RNA Methyltransferase METTL16's Protein Domains Have Differential Functional Effects on Cell Processes

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• METTL16 is a human m6A RNA methyltransferase known to modify U6 and MAT2A RNAs.
• METTL16 binds numerous RNAs, but its modification activity on these is not fully understood.
• METTL16 possesses multiple RNA-binding domains, with the functional significance of each domain remaining unclear.

Purpose of the Study:

• To investigate the functional importance of specific METTL16 RNA-binding domains.
• To determine how mutations in METTL16 domains affect RNA binding, expression, and cellular processes.
• To elucidate the role of METTL16 binding, independent of its methyltransferase activity, in cellular functions.

Main Methods:

• Site-directed mutagenesis of METTL16's N-terminal RNA-binding domain, methyltransferase domain, and C-terminal RNA-binding domain.
• Analysis of RNA-binding ability, protein and RNA expression levels in mutant cell lines.
• Flow cytometry to assess cell cycle phase occupancy (G1 and S phases).
• Cell proliferation assays to measure the impact of METTL16 mutations on cell growth.

Main Results:

• Mutations in METTL16 domains altered RNA-binding capabilities and affected protein and RNA expression.
• METTL16 mutants exhibited varying impacts on cell cycle progression, with significant changes in G1 and S phase occupancy.
• Cellular processes including metabolism, intracellular transport, and RNA processing were differentially affected by METTL16 mutations.
• Proliferative ability was significantly impacted in some, but not all, METTL16 mutant lines.

Conclusions:

• METTL16 binding, irrespective of its m6A modification status on all bound RNAs, plays a crucial role in regulating diverse cellular processes.
• Individual RNA-binding domains of METTL16 contribute uniquely to its overall cellular functions.
• The study highlights the significance of METTL16's RNA-binding interactions in maintaining cellular homeostasis and regulating cell division and metabolism.