Molecular basis of tRNA modification by the human m5C methyltransferase NSUN2

RNA 5-methylcytidine (m5C) is a prevalent modification that drives RNA stability and function. In humans, m5C is deposited on distinct RNA substrates by DNMT2/TRDMT1 and the NSUN family, to regulate diverse cellular processes, but how m5C writers recognise their substrates remains unclear. NSUN2 is a major m5C methyltransferase with broad roles in cell physiology and strong links to cancer and neurodevelopmental disorders 1. Here, we reconstitute an active human NSUN2-tRNA complex and capture its post-catalytic, tRNA-bound structure at 3.1 [A] resolution. Using an integrated approach combining biochemistry, cryo-electron microscopy, crosslinking mass spectrometry and molecular dynamics simulations, we show that NSUN2 remodels the tRNA to access the variable-loop target cytidine. Recognition is driven by RNA architecture, with NSUN2 exploiting the L-shaped tRNA scaffold to position the target base in the catalytic centre. We further show that Gly679 at the NSUN2-tRNA interface is important for the stability of the complex, providing a mechanistic basis for how the disease-associated Gly679Arg substitution can impair tRNA binding. Together, these findings establish an RNA-structure-guided mechanism for NSUN2 substrate recognition and methylation and provide general principles for m5C deposition on cellular RNAs and their fundamental role in disease.