SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency

m 6 A 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 m 6 A 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 SUPREM, which exhibits 8-fold higher expression levels, 7 °C higher thermostability, and 12-fold greater m 6 A RNA methylation activity compared with M.EcoGII. Immunofluorescent staining and quantitative 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.