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A Mitochondrial Micropeptide Is Required for Activation of the Nlrp3 A Mitochondrial Micropeptide Is Required for Activation of the Nlrp3 Inflammasome Ankit Bhatta, Maninjay Atianand, Zhaozhao Jiang, Juliet Crabtree, Juliana Blin and Katherine A. Fitzgerald This information is current as of September 27, 2021. J Immunol published online 13 December 2019 http://www.jimmunol.org/content/early/2019/12/12/jimmun ol.1900791 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/12/12/jimmunol.190079 Material 1.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 13, 2019, doi:10.4049/jimmunol.1900791 The Journal of Immunology A Mitochondrial Micropeptide Is Required for Activation of the Nlrp3 Inflammasome Ankit Bhatta,* Maninjay Atianand,† Zhaozhao Jiang,* Juliet Crabtree,* Juliana Blin,* and Katherine A. Fitzgerald* Functional peptides encoded by short open reading frames are emerging as important mediators of fundamental biological process- es. In this study, we identified a micropeptide produced from a putative long noncoding RNA (lncRNAs) that is important in controlling innate immunity. By studying lncRNAs in mice macrophages, we identified lncRNA 1810058I24Rik, which was downregulated in both human and murine myeloid cells exposed to LPS as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed that this transcript was localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 aa micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the Downloaded from mitochondrion. We thus named this gene mitochondrial micropeptide-47 (Mm47). Crispr–Cas9–mediated deletion of Mm47,as well as small interfering RNA studies in mice primary macrophages, showed that the transcriptional response downstream of TLR4 was intact in cells lacking Mm47. In contrast, Mm47-deficient or knockdown cells were compromised for Nlrp3 inflam- masome responses. Activation of Nlrc4 or Aim2 inflammasomes were intact in cells lacking Mm47. This study therefore identifies, to our knowledge, a novel mitochondrial micropeptide Mm47 that is required for the activation of the Nlrp3 inflammasome. This work further highlights the functional activity of short open reading frame–encoded peptides and underscores their importance in http://www.jimmunol.org/ innate immunity. The Journal of Immunology, 2020, 204: 000–000. ibonucleic acid molecules can be divided into two cate- These sORF-encoded peptides (SEPs) or micropeptides are now gories: mRNAs and noncoding RNAs. A large proportion being recognized for their functions in development, differen- R of the mammalian genome is transcribed as long non- tiation, transport, and other fundamental biological processes coding RNAs (lncRNAs) (1). Recent studies have demonstrated (7, 9). Indeed, a recent study has revealed that a large number of diverse physiological functions for lncRNAs, including a growing transcripts currently annotated as lncRNAs are associated with appreciation for the role of these molecules in the immune system, ribosomes and, in some cases, are translated (8). Among these, in which they control the differentiation and/or activation of im- one such transcript, Aw112010, was found to be encoded from a by guest on September 27, 2021 mune cells (2–5). In the innate immune system, our group and noncanonical open reading frame (ORF) and found to play a others have found that lncRNAs act as positive or negative role in Salmonella typhimurium infection and intestinal in- regulators of inflammatory gene expression in a variety of im- flammation (8). Similarly, humanin, an antiapoptotic 24 aa SEP mune cells, including macrophages (4, 5). However, many an- localized on the mitochondria, prevents Bax translocation from notated lncRNAs have short open reading frames (sORFs) that the cytosol to the mitochondria (10). Other SEPs, such as Dworf may encode for functional proteins or small peptides (6–8). andMyoregulin,havealsobeenshowntoregulatecalcium transport in the endoplasmic reticulum (ER) (11). Collectively, these studies highlight important roles for SEPs in fundamental *Program in Innate Immunity, Department of Medicine, University of Massachusetts biological processes, including cell death and innate immunity. † Medical School, Worcester, MA 01605; and Department of