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

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• N 6-Methyladenine (m6A) RNA methylation is crucial for cellular processes but difficult to study at individual modification sites.
• Current RNA methylation research faces challenges in isolating specific m6A modifications.
• Engineering RNA methyltransferases (RNA MTases) is vital for developing precise synthetic biology tools for RNA manipulation.

Purpose of the Study:

• To engineer a more efficient and stable RNA methyltransferase for studying N 6-Methyladenine (m6A) RNA modifications.
• To explore the potential of bacterial DNA methyltransferase EcoGII (M.EcoGII) for protein engineering targeting RNA methylation.
• To develop improved synthetic biology tools for in vivo RNA methylation and labeling.

Main Methods:

• Ancestral sequence reconstruction to explore the sequence space of M.EcoGII.
• Protein engineering to create the SUPer RNA EcoGII Methyltransferase (SUPREM).
• Comparative analysis of SUPREM and M.EcoGII using immunofluorescence, LC-MS/MS, and Nanopore direct RNA sequencing in mammalian cells.

Main Results:

• SUPREM demonstrated 8-fold higher expression, 7°C greater thermostability, and 12-fold increased m6A RNA methylation activity compared to M.EcoGII.
• In mammalian cells, SUPREM showed significantly higher RNA methylation activity and targeted a broader range of RNA methylation sites.
• Phylogenetic and mutational analyses identified a key residue responsible for SUPREM's enhanced RNA methylation activity.

Conclusions:

• SUPREM is a highly efficient, non-specific RNA methyltransferase with improved properties over M.EcoGII.
• The engineered SUPREM enzyme offers a promising tool for advanced in vivo RNA methylation and labeling applications.
• This study highlights the potential of protein engineering via ancestral sequence reconstruction for developing novel synthetic biology tools.