5-methylcytosine (m ⁵ C) RNA modification controls the innate immune response to virus infection by regulating type I interferons

Abstract

5-methylcytosine (m 5 C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m 5 C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m 5 C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m 5 C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m 5 C methylation of several abundant noncoding RNAs (ncRNAs). However, m 5 C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I–mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m 5 C methylation controls antiviral innate immunity through modulating the m 5 C methylome of ncRNAs and their expression.

Transcriptome-wide 5-methylcytosine modification profiling of long non-coding RNAs in A549 cells infected with H1N1 influenza A virus

“…By conjoint analysis of the epitranscriptomic profile and expression profile, we identified 143 ‘hyper-up’, 4 ‘hyper-down’, 81 ‘hypo-up’, and 6 ‘hypo-down’ lncRNAs. GO analysis highlighted the enrichment of target genes of DM and DE lncRNAs in nucleosome assembly, and regulation of type I interferon production pathway which is consistent with the recent report of the involvement of m 5 C methylation in the regulation of lncRNAs expression and type I interferon production [ 15 ]. KEGG pathway analyses revealed that these DM and DE lncRNAs were predominantly associated with pathogen recognition and pathogenesis pathways.…”

“…2 A). To evaluate the reliability of our data, LncRNA RPPH1, VTRNA1-1 and SCARNA2, which have been reported harboring m 5 C modification sites [ 15 , 34 ], were examined and the m 5 C peaks detected in our data were visualized by IGV (Supplementary Fig. 2A in Additional file 4 ).…”

“…m 5 C and m 6 A modifications of LncRNA NKILA have been found to facilitate cholangiocarcinoma growth and metastasis through the miR-582-3p-YAP1 axis [ 47 ]. Recently, it was reported that m 5 C methylation in lncRNAs played an important role in the regulation of type I interferons and antiviral innate immunity [ 15 ].…”

“…As a relatively common epitranscriptomic mRNA modification, m 5 C has been found to present at higher levels in retroviral transcripts than in cellular mRNAs, and the modification can regulate RNA splicing and promote the translation of viral mRNAs [ 14 ]. A mor e recent study has demonstrated that RNA m 5 C methylation can control antiviral innate immunity through modulating the m 5 C methylome of noncoding RNAs (ncRNAs) and their expression, which regulate type I interferons [ 15 ].…”

NSUN2-mediated m 5 C methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections

“…Here, firstly, we revealed a novel mechanism by which the m 5 C machinery functions in innate immune responses via the methylation of IRF3 mRNA to negatively regulate type I interferon responses [ 51 ], indicating that the m 5 C and m 6 A machineries may have different specificities with respect to regulating multiple signalling molecules involved in antiviral innate immune responses. Recently on October 2022, Zhang et al also reported that depletion of NSUN2 enhanced type I interferon response and inhibited virus replication, which is dependent on RIG-I but not MDA5 [ 52 ]. But the mechanism they reported is different from what we found, which implies the important function of m 5 C modification in innate immune regulation during various viral infections.…”