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 2

mascRNA suppresses proinflammatory cytokine transcription through inhibiting MyD88/TAK1-dependent NF-κB and MAPK signaling.

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mascRNA suppresses proinflammatory cytokine transcription through inhibi...
(A) mascRNA enhances Tnf and Il6 promoter activity. MEFs were transfected with mascRNA ASO or NC ASO. Twelve hours later, cells were again transfected with a reporter plasmid containing Tnf or Il6 promoter–driven luciferase reporter and incubated for 48 hours. Cells were then stimulated with LPS for 6 hours. Firefly luciferase activity in cell lysates was analyzed and normalized to the activity of a cotransfected Renilla luciferase plasmid. (B–E) Effects of knockdown or overexpression of mascRNA on NF-κB and MAPK signaling in RAW264.7 cells. Cells were transfected with either mascRNA ASO (B and D) or pGV-mascRNA (C and E), stimulated with LPS for the indicated times, and then harvested for immunoblot analysis. (F) Immunoblot analysis of phosphorylated TAK1 (p-TAK1) and total TAK1 in mascRNA-knockdown RAW264.7 cells treated with LPS. (G) mascRNA inhibits TAK1 activity to suppress proinflammatory cytokine expression. mascRNA-knockdown RAW264.7 cells were pretreated with or without TAK1 kinase inhibitor 5Z-7-oxozeaenol (5Z-7) for 30 minutes prior to 2-hour (Tnf) or 6-hour (Il6) LPS challenge, followed by qPCR analysis of Tnf and Il6 expression. (H) qPCR analysis of Tnf and Il6 expression in mascRNA-knockdown RAW264.7 cells stimulated with Pam3CSK4. (I) Immunoblot analysis of key molecules in the NF-κB and MAPK signaling pathway in mascRNA-knockdown RAW264.7 cells stimulated with Pam3CSK4. NC, negative control. Data shown in A, G, and H are mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001 (2-tailed Student’s t test). Data are representatives of 2 (A, F, and G) or 3 (B–E) independent experiments.