Substrate selectivity of the human RNA m5C methyltransferase NSUN2

Key Points:

• Researchers obtained a 2.57 Å resolution cryo-EM structure of human NSUN2 enzyme crosslinked with tRNALysCTT, revealing NSUN2’s four-domain architecture and extensive binding to the tRNA except for the D-arm, which shows conformational variability.
• NSUN2 induces a large conformational change in tRNA, unfolding its canonical L-shape to expose the methylation site at Cyt48; the enzyme primarily recognizes RNA duplex structures in the T- and acceptor arms through electrostatic and stacking interactions, with a preference for guanine bases near the methylation site.
• Biochemical assays demonstrated that NSUN2 requires the dual-stem structure of the tRNA’s T- and acceptor arms for efficient m5C methylation, but does not need the full tRNA tertiary fold or the D- and anticodon arms, distinguishing its substrate specificity from other tRNA methyltransferases like METTL1–WDR4.
• A minimal RNA substrate ("Mini") containing the dual-stem structure with a bulged linker was designed and shown to support NSUN2 methylation, highlighting the importance of RNA secondary structure and the CNNRR motif near the methylation site for enzyme recognition and activity.
• The study combined structural, biochemical, and mutational analyses to elucidate the catalytic mechanism of NSUN2, including the role of conserved cysteines in forming a covalent intermediate, and provided insights into how mutations affecting RNA binding or catalysis may contribute to diseases such as cancer.