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Cutting Edge: A Natural Antisense Transcript, AS-IL1 α, Controls Inducible Transcription of the Proinflammatory IL-1α This information is current as of September 25, 2021. Jennie Chan, Maninjay Atianand, Zhaozhao Jiang, Susan Carpenter, Daniel Aiello, Roland Elling, Katherine A. Fitzgerald and Daniel R. Caffrey J Immunol 2015; 195:1359-1363; Prepublished online 15 July 2015; Downloaded from doi: 10.4049/jimmunol.1500264 http://www.jimmunol.org/content/195/4/1359 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2015/07/15/jimmunol.150026 Material 4.DCSupplemental References This article cites 17 articles, 3 of which you can access for free at: http://www.jimmunol.org/content/195/4/1359.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: A Natural Antisense Transcript, AS-IL1a, Controls Inducible Transcription of the Proinflammatory Cytokine IL-1a Jennie Chan,* Maninjay Atianand,* Zhaozhao Jiang,* Susan Carpenter,*,† Daniel Aiello,* Roland Elling,* Katherine A. Fitzgerald,*,‡,1 and Daniel R. Caffrey*,1 Natural antisense transcripts (NATs) are a class of long growing literature also supports their importance in immu- noncoding RNAs (lncRNAs) that are complementary nity (5). to other protein-coding . Although thousands of Natural antisense transcripts (NATs) are a class of lncRNAs NATs are encoded by mammalian genomes, their func- defined as being complementary to one or more protein-coding

tions in innate immunity are unknown. In this study, genes (6). Approximately 50–70% of lncRNAs are classified as Downloaded from we identified and characterized a novel NAT, AS-IL1a, NATs, making them a substantial proportion of the noncoding which is partially complementary to IL-1a. Similar to genome (2, 6). Although the functions of some lncRNAs are IL-1a, AS-IL1a is expressed at low levels in resting well defined, relatively few NATs are functionally characterized. and is induced following with NATs can both activate and repress the expression of comple- Listeria monocytogenes or stimulation with TLR ligands mentary coding genes (7, 8). Similar to the broader class of (Pam CSK , LPS, polyinosinic-polycytidylic acid). In- lncRNAs, NATs probably function in a cell type–specific http://www.jimmunol.org/ 3 4 manner. For example, in Th2 cells, lincR-Ccr2-59AS regulates ducible expression of IL-1a mRNA and protein were expression of Ccr2, Ccr3, and Ccr5 and trafficking of Th2 cells significantly reduced in macrophages expressing shRNA to the lung (9). However, the molecular functions of NATs in that target AS-IL1a.AS-IL1a is located in the nucleus a innate immunity are unknown. and did not alter the stability of IL-1 mRNA. Instead, Macrophages are the first line of defense against microbial AS-IL1a was required for the recruitment of RNA poly- a . Following infection, host cells produce proin- merase II to the IL-1 promoter. In summary, our stud- flammatory . In particular, IL-1a and IL-1b are rapidly a a ies identify AS-IL1 as an important regulator of IL-1 induced and amplify inflammation via IL-1R complexes expressed transcription during the innate immune response. The on neighboring cells (10). Because these cytokines can cause sig- by guest on September 25, 2021 Journal of Immunology, 2015, 195: 1359–1363. nificant tissue damage, their production must be tightly regulated. Caspase-1–dependent inflammasomes convert IL-1b into the mature biologically active cytokine. In contrast, IL-1a is active in ecent advances in large-scale RNA sequencing (RNA- its full-length form upon release from damaged or dying cells (11). seq) have led to the identification of novel RNA In this article, we describe a functional innate immune NAT, R species encoded in the genome, many of which are AS-IL1a, encoded within the IL-1a locus. Characterization of long noncoding RNAs (lncRNAs) (1, 2). lncRNAs are ar- this NAT shows that AS-IL1a functions as an additional layer bitrarily defined as having $200 nt, which discriminates of regulation important in controlling IL-1a transcription. them from small noncoding RNAs. These RNAs are further categorized based on their genomic location relative to Materials and Methods neighboring protein-coding genes. Several comprehensive In vivo studies demonstrated that lncRNAs are important regulators C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were maintained in of cell differentiation and developmental pathways (3, 4). A specific –free conditions and used in accordance with the University of

