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N6-methyladenosine promotes induction of ADAR1-mediated A-to-I RNA editing to suppress aberrant antiviral innate immune responses

['Hideki Terajima', 'Department Of Chemistry', 'Department Of Biochemistry', 'Molecular Biology', 'Institute For Biophysical Dynamics', 'The University Of Chicago', 'Chicago', 'Illinois', 'United States Of America', 'Howard Hughes Medical Institute']

Date: None

Among over 150 distinct RNA modifications, N 6 -methyladenosine (m 6 A) and adenosine-to-inosine (A-to-I) RNA editing represent 2 of the most studied modifications on mammalian mRNAs. Although both modifications occur on adenosine residues, knowledge on potential functional crosstalk between these 2 modifications is still limited. Here, we show that the m 6 A modification promotes expression levels of the ADAR1, which encodes an A-to-I RNA editing enzyme, in response to interferon (IFN) stimulation. We reveal that YTH N 6 -methyladenosine RNA binding protein 1 (YTHDF1) mediates up-regulation of ADAR1; YTHDF1 is a reader protein that can preferentially bind m 6 A-modified transcripts and promote translation. Knockdown of YTHDF1 reduces the overall levels of IFN-induced A-to-I RNA editing, which consequently activates dsRNA-sensing pathway and increases expression of various IFN-stimulated genes. Physiologically, YTHDF1 deficiency inhibits virus replication in cells through regulating IFN responses. The A-to-I RNA editing activity of ADAR1 plays important roles in the YTHDF1-dependent IFN responses. Therefore, we uncover that m 6 A and YTHDF1 affect innate immune responses through modulating the ADAR1-mediated A-to-I RNA editing.

Funding: This work was supported by National Institutes of Health ( https://www.nih.gov/ ) HG008935 to CH. HT acknowledges support from the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship for Overseas Researchers and the Uehara Memorial Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Dysregulation in the expression of ADARs and A-to-I RNA editing is associated with a variety of physiological abnormalities including neuronal disorders [ 28 , 29 ], circadian rhythm disruption [ 30 , 31 ], and cancer progression [ 32 ]. Growing evidence shows a profound impact of ADAR1 on cancer immunotherapies through regulating IFN signaling. Loss of ADAR1 reduces cell viability in certain types of tumor cells expressing high levels of ISGs [ 33 , 34 ]. In addition, deletion of ADAR1 in tumor cells improves sensitivity to immunotherapy through enhancing IFN sensing and overcomes resistance to immune checkpoint blockage [ 35 ]. Therefore, a better insight into regulatory mechanisms of ADAR1 expression may provide further understanding to targeting ADAR1 in cancer therapies. Intriguingly, a recent study discovered a conserved m 6 A site in the ADAR1 transcript among several primates [ 36 ], implying potential roles of m 6 A-mediated regulation of ADAR1 expression. However, biological functions of m 6 A on the ADAR1 transcript have not been investigated. Here, we find that binding of YTHDF1 to m 6 A-modified mRNA enhances IFN-mediated ADAR1p150 induction. Knockdown of YTHDF1 attenuated global induction of A-to-I RNA editing upon IFN stimulation, resulting in activation of the dsRNA-sensing pathway. In the absence of YTHDF1, IFN stimulation causes enhanced innate immune responses, and virus replication in cells was suppressed by the elevated expression of ISGs. Our data uncover a new m 6 A-mediated regulation of A-to-I RNA editing and IFN response in innate immunity.

On the other hand, A-to-I RNA editing is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, ADAR1 and ADAR2, that specifically bind to double-stranded RNA (dsRNA) [ 17 , 18 ]. Among mammalian ADAR family members, editing activity of ADAR3 has not been detected. Because inosine base pairs preferentially with cytosine, A-to-I RNA editing can cause alteration in amino acid sequences [ 19 ], splicing [ 20 ], and dsRNA structures [ 21 , 22 ]. Recently, the ADAR1-mediated A-to-I RNA editing has emerged as a key regulatory factor that controls innate immune interferon (IFN) response [ 23 , 24 ]. Both ADAR1 knockout mice and knock-in mice expressing the catalytic-deficient ADAR1 (ADAR1 E861A/E861A ) exhibited embryonic lethality with elevated IFN-stimulated genes (ISGs) signature and apoptosis [ 24 , 25 ]. The lethality can be rescued by concurrent knockout of interferon induced with helicase C domain 1 (IFIH1, also called MDA5), a cytosolic sensor that recognizes viral dsRNA and induces the type I IFN-mediated antiviral immunity [ 25 , 26 ]. The molecular mechanism underlying these phenotypes is that ADAR1 unwinds dsRNA structure by RNA editing and prevents self-activation of the IFN response induced by endogenous dsRNA. Genomic mutations in ADAR1 were identified in patients with Aicardi–Goutières syndrome (AGS), a severe autoimmune disease with a high IFN signature [ 27 ]. These studies indicated that the appropriate regulation of IFN signaling by ADAR1 is essential for normal homeostasis of the innate immune system.

