N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells

Abstract

Historically, N6-methyladenosine (m6A) has been identified as the most abundant internal modification of messenger RNA (mRNA) in eukaryotes 1. Its mammalian function remained unknown until recently, when it was reported that thousands of mammalian mRNAs and long noncoding RNAs (lncRNAs) show m6A modification 2,3 and that m6A demethylases are required for mammalian energy homeostasis and fertility 4,5. As yet, the identity of m6A methyltransferases (MTase) and the molecular mechanisms regulated by m6A remains unclear. Here, we show that two proteins, the putative m6A MTase, methyltransferase-like 3 (Mettl3) 6, and a related but uncharacterized protein Mettl14, function synergistically to control m6A formation in mammalian cells. Since m6A modification is involved in cell fate determination in yeast 7,8 and embryo development in plant 9,10, we knocked down Mettl3 and Mettl14, respectively, in mouse embryonic stem cells (mESCs). The resulting cells displayed equivalent phenotypes characterized by lack of m6A RNA methylation and lost self-renewal capability. We also observed that a large number of transcripts, including many encoding developmental regulators, showed m6A methylation inversely correlated with mRNA stability and gene expression. Further analysis suggested that some of these effects were mediated through Human antigen R (HuR) and microRNA pathways. Overall our work provides first experimental evidence of mammalian m6A MTases and reveals a previously unknown gene regulatory mechanism operating in mESCs through m6A methylation. This mechanism is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

“…2b). In addition to the known MTA‐FIP37 interaction (Zhong et al ., 2008), we observed heterodimerization of the Arabidopsis MTA and MTB, consistent with the reported interaction between their mammalian orthologues METTL3 and METTL14 (Liu et al ., 2014; Ping et al ., 2014; Wang et al ., 2014b). We also found that MTB but not MTA formed homodimers in Y2H.…”

“…Orthologues of MTA and MTB interact with each other and with orthologues of FIP37 in S. cerevisiae (Agarwala et al ., 2012) and human cells (Liu et al ., 2014; Ping et al ., 2014; Schwartz et al ., 2014; Wang et al ., 2014b). In S. cerevisiae , writing of m 6 A requires orthologues of MTA (IME4), FIP37 (MUM2) and the yeast‐specific SLZ1 (Agarwala et al ., 2012).…”

“…N 6‐methyladenosine (m 6 A) is the most prevalent internal mRNA modification found in eukaryotes, and has received a burst of interest in recent years (Meyer & Jaffrey, 2014; Fray & Simpson, 2015; Yue et al ., 2015). m 6 A appears to be involved in a broad range of biological processes including mRNA export from the nucleus (Fustin et al ., 2013), regulation of splicing (Alarcón et al ., 2015b; Haussmann et al ., 2016; Lence et al ., 2016), mRNA translatability and stability (Wang et al ., 2014a,b, 2015; Bodi et al ., 2015; Zhou et al ., 2015), alternative polyadenylation site choice (Ke et al ., 2015) and other mechanisms accompanying RNA maturation (Meyer & Jaffrey, 2014; Yue et al ., 2015). m 6 A is essential for the earliest stages of pattern formation in plants (Zhong et al ., 2008; Bodi et al ., 2012; Shen et al ., 2016) and metazoans (Meyer & Jaffrey, 2014; Geula et al ., 2015; Yue et al ., 2015; Haussmann et al ., 2016; Lence et al ., 2016), linked with diseases in humans and other mammalian species (Jia et al ., 2011; Zheng et al ., 2013) and is required for meiosis in Saccharomyces cerevisiae (Clancy et al ., 2002).…”

“…The identity of any of the other proteins that act together with METTL3 remained unknown until it was shown in Arabidopsis that FIP37 (FKBP12 INTERACTING PROTEIN 37) was a partner protein of MTA (the homologue of METTL3) (Zhong et al ., 2008; Shen et al ., 2016). Following this initial discovery, the homologues of FIP37, S. cerevisiae MUM2 (Muddled Meiosis 2), and mammalian WTAP (Wilms tumour 1 associated protein), were shown to interact with METTL3 (MTA) orthologues and to be required for mRNA methylation in their respective model organisms (Agarwala et al ., 2012; Ping et al ., 2014; Wang et al ., 2014b; Liu et al ., 2015). More recently, another human methylase, METTL14, phylogenetically related to METTL3 (Bujnicki et al ., 2002), was shown to form a complex with METTL3 and WTAP, and to be required also for m 6 A formation (Ping et al ., 2014; Wang et al ., 2014b; Liu et al ., 2015).…”

Structure of the YTH domain of human YTHDF2 in complex with an m6A mononucleotide reveals an aromatic cage for m6A recognition

“…This modification is post-transcriptionally installed by m 6 A methyltransferases (METTL3-METTL14-WTAP complex) [1][2][3][4] and oxidatively removed by m 6 A demethylases (FTO and ALKBH5) [5,6]. These 'writer' and 'eraser' enzymes are required for embryo development, energy homeostasis and fertility, suggesting fundamental regulatory roles of m 6 A [1, 2,5].…”