Function And Mechanism Of RNA Methyltransferase NSUN2 In Type Ⅰ Interferon Responses

Type I interferon plays a crucial role in cellular antiviral innate immunity.After infection by viruses or other pathogenic microorganisms,type I interferon in most cell types is induced to produce a series of antiviral effects,further amplifying a kind of positive feedback signal cascade through the JAK-STAT pathway.Then the expression of interferon stimulated genes（ISGs）and other cytokines are activated to inhibit viral replication and eliminate infected cells.Interferon regulatory factor 3（IRF3）plays an important role in the induction of type I interferon by viral infection.RNA modification is an important post-transcriptional modification process.To date,more than 100 modifications have been found on various types of RNA.5-Methylcytosine（m⁵C）is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism.However,its regulatory role in antiviral innate immunity has not yet been elucidated.Here,we report that NSUN2,a typical m⁵C methyltransferase,can negatively regulate type I interferon responses during viral infection.NSUN2specifically mediates m⁵C methylation of IRF3 m RNA and accelerates its degradation,resulting in low levels of IRF3 and downstream IFN-βproduction.Knockout or knockdown of NSUN2 could enhance type I interferon responses and downstream ISG expression after viral infection in vitro.And in vivo,the antiviral innate responses is more dramatically enhanced and therefore the replication level of VSV is reduced in Nsun2^+/-mice than in Nsun2^+/+mice.Further,we identified four highly methylated cytosine sites in IRF3 m RNA by combining m⁵C-Me RIP-Seq,Bisulfite RNA sequencing and RIP-Seq.We combined in vivo and in vitro methylation experiments to demonstrate that NSUN2 could specifically methylate IRF3 m RNA through the four major cytosine sites.The mutation of the four highly m⁵C methylated cytosines could enhance the cellular IRF3 m RNA levels,which further enhanced the response of type I interferon.Moreover,we found that NSUN2 expression is decreased after infections with different viruses,including Sendai virus（Se V）,vesicular stomatitis virus（VSV）,herpes simplex virus 1（HSV-1）,Zika virus（ZIKV）,or especially SARS-Co V-2.We can therefore propose a model whereby NSUN2 is constitutively expressed in resting cells and that IRF3 expression is maintained at a relatively low level.During viral infection,endogenous NSUN2 expression levels decrease,and the IRF3 expression level would therefore be elevated to allow a stronger interferon response and the effective elimination of viruses.In conclusion,we reveal a novel mechanism by which NSUN2-mediated m⁵C methylation on IRF3 m RNA accelerates its degradation and negatively regulates the type I interferon response.We have proposed a crosstalk between m⁵C methylation and antiviral innate immunity,and this might benefit the development of efficient therapeutic interventions for infectious diseases.To move forward,further work is urgently needed to precisely demonstrate how m⁵C methylation is involved in antiviral innate immunity and other physiological processes.