*Program in Innate Immunity, Division of Infectious Diseases, Department of Medi- Address correspondence and reprint requests to Dr. Katherine A. Fitzgerald and cine, University of Massachusetts Medical School, Worcester, MA 01605; †Department Dr. Daniel R. Caffrey, Program in Innate Immunity, Division of Infectious Diseases, of Immunology and Microbiology, University of California, San Francisco, San Fran- Department of Medicine, University of Massachusetts Medical School, Worcester, cisco, CA 94143; and ‡Centre for Molecular Inflammation Research, Department of MA 01605. E-mail addresses: [email protected] (K.A.F.) and Daniel. Cancer Research and Molecular Medicine, Norwegian University of Science and Tech- [email protected] (D.R.C.) nology, 7491 Trondheim, Norway The online version of this article contains supplemental material. 1K.A.F. and D.R.C. contributed equally to this work. Abbreviations used in this article: BMDM, bone marrow–derived ; ISD, IFN- Received for publication February 12, 2015. Accepted for publication June 11, 2015. stimulatory DNA; lncRNA, long noncoding RNA; NAT, natural antisense transcript; Poly(I:C), polyinosinic-polycytidylic acid; qRT-PCR, quantitative RT-PCR; RNAPII, This work was supported by grants from the National Institutes of Health (T32 Training RNA polymerase II; RNA-seq, RNA sequencing; shRNA, short hairpin RNA. Grant A1095213 to J.C. and Grant AI067497 to K.A.F.), the American Heart Associ- ation (to M.A.), the German Research Foundation (to R.E.), and the Arthritis National Ó Research Foundation (to S.C.). Copyright 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 The sequences presented in this article have been submitted to the ArrayExpress database under accession number E-MTAB-3315 and to the Genbank database under accession number KR095173.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500264 1360 CUTTING EDGE: AS-IL1a CONTROLS INDUCIBLE TRANSCRIPTION OF IL-1a

RNA sequencing Single-cell suspensions from spleens were used to make total RNA, and 4 mgtotal RNA was used to generate libraries for RNA sequencing (RNA-seq) (Illumina unstranded RNA Kits). Samples were sized, quantified, and validated on a bio- analyzer. Libraries were sequenced on a HiSeq 2000 (Illumina, San Diego, CA) as paired-end 100 bp reads. Sequence reads were aligned to the mouse genome (NCBI m37/mm9) using TopHat. -level read counts were calculated using HTSeq and the Ensembl 64 transcript annotation file. The DESeq package was used to normalize gene counts and calculate fold-change values and p values. The Circos program was used to visualize genome-wide gene-expression changes. As previously described (3), all protein-coding genes annotated with particular IDs were classified as immune genes. All RNA-seq data are available from ArrayExpress under accession number E-MTAB-3315.

Macrophage stimulations Primary bone marrow–derived macrophages (BMDMs) were generated and infected with Listeria or stimulated with LPS (100 ng/ml), Pam3CSK4 (100 nM), polyinosinic-polycytidylic acid [Poly(I:C)] (25 mg/ml), and IFN- stimulatory DNA (ISD; 3 mM) oligonucleotides (13), as described (12). After 2 h of incubation, total RNA was harvested from all conditions

(RNeasy; QIAGEN). Downloaded from

Polysome profiling Polysome profiling was performed as described (3, 4).

Short hairpin RNA–mediated silencing

Lentiviral particles were generated using pLKO.1 TRC, as described (14). http://www.jimmunol.org/ FIGURE 1. Identification of AS-IL1a.(A) Circos plot showing differentially Knockdown efficiency was assessed by one-step quantitative RT-PCR (qRT- PCR; Bio-Rad). Sequences/primers are listed in Supplemental Table I. expressed genes in splenocytes from L. monocytogenes–infected mice. are indicated along the outer track. The middle track shows log2 fold-change values Western blot for all lncRNAs and AS-IL1a (red). The inner track shows log2 fold-change values for protein-coding genes, with immune genes shown in red. (B) Schematic diagram of Immunoblotting was performed using mouse IL-1a/IL-1F1 Ab (R&D Sys- AS-IL1a in murine macrophages. AS-IL1a chromosomal localization overlaps with tems, cat. no. AB-400NA) and anti-goat IgG (Bio-Rad, cat. no. 172-1011). IL-1a protein-coding gene on 2 (indicated by vertical bar). (C)Mac- rophages infected with L. monocytogenes for 3 h at an multiplicity of infection (MOI) NanoString analysis of 5 and 10. Data were normalized to Gapdh and fold change was calculated. Data

