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Updated: May 11, 2026
Antibody-Free Assay for RNA Methyltransferase Activity Analysis
Published on: July 9, 2019
Identification of Endogenous Sequences Interacting with METTL3/METTL14 RNA Methyltransferase
Tamaki Endoh1,2, Yiwei Ling3, Shujiro Okuda3
1Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan.
Abstract:
N6-methyladenosine (m6A) is the most abundant RNA modification in mRNA and regulates various biological processes. The RNA-binding properties of m6A writer proteins play an important role in determining RNA modification sites. METTL3 and METTL14 form the core of the m6A writer complex, with METTL3 as the catalytic methyltransferase and METTL14 as the RNA-binding scaffold. Thus far, the comprehensive RNA-binding properties of METTL3/14 remain unknown. Using RNA-capturing microsphere particles (R-CAMPs), immobilizing RNA fragments derived from endogenous RNAs of human lung carcinoma cells, RNA fragments that interacted with the METTL3/14 methyltransferase domain are isolated. Bioinformatics analysis reveals that the pool of isolated sequences contains significantly more regions with the potential to form RNA G-quadruplexes (rG4s) than the randomly extracted RNA sequence pool and that the (GGA) repeat sequences are most enriched. Circular dichroism spectroscopy, gel mobility shift assays, and methylation experiments demonstrate that METTL3/14 binds to RNA sequences containing (GGA) repeats that form rG4 structure much stronger than RNAs that do not form rG4s. This study shows the potential of RNA G-quadruplex structures as a modulator of epitranscriptomic modification of m6As.
Insights
The METTL3/14 complex preferentially binds to RNA G-quadruplex structures, particularly those with (GGA) repeats. This finding reveals RNA G-quadruplexes as key modulators of N-methyladenosine (m6A) epitranscriptomic modification.
Area of Science:
- Molecular Biology
- Epigenetics
- RNA Biology
Background:
- N-methyladenosine (m6A) is the most prevalent mRNA modification, regulating diverse biological processes.
- The m6A writer complex, comprising METTL3 and METTL14, is crucial for m6A deposition, with METTL14 acting as an RNA-binding scaffold.
- The complete RNA-binding landscape of the METTL3/14 complex remains largely uncharacterized.
Purpose of the Study:
- To comprehensively investigate the RNA-binding properties of the METTL3/14 complex.
- To identify specific RNA sequence motifs and structures that interact with METTL3/14.
- To elucidate the role of RNA structure in m6A modification.
Main Methods:
- RNA-capturing microsphere particles (R-CAMPs) were employed to isolate RNA fragments interacting with the METTL3/14 methyltransferase domain from human lung carcinoma cells.
- Bioinformatics analysis was used to identify enriched sequence features and potential secondary structures in the captured RNA fragments.
- Circular dichroism spectroscopy, gel mobility shift assays, and methylation experiments were performed to validate RNA-structure-protein interactions.
Main Results:
- Bioinformatic analysis revealed a significant enrichment of RNA G-quadruplex (rG4) forming regions, particularly (GGA) repeats, in RNA fragments bound by METTL3/14.
- Experimental validation confirmed that METTL3/14 exhibits significantly stronger binding to RNA sequences forming rG4 structures, especially those with (GGA) repeats, compared to non-rG4 forming sequences.
- The study identified specific RNA structural motifs that dictate METTL3/14 binding.
Conclusions:
- RNA G-quadruplex structures, particularly those containing (GGA) repeats, are key determinants of METTL3/14 binding specificity.
- These findings highlight the potential of RNA G-quadruplexes to modulate epitranscriptomic modifications by influencing m6A deposition.
- This work provides novel insights into the regulatory mechanisms of m6A modification at the RNA sequence and structure level.
