N6-methyladenosine (m6A), the most frequent internal modification of mRNA, is required to maintain the properties of mouse embryonic stem (ES) cells, according to new research published in Nature Cell Biology. This is one of the first demonstrations of a mammalian function for m6A modification, through effects on mRNA stability.

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Two mammalian m6A methyltransferases, METTL3 and METTL14, have been predicted on the basis of sequence analysis. Using short hairpin RNAs to target Mettl3 or Mettl14, the authors generated knockdown ES cell lines and showed that m6A levels were decreased, as compared with controls, in cells depleted of either methyltransferase. A direct RNA methylation assay confirmed that METTL3 and METTL14 mediate m6A formation individually and can function synergistically to increase m6A modification.

Genome-wide analysis identified 4,766 genes in Mettl3 knockdown ES cells and 4,749 genes in Mettl14 knockdown ES cells that had significantly decreased m6A mRNA levels compared with controls. There was substantial overlap between these target genes, and METTL3 and METTL14 were shown to interact in vivo, which supports the idea of functional synergy between these m6A methyltransferases. Furthermore, METTL3 was almost absent in Mettl14 knockdown ES cells, despite only a small decrease in Mettl3 mRNA (and vice versa), which indicates that METTL3 and METTL14 might stabilize each other at the protein level.

m6A modification seems to increase mRNA decay

Microarray analysis and genome-wide gene-set enrichment analysis of the Mettl3 and Mettl14 knockdown ES cell lines showed that the expression of most pluripotency factors was downregulated compared with control cells, whereas the expression of some developmental regulators was significantly upregulated. Thus, m6A modification seems to be required to maintain the 'ground' state of ES cells; indeed, m6A levels in the RNA encoding five out of eight developmental regulators were significantly decreased by day 12 of ES cell differentiation.

So, how might m6A maintain pluripotency? Loss of m6A modification in knockdown cells was more significantly associated with gene upregulation than downregulation, and the stability of methyltransferase RNA targets was increased in knockdown cells compared with controls. Hence, m6A modification seems to increase mRNA decay; this destabilizing effect was shown to target developmental regulators in particular, which are more significantly enriched for METTL3 and METTL14 target sequences than are pluripotency genes.

The RNA stabilizer protein Hu-antigen R (HUR; also known as ELAVL1) showed increased binding to demethylated RNA from knockdown cells compared with m6A-modified RNA from control cells, which could explain how loss of m6A modification increases mRNA stability and gene expression. For example, the expression of the METTL3 and METTL14 target gene Igfbp3 (insulin-like growth factor-binding protein 3) is increased in knockdown ES cells; m6A demethylation of Igfbp3 in these cells is associated with increased HUR binding and increased stability of Igfbp3 mRNA, whereas Igfbp3 stability is decreased to control levels in methyltransferase-knockdown ES cells that also lack HUR. Finally, the authors showed that HUR increases the stability of Igfbp3 in methyltransferase-knockdown cells by blocking binding of the RNA-induced silencing complex (RISC).

Together, the data indicate that the pluripotency of ES cells is maintained, in part, through an RNA regulatory mechanism involving the m6A modification of developmental regulators, which blocks HUR binding, increases RISC binding and decreases mRNA stability to decrease gene expression. As thousands of mammalian mRNAs and long non-coding RNAs show m6A modification, this mechanism might have more widespread implications in various cell types.