Protein Engineering and Evolution Unit, Okinawa Institute of Science and Technology Graduate University (OIST), 1919-1 Tancha, Onna, Kunigami District, Okinawa 904-0495, Japan.
Molecular Neuroscience Unit, Okinawa Institute of Science and Technology Graduate University (OIST), 1919-1 Tancha, Onna, Kunigami District, Okinawa 904-0495, Japan.
Nucleic Acids Res. 2024 Nov 11;52(20):12158-12172. doi: 10.1093/nar/gkae887.
N 6-Methyladenine (m6A) RNA methylation plays a key role in RNA processing and translational regulation, influencing both normal physiological and pathological processes. Yet, current techniques for studying RNA methylation struggle to isolate the effects of individual m6A modifications. Engineering of RNA methyltransferases (RNA MTases) could enable development of improved synthetic biology tools to manipulate RNA methylation, but it is challenging due to limited understanding of structure-function relationships in RNA MTases. Herein, using ancestral sequence reconstruction, we explore the sequence space of the bacterial DNA methyltransferase EcoGII (M.EcoGII), a promising target for protein engineering due to its lack of sequence specificity and its residual activity on RNA. We thereby created an efficient non-specific RNA MTase termed SUPer RNA EcoGII Methyltransferase (SUPREM), which exhibits 8-fold higher expression levels, 7°C higher thermostability and 12-fold greater m6A RNA methylation activity compared with M.EcoGII. Immunofluorescent staining and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed SUPREM's higher RNA methylation activity compared with M.EcoGII in mammalian cells. Additionally, Nanopore direct RNA sequencing highlighted that SUPREM is capable of methylating a larger number of RNA methylation sites than M.EcoGII. Through phylogenetic and mutational analysis, we identified a critical residue for the enhanced RNA methylation activity of SUPREM. Collectively, our findings indicate that SUPREM holds promise as a versatile tool for in vivo RNA methylation and labeling.
N6-甲基腺嘌呤（m6A）RNA 甲基化在 RNA 加工和翻译调控中发挥着关键作用，影响着正常的生理和病理过程。然而，目前研究 RNA 甲基化的技术难以分离单个 m6A 修饰的影响。RNA 甲基转移酶（RNA MTases）的工程化可以开发出改进的合成生物学工具来操纵 RNA 甲基化，但由于对 RNA MTases 的结构-功能关系的理解有限，这一过程具有挑战性。在此，我们利用祖先序列重建，探索了细菌 DNA 甲基转移酶 EcoGII（M.EcoGII）的序列空间，由于其缺乏序列特异性和在 RNA 上的残留活性，它是蛋白质工程的一个很有前途的目标。我们因此创建了一种高效的非特异性 RNA MTase，称为 SUPer RNA EcoGII Methyltransferase（SUPREM），与 M.EcoGII 相比，它的表达水平提高了 8 倍，热稳定性提高了 7°C，m6A RNA 甲基化活性提高了 12 倍。免疫荧光染色和定量液相色谱-串联质谱（LC-MS/MS）分析证实，与 M.EcoGII 相比，SUPREM 在哺乳动物细胞中的 RNA 甲基化活性更高。此外，纳米孔直接 RNA 测序突出表明，SUPREM 能够甲基化比 M.EcoGII 更多的 RNA 甲基化位点。通过系统发育和突变分析，我们确定了 SUPREM 增强 RNA 甲基化活性的关键残基。总之，我们的研究结果表明，SUPREM 有望成为一种用于体内 RNA 甲基化和标记的多功能工具。
