Regulation and Site-Specific Covalent Labeling of NSUN2 via Genetic Encoding Expansion.

Jizhong Zhao,Hongmei Hu,Li Wang,Sheng Wang,Rui Wang

doi:10.3390/genes12101488

Abstract

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 the NSUN enzyme 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 the MbPylRS-tRNACUA unnatural lysine substitution system, which specifically recognizes lysine with a defined group. Moreover, non-natural lysine substitution at C271 of NSUN2 active site and the subsequent fluorescent labeling was realized through the click reaction. Then, the function of the NSUN2 mutant and its upregulated CDK1 gene as well as its effect on cell proliferation were evaluated. Efficient labeling and regulation of NSUN2 was achieved, laying the basis for further studies on the function and regulatory mechanism of upregulated genes.

Highlights

In living organisms, ribonucleic acids regulate gene expression, cell migration, differentiation, and cell death [1,2]
[23] substrate shows that Lys-N3 (S3) was restored to its native form through rapid addition of tricarboxyethyl phosphine (TCEP) [26]
Our findings show that biological orthogonal reactions of unnatural lysine with the azido group together with genetic encoding expansions enable specific NSUN2 protein labeling and tracking (Figure 3C,D)

Summary

Introduction

Ribonucleic acids regulate gene expression, cell migration, differentiation, and cell death [1,2]. To achieve the regulation purpose, RNA modifying enzymes play essential roles, adding a wide range of chemical modifications into target RNAs. Methylation is heavily associated with intrinsic RNAs in various species [6,7,8,9,10,11,12]. Based on the roles of NSUN2 enzymes in m5C-associated RNA biological activities in living organisms, including cellular proliferation, senescence, migration, differentiation, mRNA nuclear export, enhanced mRNA translation, tRNA stabilization, and cleavage [16], achieving site-specific labeling and modulation of NSUN2 is, of high importance. Precise regulation enhanced mRNA translation, tRNA stabilization, and cleavage [16], achieving sitelabeling and modulation of NSUN2 is, of high importance. Regulation of nuclear-cytoplasmic shuttling of endogenous RNAs by manipulating tivity of NSUN2 remains elusive

Methods

Results

Conclusion