Liu Jianheng, Huang Tao, Zhang Yusen, Zhao Tianxuan, Zhao Xueni, Chen Wanying, Zhang Rui
MOE Key Laboratory, of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510275, China.
Natl Sci Rev. 2020 Oct 31;8(6):nwaa273. doi: 10.1093/nsr/nwaa273. eCollection 2021 Jun.
mRNA mC, which has recently been implicated in the regulation of mRNA mobility, metabolism and translation, plays important regulatory roles in various biological events. Two types of mC sites are found in mRNAs. Type I mC sites, which contain a downstream G-rich triplet motif and are computationally predicted to be located at the 5 end of putative hairpin structures, are methylated by NSUN2. Type II mC sites contain a downstream UCCA motif and are computationally predicted to be located in the loops of putative hairpin structures. However, their biogenesis remains unknown. Here we identified NSUN6, a methyltransferase that is known to methylate C72 of tRNA and tRNA, as an mRNA methyltransferase that targets Type II mC sites. Combining the RNA secondary structure prediction, miCLIP, and results from a high-throughput mutagenesis analysis, we determined the RNA sequence and structural features governing the specificity of NSUN6-mediated mRNA methylation. Integrating these features into an NSUN6-RNA structural model, we identified an NSUN6 variant that largely loses tRNA methylation but retains mRNA methylation ability. Finally, we revealed a weak negative correlation between mC methylation and translation efficiency. Our findings uncover that mRNA mC is tightly controlled by an elaborate two-enzyme system, and the protein-RNA structure analysis strategy established may be applied to other RNA modification writers to distinguish the functions of different RNA substrates of a writer protein.
信使核糖核酸（mRNA）上的5-甲基胞嘧啶（mC）最近被认为参与了mRNA迁移、代谢及翻译的调控，在多种生物学事件中发挥着重要的调节作用。在mRNA中发现了两种类型的mC位点。I型mC位点含有一个下游富含鸟嘌呤的三联体基序，通过计算预测位于假定发夹结构的5'端，由NSUN2甲基化。II型mC位点含有一个下游UCCA基序，通过计算预测位于假定发夹结构的环中。然而，它们的生成机制仍然未知。在这里，我们鉴定出NSUN6，一种已知可甲基化转运RNA（tRNA）和5-甲基胞嘧啶转运核糖核酸（tRNA）的C72的甲基转移酶，是一种靶向II型mC位点的mRNA甲基转移酶。结合RNA二级结构预测、甲基化胞嘧啶单核苷酸分辨率交联免疫沉淀（miCLIP）以及高通量诱变分析的结果，我们确定了决定NSUN6介导的mRNA甲基化特异性的RNA序列和结构特征。将这些特征整合到一个NSUN6-RNA结构模型中，我们鉴定出一种NSUN6变体，其在很大程度上丧失了tRNA甲基化能力，但保留了mRNA甲基化能力。最后，我们揭示了mC甲基化与翻译效率之间存在微弱的负相关。我们的研究结果揭示，mRNA mC受到一个精细的双酶系统的严格控制，并且所建立的蛋白质-RNA结构分析策略可能适用于其他RNA修饰酶，以区分一种酶蛋白的不同RNA底物的功能。