The profound impact of m 6 A modification on diverse biological functions and diseases have only recently been demonstrated [ 4 , 5 ]. The m 6 A modification exhibits biological functions through a series of m 6 A effector proteins generally referred to as writers, readers, and erasers, which deposit, recognize, and remove m 6 A, respectively. In mammals, the deposition of m 6 A on most mRNA is catalyzed by a large methyltransferase complex (writers) containing the core heterodimer of methyltransferase-like 3 (METTL3) and METTL14 [ 6 – 8 ]. m 6 A modification recruits m 6 A-binding proteins (readers) including several members of YTH family [ 9 ] that specifically recognize m 6 A to modulate various aspects of RNA metabolism. For example, YTH N 6 -methyladenosine RNA binding protein 1 (YTHDF1) promotes translation efficiency of m 6 A-modified mRNAs through recruiting translation initiation factors [ 10 , 11 ]. Another members of YTH family, YTHDF2, interacts with the CCR4-NOT deadenylase complex and facilitates degradation of its target mRNAs [ 12 , 13 ]. The removal of m 6 A is carried out by 2 demethylases (erasers), Fat mass and obesity-associated gene (FTO) [ 14 ], and alkB homolog 5 RNA demethylase (ALKBH5) [ 15 ]. The orchestration of writers and erasers functions suggests m 6 A as a reversible modification that can control dynamics of physiological processes [ 16 ].

Posttranscriptional regulation plays a pivotal role in ensuring proper gene expression in almost all organisms. An emerging new mechanism of posttranscriptional regulation is conducted through various RNA modifications. With over 150 distinct chemical modifications known to exist in different RNA species [ 1 ], new scientific discoveries and technological advances have prompted rapid development of the field of epitranscriptomics [ 2 , 3 ]. Among mRNA modifications, 2 of the most well-characterized are N 6 -methyladenosine (m 6 A) and adenosine-to-inosine (A-to-I) RNA editing.

