Identification of Endogenous Sequences Interacting with METTL3/METTL14 RNA Methyltransferase

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.