nCounter CodeSets were constructed for detecting selected mouse-specific by guest on September 25, 2021 are representative of two independent experiments. (D)PolysomeprofilingofGapdh, immune genes, and levels of RNA were measured using the nCounter Dig- IL-1a,andAS-IL1a transcripts. Immortalized macrophages were stimulated with ital Analyzer, as described (3, 15). LPSandtreatedwithcycloheximidealoneor harringtonine prior to cycloheximide treatment to determine the translational potential of AS-IL1a. Cell fractionation Cytosolic and nuclear fractions were prepared as described (16). The cell lysis Massachusetts Medical School Institutional Animal Care and Use Committee. buffer contained 0.15% Nonidet P-40; the sucrose cushion did not contain Listeria monocytogenes (clinical isolate 10403s) was from V. Boyartchuk (Norwe- detergent. After fractionation, cytoplasmic and nuclear RNA were purified using gian University of Science and Technology). Mice were infected as described (12) QIAGEN RNeasy columns. Gapdh and 7SK were used for cytoplasmic and for 24 h before harvesting the spleen. PBS was used as a control. nuclear controls, respectively. Primers are listed in Supplemental Table I.

FIGURE 2. Inducibility of AS-IL1a by TLR ligands. (A) Schematic diagram of AS-IL1a and IL-1a and qRT-PCR primers for experiments in (B)–(E)(nottoscale).(B) LPS, Pam3CSK4, Poly(I:C), or ISD were stimulated on immortalized BMDMs for 0, 1, 2, or 6 h, and AS-IL1a expression was measured by qRT-PCR. (C) Wild-type and IL- 1a–knockout macrophages were stimulated with LPS for 6 h. AS-IL1a expression was induced, even in the absence of IL-1a.(D) Wild-type (WT) and MyD88/Trif double- a , knockout (dKO) macrophages were treated or not treated (NT) with LPS, Pam3CSK4, or Poly(I:C) [p(IC)] for 6 h. AS-IL1 expression was measured by qRT-PCR. ****p 0.0001, **p , 0.01, one-way ANOVA. (E)AnNF-kB inhibitor, Bay11-7082, was used to treat cells 1 h prior to LPS stimulation (6 h), and AS-IL1a expression was measured by qRT-PCR. Data were normalized to Gapdh, and fold change was calculated. Data are mean + SD and are representative of three replicates. ***p , 0.001, two-tailed t test. The Journal of Immunology 1361

Chromatin immunoprecipitation TRIF or following inhibition of NF-kB, LPS failed to in- a Cells were stimulated, as described, fixed in 1% formaldehyde, lysed, and duce AS-IL1 (Fig. 2D, 2E). sonicated. A total of 5 mg chromatin was immunoprecipitated with Dyna- beads Protein G (Novex/Life Technologies, cat. no. 10009D) and Abs to Knocking down AS-IL1a inhibits inducible IL-1a expression RNA polymerase II (RNAPII; Active Motif, cat. no. 102660), H3K9-Ac (Abcam, cat. no. AB4441), or IgG isotype (Abcam, cat no. AB37415). Pu- We next generated macrophage cell lines in which AS-IL1a rified chromatin DNA was used for quantitative PCR amplification at the IL- expression was specifically silenced by short hairpin RNA 1a, IP-10, or Gapdh promoter. (shRNA). We made two independent shRNAs that targeted AS-IL1a exons that did not overlap with those of IL-1a Statistical analysis (Fig. 3A). We confirmed that AS-IL1a was significantly si- Statistical analysis was performed using a Student t test with GraphPad Prism lenced in LPS-stimulated cell lines that expressed these software for bivariate studies and one-way ANOVA for multivariate studies. shRNA (Fig. 3B). In shRNA-GFP lines, LPS strongly in- The p values # 0.05 were considered statistically significant. duced IL-1a mRNA levels, whereas LPS only induced low levels of IL-1a mRNA in cells expressing shRNA–AS-IL1a Results (Fig. 3C). LPS also failed to induce the high levels of IL-1a Identification of AS-IL1a, a natural antisense noncoding RNA in the protein normally detected by immunoblotting (Fig. 3D). IL-1a locus These results indicate that AS-IL1a is required for the in- To assess lncRNA expression during an active infection, we ducible expression of IL-1a. We subsequently used RNA infected C57BL/6 mice with L. monocytogenes and prepared profiling (NanoString) to simultaneously analyze mRNA Downloaded from libraries for RNA-seq from splenocytes isolated from infected and noninfected mice (24 h). Consistent with previous reports (17), Listeria induced expression of a wide range of protein-coding immune genes (Fig. 1A, inner track). We also detected many lncRNAs that were differentially expressed following Listeria infection (Fig. 1A, outer track). http://www.jimmunol.org/ Strikingly, one of these lncRNAs (hereafter referred to as AS- IL1a) was induced 14-fold and was encoded by the opposite strand of the IL-1a locus on . We used PCR to confirm the orientation and gene structure of AS-IL1a (Fig. 1B, sequence accession KR095173 http://www.ncbi.nlm.nih.gov/ genbank). Similar to the in vivo infection, AS-IL1a was in- duced in macrophages infected with Listeria (Fig. 1C). The

