Understanding the source of METTL3-independent m⁶A in mRNA

Abstract

N6-methyladenosine (m6A) is a highly prevalent mRNA modification which promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30–80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we re-examined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines which continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3’s role in synthesizing m6A using a genomic deletion of Mettl3, and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

m6A-ELISA, a simple method for quantifyingN6-methyladenosine from mRNA populations

“…Also, we measured relative m6A levels in WT mESC and a Mettl3 knockout mESC line (Figure 3C). M6A levels were reduced over 3-fold in the Mettl3 knockout cell line, which is consistent with the idea that METTL3 is the major m6A methyltransferase for mRNAs in mammals (Poh et al 2021). Furthermore, we found that low technical variability could be achieved when using as little as 25 ng mammalian mRNA (Figure 3C and Supplementary Figure 2).…”

scm6A-seq reveals single-cell landscapes of the dynamic m6A during oocyte maturation and early embryonic development

“…Our data proved that METTL3 is a critical m 6 A methyltransferase for mouse oogenesis. Although it was reported that METTL3 has been previously reported to be critical for more than 90% of m 6 A marks in mESCs 62 , we detected m 6 A distribution on the mRNAs of the Mettl3 cKO GV oocytes, suggesting the presence of other methyltransferases critical for m 6 A deposition in mouse GV oocytes.…”

Selective detection of m6A derived from mRNA using the Phospho-tag m6A assay

“…We therefore prepared total RNA from wild-type mouse embryonic stem (mES) cells as well as Mettl3 knockout mES cells 32 . We used the Mettl3 knockout mES cells generated by Hanna and colleagues, which exhibit a >99% reduction in m 6 A levels in highly purified mRNA 32,33 . Thus, Mettl3 knockout mES cells are an ideal system to establish whether any of the m 6 A signal in the phospho-tag assay derives from mRNA.…”