Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia.
Int J Mol Sci. 2022 Aug 28;23(17):9740. doi: 10.3390/ijms23179740.
The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.
RNA 胞嘧啶 C5 甲基转移酶 NSUN2 具有多种 RNA 底物，在 mRNA 代谢中发挥重要作用。NSUN2 结合到富含外泌体 mRNA 的特定序列中，表明其可能参与 mRNA 分选到外泌体中。我们应用光活化.4-硫代尿嘧啶增强交联和免疫沉淀结合高通量 RNA 测序（RNA-seq）分析方法，鉴定 HEK293T 细胞中的 NSUN2 mRNA 靶标。NSUN2 交联位点存在于 100 多个相对丰富的 mRNA 中，这些 mRNA 具有高 GC 含量和明显的二级结构。然后，利用 RNA-seq 分析 HEK293T 细胞中总 mRNA 和多核糖体相关 mRNA，分别在敲低 NSUN2 前后进行，我们鉴定了差异表达基因以及翻译效率改变的基因（GATEs）。结果表明，上调的 GATE mRNA 平均比下调的 GATE mRNA 短，GC 含量更高；此外，它们包含 C 残基位于富含 GC 环境中的基序。我们的研究结果揭示了使它们成为 NSUN2 潜在靶标的 mRNA 的特定特征，并扩展了我们对 NSUN2 在控制翻译以及可能通过胞嘧啶残基甲基化实现 mRNA 分选到外泌体中的作用的理解。
