For stable transfection, RAW264.7 cells were transfected with pGV-mascRNA or empty vector (pGV-NC) by means of jetPEI-Macrophage (Polyplus, Cat# 103-05N) according to the manufacturers’ instruction. 24 h after transfection, cells were screened by G-418 (400 ng/mL) for 10 d. The stably transfected cell pools were then split and plated for subsequent experiments.

mascRNA is a highly conserved 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 Toll-like receptor (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 lipopolysaccharide (LPS), a TLR4 ligand. Furthermore, exogenous mascRNA alleviated LPS-induced lung inflammation. On the contrary, mascRNA potentiated the phosphorylation of IRF3 and STAT1 and the transcription of interferon-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 interferon signaling. Additionally, 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.