5-methylcytosine (m5C) RNA modification controls the innate immune response to virus infection by regulating type I interferons

Cytosine-5 methylation (m5C) 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 m5C methylation in innate immunity remains elusive. Here we show that depletion of NSUN2, an m5C 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 m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant non-coding RNAs (ncRNAs). However, m5C 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 upregulated, leading to an increased level in unshielded 7SL RNA in cytoplasm, which served as direct ligands 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 m5C methylation controls antiviral innate immunity through modulating m5C methylome of ncRNAs and their expression. [dataset1] 16 samples (Sample 1-16): Duplicates for bisulfite-seq for RNA m5C detection in total RNA from wild-type and NSUN2-knockout A549 cells, under RSV or VSV infection. [datase2] 16 samples (Sample 21-36): Duplicates for RIP-seq of RIG-I IP-enriched RNA from wild-type and NSUN2-knockout A549 cells, under RSV or VSV infection. [datase3] 12 samples (Sample 37-48): Triplicates for RNA-seq of "RNA input" and "anti-dsRNA antibody IP-enriched dsRNA" from wild-type and NSUN2-knockout A549 cells.