Elucidating the Kinetic Mechanism of Human METTL16

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Methylation

Background:

• Methyltransferase-like protein 16 (METTL16) is a key human m⁶A RNA methyltransferase.
• METTL16 is known to methylate U6 small nuclear RNA (U6 snRNA) and MAT2A mRNA.
• METTL16 also interacts with other RNAs, including the MALAT1 transcript.

Purpose of the Study:

• To investigate the kinetic mechanism and biochemical properties of METTL16.
• To determine the binding affinities of METTL16 to U6 snRNA and MALAT1 RNA.
• To elucidate the order of substrate binding and catalytic steps in METTL16-mediated methylation.

Main Methods:

• Enzyme kinetics assays (steady-state and single-turnover).
• Isotope partitioning and preincubation experiments.
• RNA binding studies using various RNA substrates.

Main Results:

• METTL16 exhibits distinct binding affinities for U6 snRNA (18 nM) and methylated U6 snRNA (1.1 μM).
• An ordered-sequential mechanism was identified, with METTL16 binding U6 snRNA prior to S-adenosylmethionine (SAM).
• The methyltransferase domain of METTL16 shows specific catalytic parameters (kcat, kchem) for U6 snRNA methylation.

Conclusions:

• METTL16 functions as a monomer and follows an ordered kinetic mechanism for U6 snRNA methylation.
• The MALAT1 triple helix is a binding target but not a direct substrate for METTL16 in vitro.
• Understanding METTL16's catalytic mechanism provides insights into its biological roles in RNA modification.