Regulation and Site-Specific Covalent Labeling of NSUN2 via Genetic Encoding Expansion
In living organisms, RNA regulates gene expression, cell migration, differentiation, and cell death. 5-Methylcytosine is a post-transcriptional RNA modification in a wide range of RNA species, including messenger RNAs. The addition of m5C to RNA cytosines is enabled by theNSUNenzyme family, a critical RNA methyltransferase. In this study, natural lysines modified with special groups were synthesized. Through two rounds of positive screening and one round of negative screening, we evaluated and identified theMbPylRS-tRNACUAunnatural lysine substitution system, which specifically recognizes lysine with a defined group. Moreover, non-natural lysine substitution at C271 ofNSUN2active site and the subsequent fluorescent labeling was realized through the click reaction. Then, the function of theNSUN2mutant and its upregulatedCDK1gene as well as its effect on cell proliferation were evaluated. Efficient labeling and regulation ofNSUN2was achieved, laying the basis for further studies on the function and regulatory mechanism of upregulated genes.Keywords:RNA modification;m5C;NSUN enzyme;regulation;labeling
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