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Related Experiment Video

Updated: Jul 21, 2025

Antibody-Free Assay for RNA Methyltransferase Activity Analysis
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RNA Methyltransferase METTL16's Protein Domains Have Differential Functional Effects on Cell Processes.

Emily S Talic1, Ashley Wooten2, Tonya N Zeczycki1,2

  • 1Biochemistry and Molecular Biology Department, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA.

Current Issues in Molecular Biology
|July 28, 2023
PubMed
Summary

The METTL16 methyltransferase binds many RNAs, but its modification role is unclear. Mutating METTL16 domains reveals its binding impacts cell cycle, metabolism, and proliferation.

Keywords:
METTL16N6-methyladenosineRNA-binding proteincell cyclem6Amethyltransferase

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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.