The small RNA mascRNA differentially regulates TLR-induced proinflammatory and antiviral responses
Tao Sun, Chunxue Wei, Daoyong Wang, Xuxu Wang, Jiao Wang, Yuqing Hu, Xiaohua Mao
Tao Sun, Chunxue Wei, Daoyong Wang, Xuxu Wang, Jiao Wang, Yuqing Hu, Xiaohua Mao
View: Text | PDF
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

×

Figure 3

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

Options: View larger image (or click on image) Download as PowerPoint
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.