Mutational Analysis Reveals Functional Roles of METTL16 Domains and Residues

Human methyltransferase-like protein 16 (METTL16) installs <i>N</i>⁶-methyladenosine on U6 small nuclear RNA (snRNA) and other RNAs. Multiple X-ray crystal structures of METTL16 have been published; however, we do not yet fully understand the structure-function relationships of specific residues. We designed 38 mutants, including seven cancer-associated mutants, and used electrophoretic mobility shift assays and single-turnover kinetic assays to better understand the functional roles of specific domains and amino acid residues in binding to U6 snRNA, formation of the METTL16•U6 snRNA•<i>S</i>-adenosylmethionine (SAM) complex, and the rate of methylation. While point mutations in the methyltransferase domain mildly weaken the binding of METTL16 to U6 snRNA, the C-terminal vertebrate conserved regions (VCRs), particularly the arginine-rich region (R382 to R388), mediate cooperative binding and contribute more to RNA binding. All METTL16 K-loop mutants displayed tighter SAM binding, suggesting that the K-loop blocks SAM binding. In addition, residues E133 and F227 are critical for stabilizing SAM binding. Mutations in the ₁₈₄NPPF₁₈₇ catalytic core and R282A abolished methyltransferase activity. Two METTL16 somatic cancer-associated mutants (G110C and R241Dfs*2) displayed reduced methylation activity. This mutational analysis expands our understanding of how specific domains and residues contribute to substrate-binding activity and methylation of U6 snRNA catalyzed by METTL16.