA extraction and RT-PCR. RAW264.7 cells were transfected with siRNA or plasmid(s). At 48 h after transfection, total RNA was extracted via TRIZOL reagent (Invitrogen). Nucleo-cytoplasmic fractionation of RNA was performed as follows: cells were washed once with PBS buffer and were lysed for 5 min on ice in lysis buffer (10 mM Tris-HCl, 140 mM NaCl, 1.5 mM MgCl2, 10 mM EDTA, 0.5% NP-40 and 40 U/ml RNasin, pH 7.4). After centrifugation at 12,000g for 5 min, the supernatants containing the cytoplasmic fraction of RNA were collected. The remaining nuclear pellets were rinsed with lysis buffer twice and were finally centrifuged as the nuclear fraction. Cytoplasmic and nuclear RNA were then extracted by TRIZOL reagent. RNA was reversed-transcribed using the Reverse Transcription System from Toyobo (Osaka, Japan). The reverse-transcription products from samples in the various treatment conditions were amplified by real-time PCR and analyzed as described previously44. The primer sequences for qPCR analysis are listed in Supplementary Table 5.

doi:10.1038/ni.3830 

Molecular cloning of related genes. The genes studied here were obtained from mouse macrophages by RT-PCR and were subsequently cloned into pcDNA vectors. Each construct was confirmed by sequencing. The corresponding primers used in this study are listed in Supplementary Tables 6 and 7. Fluorescence in situ hybridization and confocal microscopy. RAW264.7 cells were transfected with siRNA or plasmid(s) and were fixed in 4% paraformaldehyde 48 h after transfection. Fluorescence in situ hybridization (FISH) was performed as previously described21, and images were obtained with a Leica TCS SP2 confocal laser microscope LSM 510. Probes of Mavs transcripts with 5′-biotin modification were designed by Stellaris: 5′-CAA ATG CAG AGG GTC CAG AA-3′; 5′-TGC TAT GGG AAC AGA GGC AA-3′; 5′-GAC AAG AGG TTT GTC CTC AG-3′; 5′-TGA AAC AGG TGG CAG CTT TG3′; 5′-ATT GGT GAG CAC AGT AGA CG-3′; 5′-AGG AAT ACA GCC AAG AGT GC-3′; 5′-GTC AGG AGC AAT GGA GGT AA-3′; 5′-TCA GAA GAT CTG GGC GAG AT-3′; 5′-AAG AGT CCA CAG AGA ACC TC-3′; 5′-GCT AGA CAT CTG CTA AAG GC-3′. Probes of Tbk1 transcripts with 5′-biotin modification were designed by Stellaris: 5′-GCC GTT CTC TCG GAG ATG AT-3′; 5′-ACA GAG ACA CAA ACT GCT CA-3′; 5′-ATG GTA GAA TGT CAC TCC AA-3′; 5′-TTC TGG AAG TCC ATA CGC AT-3′; 5′-TGA CAG CAT AGA GAT CAC CA-3′; 5′-TGT AAA TCT TAT GCG CCG TC-3′; 5′CTG GCA GTT CTG CAG GCG TA-3′; 5′-TGC TGA TGT CCT GAA GAC TG-3′; 5′-TCA TTA TAA GCT AGT CTG CG-3′; 5′-GAG GCA GAG TTT CTT GTA AC-3′. Luciferase reporter assays. HEK293T cells (1 × 105) were plated in 24-well plates, then were transfected with a mixture of IFN-β luciferase reporter plasmid and pRL-TK-Renilla luciferase plasmid, together with various amounts of the appropriate control or protein-expressing plasmid(s). An empty pcDNA3.1 vector was used to maintain an equal amount of DNA among wells. Cells were collected at 24 h after transfection, and luciferase activity was measured with a Dual-Luciferase Assay (Promega) with a Luminoskan Ascent Luminometer (Thermo Scientific) as described previously 44. Reporter gene activity was determined by normalization of firefly luciferase activity to renilla luciferase activity. Immunoblot and immunoprecipitation. Cells were lysed using Cell Lysis Buffer (Cell Signaling Technology) supplemented with cocktail protease inhibitor (Calbiochem). Protein concentrations of the extracts were measured using a BCA assay (Pierce) and were equalized with the extraction reagent. Equivalent amounts of extract were loaded onto gels and separated by SDSPAGE and then transferred onto nitrocellulose membranes, then were blotted as described previously45 (antibodies identified above). Nanospray liquid chromatography-tandem mass spectrometry analysis of DDX46-associated protein and post-translational modifications. Macrophages were infected with or without VSV (6 h), then were lysed for immunoprecipitation with anti-DDX46 (identified above). After silver staining, the DDX46-specific band and the bands with a more-intense signal at 6 h after infection than in uninfected cells were cut and digested, followed by analysis by reverse-phase nanospray liquid chromatography–tandem mass spectrometry. The spectra from tandem mass spectrometry were automatically used for searching against the nonredundant International Protein Index mouse protein database (version 3.72) with the Bioworks browser (rev.3.1). iCLIP assay and data analysis. We followed the previously reported protocol46 with the following modifications. In brief, RAW264.7 cells were infected with or without VSV for 6 h, then were subjected to crosslinking with 0.4 J/cm2 of 254 nm UV light in a crosslinker HL-2000 (UVP). Cells were lysed with NP-40 lysis buffer on ice for 30 min and treated with RNAase I (0.2U/ml) for 5 min. After removal of cell debris, the crude lysates were incubated with anti-DDX46 or IgG (identified above) overnight at 4 °C. After immunoprecipitation, a minority of the beads (less than half) were left for linking biotin-labeled linker for further detection, while the majority of the beads (more than half) were linked with non-radioactive linker. After being run on a 4–12% NuPAGE gel (Invitrogen NP0321B0X), the protein–RNA complexes were transferred to