Results

YTHDF1 enhances interferon responses Recent studies have shown that ADAR1-mediated A-to-I RNA editing disrupts secondary structures of dsRNA to prevent self-activation of cytosolic viral dsRNA sensors that induce IFN production and downstream ISGs, contributing to antiviral immunity [23]. These observations prompted us to explore potential roles of YTHDF1 in the IFN signaling. YTHDF1 knockdown significantly enhanced the induction of not only interferon beta 1 (IFNB1) but also interferon lambda 1 (IFNL1) and IFNL3 mRNAs following stimulation with IFN-α (Fig 3A). Consistent with this, the IFN-α–induced secretion of the IFN-β protein was higher in YTHDF1-deficient cells than that in control cells, whereas IFN-β was not detected in unstimulated cells (Fig 3B). IFNB1 mRNA is known to be m6A modified, and its stability could be affected by m6A in certain cells [47,48]. However, decay rates of IFNB1 and IFNL1 transcripts were not altered with YTHDF1 knockdown in A172 cells (S2A Fig). Thus, the up-regulation of IFN-β is not caused by the m6A-dependent regulation of the IFNB1 mRNA stability, but rather by signaling pathways upstream of IFNB1 transcription in this cell line. During dsRNA-sensing response, the transcription of IFNB1 is under the control of the transcription factor IFN regulatory factor 3 (IRF3) that is activated by TANK-binding kinase 1 (TBK1). We found that phosphorylation of TBK1, a hallmark of TBK1 activation, was drastically increased by YTHDF1 knockdown following stimulation with IFN-α (Figs 3C and S2B), demonstrating that YTHDF1 depletion could activate dsRNA-sensing pathway that induces IFNB1 transcription. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. YTHDF1 knockdown induces IFN responses. Cells were treated with mock (−) or IFN-α (+). (A) RT-qPCR showing significantly elevated expression of IFN genes upon YTHDF1 knockdown and IFN stimulation. The signals were normalized to control siRNA samples. (B) Spontaneous IFN-β secretion by cells transfected with control or YTHDF1-specifc siRNA as quantified by ELISA after stimulation of IFN-α. (C) Immunoblot analyses of TBK1 and STAT1 phosphorylation levels. Immunoblot images are representative of 3 biological replicates. (D) RT-qPCR showing significantly elevated expression of ISGs upon YTHDF1 knockdown and IFN stimulation. The signals were normalized to mock treatment samples. (E, F) Heatmaps of expression levels of ISGs (E) and NF-κB–inducible genes (F) from RNA-seq data. (G) RT-qPCR showing significantly elevated expression of NF-κB–inducible genes upon YTHDF1 knockdown and IFN stimulation. The signals were normalized to mock treatment samples except for CXCL11. (H) Immunoblot analyzing of IκBα and NF-κB p65 phosphorylation levels. Immunoblot images are representative of 3 biological replicates. (A, D, G) The signals were normalized to GAPDH. (A, B, D, G) Two-tailed Student t tests were performed to assess the statistical significance of differences between groups, *p < 0.05, **p < 0.01, ***p < 0.001, N.D. means not detected. n = 3 for all experiments. Data are presented as the mean ± SEM. The numerical values for this figure are available in S1 Data. IFN, interferon; ISG, IFN-stimulated gene; RT-qPCR, quantitative reverse transcription PCR; SEM, standard error of the mean; siRNA, small interfering RNA; TBK1, TANK-binding kinase 1. https://doi.org/10.1371/journal.pbio.3001292.g003 The type I IFNs including IFN-α and IFN-β trigger STAT1 phosphorylation and subsequently initiate transcription of various ISGs. Our RT-qPCR analysis and RNA-seq data revealed that the expression levels of ISGs transcripts were highly induced by IFN-α treatment in YTHDF1 knockdown cells (Fig 3D and 3E, S3 Table), as a result of high IFN-β secretion. As expected, enhanced induction of STAT1 phosphorylation by IFN-α treatment was observed in YTHDF1 knockdown cells (Figs 3C and S2B). The cytosolic dsRNA-sensing pathway also causes activation of NF-κB, which induces transcription of a variety of genes involved in immune and inflammatory responses. We found that the expression levels of several NF-κB–regulated genes were increased by YTHDF1 knockdown with IFN-α stimulation in our RNA-seq data (Fig 3F and S3 Table) and RT-qPCR analysis (Fig 3G). The transcriptional activation of NF-κB is regulated by its interaction with inhibitory modulator IκBα that retains NF-κB in the cytoplasm. In response to various pathways including dsRNA signaling, IκBα is phosphorylated and degraded via the ubiquitin–proteasome system. Phosphorylation of NF-κB is also known to contribute to its activation. The phosphorylation of IκBα and NF-κB were increased in YTHDF1 knockdown cells following stimulation with IFN-α (Figs 3H and S2B), indicating that NF-κB signaling pathway is also activated by YTHDF1 deficiency under the condition of IFN treatment.