Immunology, University Macrophages represent the first line of defense against infection of Pittsburgh, Pittsburgh, PA 15261 by detecting pathogens through germline-encoded pattern recog- ORCIDs: 0000-0003-4921-0087 (J.C.); 0000-0002-9628-8920 (J.B.). nition receptors. These pattern recognition receptors recognize Received for publication July 11, 2019. Accepted for publication November 10, specific pathogen-associated molecular patterns (PAMPs) such as 2019. LPS and trigger the expression of proinflammatory cytokines and This work was supported by grants from the National Institutes of Health (AI067497 to K.A.F.) and a T32 Training Grant (T32 AI095213 to A.B.). type I IFNs through the activation of the transcription factors k Address correspondence and reprint requests to Dr. Katherine A. Fitzgerald, University of NF- B and IRF3 (12, 13). Macrophages also upregulate the Massachusetts Medical School, Aaron Lazare Medical Research Building, Room 208, 364, expression of Nlrp3 and IL-1b, both of which are critical for Plantation Street, Worcester, MA 01605. E-mail address: kate.fi[email protected] mounting rapid immune responses during infection (14). Nlrp3 The online version of this article contains supplemental material. forms a multiprotein complex known as the inflammasome. Abbreviations used in this article: BMDM, bone marrow–derived macrophage; ER, Inflammasomes are cytoplasmic supramolecular complexes that endoplasmic reticulum; ETC, electron transport chain; Fwd, forward; Gsdmd, form in response to microbial as well as endogenous damage or gasdermin D; KO, knockout; lincRNA, intergenic lncRNA; lncRNA, long non- coding RNA; MEF, mouse embryonic fibroblast; Mm47, mitochondrial micropep- danger signals (13, 15–17). Inflammasomes activate inflamma- tide-47; mtROS, mitochondrial reactive oxygen species; NTC, nontargeting tory caspases such as caspase-1, which in turn controls the control; OCR, oxygen consumption rate; ORF, open reading frame; PAMP, b pathogen-associated molecular pattern; Poly(dA:dT), poly(deoxyadenylic-deoxy- protolytic maturation of the proinflammatory cytokines IL-1 thymidylic); Poly(I:C), polyinosinic-polycytidilic acid; qRT-PCR, quantitative RT- and IL-18. Caspase-1 also cleaves gasdermin D (Gsdmd) to PCR; Rev, reverse; SEP, sORF-encoded peptide; sgRNA, single-guide RNA; generate an N-terminal pore-forming fragment that facilitates siRNA, small interfering RNA; sORF, short open reading frame. cytokine release and pyroptotic cell death (18–20). The Nlrp3 Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 inflammasome is activated in response to a wide variety of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900791 2 INFLAMMASOME REGULATION BY sORF-ENCODED PEPTIDES microbial and endogenous danger signals, including nigericin, Reverse Transcription Supermix (1708840; Bio-Rad Laboratories). uric acid crystals, amyloid-b fibrils, and extracellular ATP. The Quantitative PCR on the cDNA was performed in Bio-Rad CFX96 Touch precise mechanisms leading to the formation of the Nlrp3 Real-time PCR using gene-specific primers. The genes were normalized to the housekeeping gene Gapdh. Mouse primers used are GAPDH Fwd 59- inflammasome in response to these diverse ligands are still TGGCAAAGTGGAGATTGTTGC-39, GAPDH Rev 59-AAGATGGTGA- unclear, although K+ efflux and, in some cases, mitochondria are TGGGCTTCCCG-39,MALAT1Fwd59-TTGGGACAGTGGACGTGTGG-39, important (15, 21, 22). MALAT1 Rev 59-TCAAGTGCCAGCAGACAGCA-39,Mm47Fwd59-GCT- 9 9 In this study, we have identified a SEP encoded from an an- CAGAACTATGAAATGCCAAAC-3 ,Mm47Rev5-GGTCTCAGAAGCA- GGTGGAC-39,IL-1b Fwd 59-GCCACCTTTTGACAGTGATGAG-39,IL-1b notated intergenic lncRNA (lincRNA) transcript that is regulated Rev 59-GTTTGGAAGCAGCCCTTCATC-39,Nlrp3Fwd59-CATGTTGCCT- in macrophages exposed to LPS. Loss of function studies identify GTTCTTCCAGAC-39,andNlrp3Rev59-CGGTTGGTGCTTAGACTTGAGA- a key role for this mitochondrial micropeptide-47 (Mm47) in 39.HumanprimersusedwereGAPDHFwd59-TGCAACAACCAACTGCTTA- controlling activation of the Nlrp3 inflammasome. CRISPR– 39, GAPDH Rev 59-AGAGGCAGGGATGATGTTC-39,Mm47Fwd59-CA- 9 9 Cas9 knockout (KO) of Mm47 or small interfering RNA (siRNA)– CCGACATCATGCTCGAGT-3 , and Mm47 Rev 5 -GCCAGATACATTCCA- ACCACGT-39.
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