Fig 1.
m6A methylation requires a multi-protein writer complex.
The m6A writer complex is a multi-component assembly that deposits N6-methyladenosine (m6A) on mRNA in a transcriptome-wide yet site-selective manner. In the writer complex, only METTL3 contains the catalytic domain, but optimal methylation requires additional non-catalytic auxiliary proteins. Unlike other RNA modifications that are catalyzed by single enzymes, m6A methylation requires a multi-protein complex. By contrast, NSUN2 (a cytosine-5 RNA methyltransferase) and pseudouridine synthase (PUS) enzymes typically function as single-protein modifiers that act on defined RNA substrates. NSUN2 catalyzes 5-methylcytosine (m5C) deposition primarily on tRNAs, but also on vault RNAs, 7SK RNA and some mRNAs, where it regulates the RNA’s stability and translation. PUS enzymes convert uridine to pseudouridine (Ψ) on tRNAs, rRNAs, snRNAs and a subset of mRNAs in a site-specific manner. The necessity for a multi-protein complex for m6A deposition and the roles of each writer complex component remains a mystery. Created in BioRender. Jaffrey, S. (2025) https://BioRender.com/hffsuxx.
Fig 2.
Potential functions of the components of the m6A writer complex. This schematic summarizes the proposed potential roles of METTL3, METTL14, WTAP, VIRMA, ZC3H13, RBM15/15B and HAKAI in the m6A writer complex. METTL3 serves as the catalytic subunit, while METTL14 stabilizes METTL3 and contributes to RNA binding. WTAP functions as a scaffold for nuclear localization and complex assembly. VIRMA is implicated in transcript selectivity and preferential 3′ UTR methylation. ZC3H13 anchors the complex to the nuclear matrix and may regulate its activity state. RBM15 is thought to act as adapter proteins that recruit the m6A writer complex to specific RNA substrates, particularly to U-rich regions of transcripts. HAKAI, a putative E3 ubiquitin ligase, may modulate complex stability or respond to cellular stress, although its exact function remains unclear. Together, these components coordinate methylation in a transcript- and context-specific manner.
Fig 3.
Auxiliary proteins may act as rate-limiting factors of the m6A writer complex.
Here we use the term ‘rate-limiting’ to describe the functionally limiting component, based on knockdown correlations rather than direct kinetic measurements. About 50% of m6A remained when METTL3 was reduced to 5%, showing that only a small amount of METTL3 is required to maintain physiological levels of m6A in mRNA. This suggests that METTL3 is not the rate-limiting factor for m6A methylation. By contrast, reducing the level of the auxiliary protein WTAP (to 15%) caused a proportional decrease in m6A (to 15%), indicating a rate-limiting role of WTAP in m6A deposition. The abundance of each protein and its dynamic assembly into the m6A writer complex may regulate sensitivity to METTL3 depletion. Further work is needed to identify the relationships between METTL3 and other auxiliary proteins. Created in BioRender. Jaffrey, S. (2025) https://BioRender.com/alkhfzt.