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 8

hnRNP H/F promote mascRNA degradation to exert a proinflammatory effect.

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hnRNP H/F promote mascRNA degradation to exert a proinflammatory effect....
(A) qPCR analysis of Tnf and Il6 mRNA abundance in hnRNP H– or/and hnRNP F–knockdown RAW264.7 cells stimulated with LPS for 2 hours. (B) Immunoblot analysis of cellular TNF and IL-6 precursors in hnRNP H– or hnRNP F–knockdown RAW264.7 cells stimulated with LPS for the indicated times. (C) hnRNP H and F enhance Tnf and Il6 promoter activity. MEFs were transfected with a Tnf or Il6 promoter–luciferase reporter construct and an indicated siRNA. Forty-eight hours after transfection, cells were stimulated with LPS for 6 hours. Firefly luciferase activity was analyzed in cell lysates and normalized to the activity of a cotransfected Renilla luciferase plasmid. (D) Immunoblot analysis of key molecules in NF-κB and MAPK signaling pathways in hnRNP H– or hnRNP F–knockdown RAW264.7 cells stimulated with LPS for the indicated times. (E) qPCR analysis of mascRNA abundance in hnRNP H– or hnRNP F–knockdown RAW264.7 cells treated with LPS for the indicated times. (F) mascRNA mediates the role of hnRNPs H or F in the regulation of inflammatory responses. Mouse peritoneal macrophages were cotransfected with NC siRNA, hnRNP H siRNA, hnRNP F siRNA, NC ASO, or mascRNA ASO in combination as indicated. Thirty-six hours after transfection, cells were stimulated with LPS for 2 (Tnf) or 6 (Il6) hours, followed by qPCR analysis of Tnf and Il6 expression. NC, negative control. Data are mean ± SD of triplicate wells (A, C, E, and F). *P < 0.05; **P < 0.01; ***P < 0.001 (2-tailed Student’s t test). Data are representatives of 2 (C, D, and E) or 3 (A and B) independent experiments.