The small RNA mascRNA differentially regulates TLR-induced proinflammatory and antiviral responses
Tao Sun, … , Yuqing Hu, Xiaohua Mao
Tao Sun, … , Yuqing Hu, Xiaohua Mao
Published September 28, 2021
Citation Information: JCI Insight. 2021;6(21):e150833. https://doi.org/10.1172/jci.insight.150833.
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Research Article Immunology

The small RNA mascRNA differentially regulates TLR-induced proinflammatory and antiviral responses

Abstract

MALAT1-associated small cytoplasmic RNA (mascRNA) is a highly conserved transfer RNA–like (tRNA-like) noncoding RNA whose function remains largely unknown. We show here that this small RNA molecule played a role in the stringent control of TLR-mediated innate immune responses. mascRNA inhibited activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling and the production of inflammatory cytokines in macrophages stimulated with LPS, a TLR4 ligand. Furthermore, exogenous mascRNA alleviated LPS-induced lung inflammation. However, mascRNA potentiated the phosphorylation of IRF3 and STAT1 and the transcription of IFN-related genes in response to the TLR3 ligand poly(I:C) both in vitro and in vivo. Mechanistically, mascRNA was found to enhance K48-linked ubiquitination and proteasomal degradation of TRAF6, thereby negatively regulating TLR-mediated MyD88-dependent proinflammatory signaling while positively regulating TRIF-dependent IFN signaling. Additionally, heterogeneous nuclear ribonucleoprotein H (hnRNP H) and hnRNP F were found to interact with mascRNA, promote its degradation, and contribute to the fine-tuning of TLR-triggered immune responses. Taken together, our data identify a dual role of mascRNA in both negative and positive regulation of innate immune responses.

Authors

Tao Sun, Chunxue Wei, Daoyong Wang, Xuxu Wang, Jiao Wang, Yuqing Hu, Xiaohua Mao

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Figure 3

mascRNA attenuates TLR4 signaling by promoting degradation and K48-linked ubiquitination of TRAF6.

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mascRNA attenuates TLR4 signaling by promoting degradation and K48-linke...
(A and B) Immunoblot analysis of TLR4, MyD88, IRAK1, and TRAF6 protein abundance in mascRNA knockdown (A) and mascRNA-overexpressing (B) RAW264.7 cells stimulated with LPS for the indicated times. (C) mascRNA promotes TRAF6 protein degradation. mascRNA-overexpressing RAW264.7 cells were treated with 40 ng/mL cycloheximide (CHX) for the indicated times, followed by immunoblot analysis. (D) mascRNA promotes TRAF6 protein degradation via ubiquitin-proteasome pathway. mascRNA-overexpressing RAW264.7 cells were treated for 8 hours with 10 μM MG132, 10 mM 3-MA, 20 mM NH4Cl, or DMSO, followed by immunoblot analysis. TRAF6 levels were normalized to β-actin and quantified (n = 3 independent experiments, mean ± SEM). (E) mascRNA increases K48-linked ubiquitination of TRAF6. mascRNA-overexpressing or control RAW264.7 cells were stimulated with LPS for 1 hour, immunoprecipitated with an anti-TRAF6 antibody, and followed by immunoblot analysis with anti-ubiquitin, anti–K48-linked ubiquitin, anti–K63-linked ubiquitin, or anti-TRAF6. Bottom, immunoblot analysis of TRAF6 and β-actin in lysates without immunoprecipitation. Ubiquitination levels were normalized to β-actin and quantified (n = 3 independent experiments, mean ± SEM). (F) mascRNA inhibits Tnf and Il6 expression via downregulating TRAF6. RAW264.7 cells were cotransfected with NC siRNA, TRAF6 siRNA, NC ASO, and mascRNA ASO in combination as indicated. Thirty-six hours after transfection, cells were stimulated with LPS for 2 (Tnf mRNA) or 6 (Il6 mRNA) hours, followed by qPCR analysis. Data are mean ± SD of triplicate wells. (G) mascRNA suppresses LPS-induced NF-κB and MAPK signaling via downregulating TRAF6. RAW264.7 cells were transfected as in F and stimulated with LPS for 30 minutes, followed by immunoblot analysis. NC, negative control. *P < 0.05; **P < 0.01; ***P < 0.001 (2-tailed Student’s t test). Data shown in A–C, F, and G are representatives of 2 independent experiments.