Coding Potential Calculator (http://cpc.cbi.pku.edu.cn/) classi- by guest on September 25, 2021 fied AS-IL1a as noncoding (score = 20.87), which was also supported by polysome profiling. Cells were treated with cy- cloheximide, which traps ribosomes along their RNA strands, and these RNA were detected in the heavier fractions of a su- crose density gradient. In parallel, cells were treated with har- ringtonine prior to cycloheximide, which inhibits translation and polysome formation by causing ribosomes to accumulate at their initiation sites, and is detected in the lighter fractions of a sucrose density gradient. As expected, harringtonine prevented polysome formation in both Gapdh and IL-1a mRNA (Fig. 1D),andbothmRNAsshiftedtolighterfractionsinthesucrose gradient. In contrast, AS-IL1a was largely unaffected by har- ringtonine treatment, indicating that it is unlikely to associate with ribosomes and encode a protein product.

AS-IL1a is inducible in macrophages exposed to TLR ligands via NF-kB We next examined the expression of AS-IL1a in BMDMs stimulated with ligands for TLRs and other sensors. Fig. 2A FIGURE 3. AS-IL1a regulates IL-1a expression. (A) shRNA 1 and shRNA depicts the location of our qRT-PCR primers, which do not a a a a 2 target AS-IL1 in red regions (not to scale) that do not overlap with IL-1 . overlap with IL-1 itself. AS-IL1 was induced by LPS (B) AS-IL1a RNA levels (qRT-PCR) were reduced in both shRNA 1 and (TLR4), Pam3CSK4 (TLR1/2), and Poly(I:C) (TLR3), but shRNA 2 cell lines. shRNA GFP is a control that targets the GFP gene. (C) not ISD, which activates cGAS (Fig. 2B). We also measured IL-1a mRNA levels (qRT-PCR) were reduced in shRNA 1 and shRNA 2 cell expression of AS-IL1a in macrophages from IL-1a–deficient lines. (D) IL-1a p33 protein expression (Western blot) was decreased in mice. In these cells, LPS induced expression of AS-IL1a but shRNA 1 and shRNA 2 cell lines stimulated with LPS. b-actin was used as E not IL-1a, indicating that transcription of AS-IL1a is regu- a loading control. ( ) NanoString analysis was performed on the cell lines to determine whether knocking down AS-IL1a affects the expression of other lated independently of IL-1a (Fig. 2C). We next compared a immune genes. Immune genes with a 15-fold change in expression following LPS-inducible levels of AS-IL1 in wild-type and MyD88/ LPS stimulation are shown. Data were calculated as fold-change relative to TRIF double-knockout cells and cells treated with Bay11- Gapdh and are representative of three biological replicates. ****p , 0.0001, 7085, an inhibitor of NF-kB. In the absence of MyD88/ one-way ANOVA. 1362 CUTTING EDGE: AS-IL1a CONTROLS INDUCIBLE TRANSCRIPTION OF IL-1a