Enzymatic activity of ADAR1 is required to enhanced interferon responses in YTHDF1-deficient cells To test whether the enhanced IFN responses in YTHDF1 knockdown cells were caused by the reduction of ADAR1p150 expression, we generated stable A172 cell lines that express control EGFP, ADAR1p150, and catalytically inactive mutant of ADAR1p150E912A, which is previously established [49] (Fig 4A), respectively. Overexpression of wild-type ADAR1p150 exhibited significantly enhanced A-to-I RNA editing activity, while overexpression of ADAR1p150E912A did not increase editing activity (S2C Fig). Strikingly, the enhanced IFNB1 induction was completely abolished by overexpression of ADAR1p150 (Fig 4B). Similarly, overexpression of ADAR1p150 suppressed IFNL1, IFNL3, and other ISGs following stimulation with IFN (Fig 4B). These inhibitory effects were abrogated by overexpression of a catalytic inactive mutant ADAR1p150E912A (Fig 4B). These data indicate that the aberrant activation of IFN pathways by YTHDF1 knockdown following IFN treatment is largely due to the defects of ADAR1p150 induction and its A-to-I RNA editing activity. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. ADAR1p150 A-to-I RNA editing activity is required for the YTHDF1-dependent IFN responses. (A) Immunoblot analysis in stable cell lines with lentivirus expressing control EGFP, wild-type ADAR1p150, or catalytically inactive mutant of ADAR1p150E912A, respectively. (B) RT-qPCR showing knockdown effect of YTHDF1 on IFN genes, ISGs, and NF-κB–inducible genes in stable cell lines following IFN-α stimulation. The enhanced IFN responses were attenuated by overexpression of ADAR1p150, but not by catalytic inactive mutant ADAR1p150E912A. (C) RT-qPCR showing knockdown effect of YTHDF1 on IFN genes, ISGs, and NF-κB–inducible genes in A172 cells that were pretreated with the BX759 inhibitor for 1 h and then treated with IFN-α. The enhanced IFN responses were attenuated by the BX759. (D) RT-qPCR showing that concomitant knockdown of YTHDF1 and MDA5 attenuated the enhanced expression of IFN genes, ISGs, and NF-κB–inducible genes upon IFN-α stimulation. (B–D) The signals were normalized to GAPDH and then normalized to control siRNA samples. Two-tailed Student t tests were performed to assess the statistical significance of differences between groups, *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 for all experiments. Data are presented as the mean ± SEM. The numerical values for this figure are available in S1 Data. A-to-I RNA editing, adenosine-to-inosine RNA editing; IFN, interferon; ISG, IFN-stimulated gene; RT-qPCR, quantitative reverse transcription PCR; SEM, standard error of the mean; siRNA, small interfering RNA. https://doi.org/10.1371/journal.pbio.3001292.g004 Furthermore, we investigated the contribution of downstream components of ADAR1-regulated signaling pathway. It is known that ADAR1 destabilizes secondary structure of endogenous RNA to prevent self-activation of the dsRNA sensors, retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs) such as MDA5 [23]. RLR activation recruits and activates mitochondrial antiviral-signaling protein (MAVS), leading to phosphorylation of TBK1 and downstream transcription of IFN genes. To examine the effect of this signaling pathway, we used a TBK1 inhibitor (BX795) that effectively inhibits the induction of IFNs genes upon stimulation with synthetic analog of dsRNA, poly (I:C), in A172 cells (S2D Fig). Pretreatment of YTHDF1 knockdown cells with BX795 attenuated the induction of IFNs genes and downstream ISGs expression following IFN-α stimulation (Fig 4C). In addition, concomitant knockdown of MDA5 and YTHDF1 markedly suppressed the induction of IFNs genes and ISGs (Figs 4D and S2E). Taken together, our results indicate that the YTHDF1-mediated induction of ADAR1p150 and its editing activity are critical for preventing undesirable activation of MDA5, which could cause phosphorylation of TBK1 and excessive downstream IFN production during IFN response.

YTHDF1 affects cellular interferon-inducible cell growth and apoptosis To elucidate the role of YTHDF1 in cellular response to IFN stimulation, YTHDF1 was stably knocked down by short hairpin RNA (shRNA) in A172 cells. The established cell line showed reduced ADAR1p150 induction and enhanced gene expression of IFNs in response to IFN-α as same phenotypes as those in transient knockdown cells (S3A and S3B Fig). IFN-α treatment reduced cell proliferation rates of the YTHDF1 knockdown cells, whereas mock treatment showed no noticeable differences in cell proliferation between the control cells and the YTHDF1-deficient cells (S3C Fig). Consistent with the previous observation (Fig 4B), overexpression of wild-type ADAR1p150 partially attenuated the reduced cell proliferation upon YTHDF1 knockdown compared with the control cells (S3C Fig). The effect was not observed by overexpression of ADAR1p150E912A (S3C Fig), suggesting that A-to-I RNA editing activity of ADAR1p150 are partially responsible for the YTHDF1 knockdown effect on cell proliferation following IFN treatment. In addition, stable knockdown of MDA5 (S3D Fig) also attenuated the inhibitory effect on cell proliferation (S3E Fig). Meanwhile, the YTHDF1-deficient cells were more susceptible to apoptosis induction by IFN than were the control cells (S3F Fig). MDA5 knockdown diminished the elevated apoptosis signals in YTHDF1 knockdown cells (S3G Fig), suggesting the importance of the dsRNA-sensing pathway in these YTHDF1 function. These results demonstrate that the enhanced IFN responses in YTHDF1 knockdown cells upon IFN stimulation decrease cellular growth and increase apoptosis.

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