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PVDF membrane. Biotin-labeled RNA was detected and visualized according to the instructions of the chemiluminescent nuclei acid kit (Thermo 89880). Unlabeled protein–RNA complexes were cut from the membrane corresponding to the visualized size of DDX46. RNA was isolated from the solution with phenol-chloroform and subjected to small-RNA library construction. iCLIP libraries were sequenced by standard Illumina protocols with 50-nucleotide single end runs. Bioinformatics and motif-enrichment analysis were performed as described previously47.

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RNA-binding protein immunoprecipitation (RIP) qRT-PCR. This procedure was adapted from a published report48. For RIP qRT-PCR, it was performed as described for the published RIP-seq assay48, but without the generation of a cDNA library. For comparison of DDX46-binding ability, relative enrichment was first normalized to input and then analyzed by comparison to the data from the sample immunoprecipitated with anti-DDX46. All samples were analyzed in triplicate for qPCR. m6A qRT-PCR. This procedure was adapted from a published report 25. For m6A qRT-PCR, total RNA was first subjected to mRNA purification by poly(A) selection (FastTrack MAG Micro mRNA isolation kit, invitrogen), but not randomly fragmented to facilitate reverse transcription with oligo(dT) and PCR amplification. Then, the analysis was performed as described for the published m6A-seq assay25 but without the generation of a cDNA library. For comparison of the change in abundance of m6A, relative enrichment was first normalized to inputs then analyzed by comparison of the data from the sample immunoprecipitated with anti-m6A. All samples were analyzed in triplicate for qPCR. RNA-seq and data analysis. Total RNA was isolated using TRIZOL Reagent (Invitrogen). cDNA library was constructed using TruSeq RNA Sample Prep Kit and then sequenced with HiSeq 2000 system (Illumina Inc.). The expression of transcripts was quantified as reads per kilobase of exon model per million mapped reads (RPKM). The change in splicing isoforms was analyzed using MISO software with the annotation of all known alternative splicing events, and we filtered the results based on the ‘percent spliced in’ values (selected by bayes_factor> = 2 and diff> = 0.2 or diff< = -0.2)49. CRISPR-Cas9–mediated depletion of ALKBH5. For the depletion of ALBKH5 in RAW264.7 cells, PX330-GFP plasmid (encoding guide RNA, the endonuclease Cas9 and green fluorescent protein) with guide RNA specific to Alkbh5 was constructed by Shanghai Biomodel Organism Science & Technology Development Company (Shanghai, China), After transfection of the plasmid into RAW264.7 cells, cells with green fluorescence were then sorted by using a Gallios Flow Cytometer (Beckman Coulter). Sorted cells were cultured for 3–5 d, and clones propagated from single cells were ‘picked out’. The depletion of Alkbh5 was confirmed by both immunoblot analysis

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(antibodies identified above) and DNA sequencing. The selected clones demonstrating unchanged ALKBH5 expression were used as wild-type clones, while the clones deficient in Alkbh5 were used as Alkbh5−/− clones. The target sequences for guide RNA were as follows: Alkbh5 guide RNA 1: 5′-GAC GTC CCG GGA CAA CTA CA-3′, Alkbh5 guide RNA 2: 5′-TTC GGC GAG GGC TAC ACG TA-3′. Generation of DDX46 knockout mouse mehydiated by TALEN. The DDX46knockout mouse was constructed by TALEN using microinjection technology as described previously50. Then, founder mice were identified by PCR assay and their DNA was sequencing using genotype-specific primers (Ddx46−/− primers) for analysis of genomic DNA obtained from mouse tails. Founder mice were hybridized with wild-type C57BL6/J mice to produce the mice identified and used for experiments. Statistical analysis. All experiments were independently repeated at least three times. Comparisons between two groups were performed using Student’s t-test. Data were analyzed with GraphPad Prism Software. Statistical values achieving a P value of