FIGURE 4. AS-IL1a regulates IL-1a transcription. (A) Localization of AS-IL1a in LPS-induced BMDMs. Cytosolic and nuclear fractions were separated Downloaded from via a sucrose gradient, and qRT-PCR was performed on fractionated RNA. Gapdh (cytosolic), IL-1a (cytosolic), and 7SK (nuclear) were also measured as controls. Data are the percentage of the cytosolic fraction or the nuclear fraction over cytosolic+nuclear (100%) total RNA. (B)qRT-PCRwasperformedon shRNA-GFP and two AS-IL1a–knockdown cell lines. Schematic diagram shows regions to be amplified by primers in mature IL-1a mRNA (exon–exon junction) and prespliced IL-1a mRNA. RNA levels are shown as a percentage of the expression in shRNA-GFP (representative of two experiments). (C)A comparison of poly-d(T) and random hexamer priming for reverse transcription to an AS-IL1a–specific primer to demonstrate specificity of the amplified region prior to qRT-PCR. (D) Chromatin immunoprecipitation was performed on the cell lines. Abs against RNAPII or IgG isotype control were used. Data http://www.jimmunol.org/ were normalized with input chromatin before immunoprecipitation. Regions of the IL-1a gene, near the transcription start site (+22), were measured for RNAPII binding. RNAPII recruitment was significantly less in shRNA-AS-IL1a cell lines than shRNA-GFP cell lines (p 5 0.0298, t test). RNAPII re- cruitment did not decrease in the respective shRNA cell lines for CXCL10/IP-10 (p 5 0.127) or Gapdh promoter (p 5 0.123) relative to shRNA-GFP. expression levels of a broader selection of immune genes pected, LPS treatment in the shRNA-GFP control cells resulted following LPS stimulation (6 h) in control and AS-IL1a– in robust RNAPII binding at this region. This was greatly re- silenced macrophages (Fig. 3E). IL-1a was the most signif- duced in macrophages expressing AS-IL1a shRNA (Fig. 4D). b icantly altered gene in the shRNA lines; IL-1 levels were We observed similar results when we measured the epigenetic by guest on September 25, 2021 decreased, albeit to a lesser extent. mark H3K9-acetylation, an indicator of active transcription at this same locus (Supplemental Fig. 1). The effect of AS-IL1a AS-IL1a is localized to the nucleus and enhances IL-1a expression at shRNA was specific to the IL-1a locus, because recruitment of the transcriptional level RNAPII to Cxcl10 or Gapdh was unaffected in the knockdown To understand how AS-IL1a might regulate IL-1a, we first line. These results indicate that AS-IL1a is required to facilitate examined its localization by performing subcellular fraction- RNAPII recruitment to the IL-1a locus during LPS-induced ation of nuclear and cytosolic compartments and analyzing transcription. RNA levels by qRT-PCR. We also measured levels of Gapdh, IL-1a, and the nuclear RNA 7SK. As expected, the mature Discussion IL-1a and Gapdh transcripts were localized to the cytosol, lncRNAs are emerging as important regulators of gene ex- whereas 7SK RNA was confined to the nucleus. AS-IL1a was pression in diverse biological contexts, including immunity. LPS primarily nuclear (Fig. 4A). was shown to induce widespread changes in lncRNA expression Because AS-IL1a was localized to the nucleus, we hypoth- in immune cells (18). lncRNAs also were shown to promote or esized that it regulated the transcription of IL-1a.Weused repress inflammatory in immune cells (19). The identification of AS-IL1a as a natural antisense lncRNA qRT-PCR primers that targeted intron-containing sequences of a the IL-1a pre-mRNA, as well as primers targeting spliced IL- that enhances IL-1 gene transcription adds to our under- 1a transcripts. Consistent with an effect on transcription, both standing of cytokine-mediated inflammation. Indeed, disrup- a tion of AS-IL1a function could limit IL-1a transcription and spliced and unspliced pre-mRNA IL-1 levels were decreased a in the three AS-IL1a shRNA lines (Fig. 4B). We also made potentially alleviate the damaging effects of excessive IL-1 reverse-transcription primers that targeted AS-IL1a only, and levels during infection and inflammatory disease. compared the results with our normal method that uses poly-d(T) and random hexamer primers to confirm specific amplifi- Disclosures cation of our qRT-PCR amplicon from AS-IL1a cDNA. The The authors have no financial conflicts of interest. trends were comparable, indicating that the abrogated AS-IL1a expression levels were specific to AS-IL1a andnottheresultof References promiscuous priming (Fig. 4C). 1. RIKEN Genome Exploration Research Group and Genome Science Group (Ge- We also performed chromatin immunoprecipitation–PCR nome Network Project Core Group) and the FANTOM Consortium, S. Katayama, Y. Tomaru, T. Kasukawa, K. Waki, M. Nakanishi, M. Nakamura, H. Nishida, assays to measure RNAPII occupancy at the region surrounding C. C. Yap, M. Suzuki, et al. 2005. Antisense transcription in the mammalian the transcription start site of the IL-1a locus (+22 nt). As ex- transcriptome. Science 309: 1564–1566. The Journal of Immunology 1363

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