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. Overall design: [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.

胞嘧啶-5甲基化（Cytosine-5 methylation，m5C）是RNA最普遍的修饰类型之一，在RNA代谢、核输出与翻译过程中发挥关键作用。然而，RNA m5C甲基化在天然免疫中的潜在功能仍有待阐明。本研究发现，敲除m5C甲基转移酶NSUN2后，可显著抑制多种RNA与DNA病毒的复制及基因表达。值得注意的是，该抗病毒效应主要由增强的I型干扰素（type I interferon，IFN）应答所介导。该抗病毒信号通路依赖于胞质RNA感受器RIG-I，而非MDA5。
对人A549细胞中NSUN2敲除后的m5C进行全转录组定位分析发现，多种高丰度非编码RNA（non-coding RNAs，ncRNAs）的m5C甲基化水平显著降低。不过，病毒RNA的m5C甲基化水平并未因NSUN2敲除而发生明显变化。
在NSUN2敲除的细胞中，宿主RNA聚合酶III（RNA polymerase III，Pol III）转录的非编码RNA（尤其是RPPH1与7SL RNA）显著上调，导致胞质中未被遮蔽的7SL RNA水平升高，该RNA可作为RIG-I介导的IFN应答的直接配体。在NSUN2敲除的细胞中，抑制Pol III转录或敲低RPPH1与7SL RNA可削弱IFN信号通路，部分恢复病毒的复制与基因表达。
最后，在离体人肺模型与小鼠模型中敲除NSUN2，可抑制病毒复制并减轻发病程度，同时伴随I型IFN应答的增强。
综上，本研究数据表明，RNA m5C甲基化可通过调控非编码RNA的m5C甲基化组及其表达水平，从而调控抗病毒天然免疫。
实验设计：
[数据集1] 共16份样本（样本1-16）：针对野生型与NSUN2敲除A549细胞在呼吸道合胞病毒（Respiratory Syncytial Virus，RSV）或水疱性口炎病毒（Vesicular Stomatitis Virus，VSV）感染条件下的总RNA进行RNA m5C检测的亚硫酸氢盐测序（bisulfite-seq）重复样本。
[数据集2] 共16份样本（样本21-36）：针对野生型与NSUN2敲除A549细胞在RSV或VSV感染条件下的RIG-I免疫沉淀（IP）富集RNA进行RNA免疫沉淀测序（RIP-seq）的重复样本。
[数据集3] 共12份样本（样本37-48）：针对野生型与NSUN2敲除A549细胞的"RNA input"与"anti-dsRNA antibody IP-enriched dsRNA"进行RNA-seq的三重复样本。