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.

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a ''CAAX protein'' that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by 2 statistic). These studies suggest a possible treatment strategy for HGPS.aging ͉ lamin A͞C ͉ laminopathy H utchinson-Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by a host of aging-like phenotypes, including a wizened appearance of the skin, osteoporosis, alopecia, and premature atherosclerosis (1). Children with HGPS die at the mean age of 13, generally from myocardial infarctions or strokes (1). This disease is caused by the accumulation of a mutant form of prelamin A that cannot be processed to mature lamin A (1). In normal cells, wild-type prelamin A is virtually undetectable because it is fully converted to mature lamin A, a structural protein of the nuclear lamina (2, 3). The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane that provides structural support for the nucleus (2, 3).Prelamin A contains a nuclear localization signal and terminates with a CAAX motif (2), in which C is a cysteine, A residues are usually aliphatic amino acids, and X can be one of many different residues. CAAX motifs are also found on lamin B1, lamin B2, the Ras family of proteins, and many other cellular proteins. The CAAX motif triggers three sequential enzymatic posttranslational modifications, beginning with protein prenylation. In the case of prelamin A, the first processing step is carried out by protein farnesyltransferase (FTase) and involves the addition of a 15-carbon farnesyl lipid to the thiol group of the cysteine within the CAAX motif. Second, the last 3 aa of the protein (i.e., ϪAAX) are removed by a prenylprotein-specific endoprotease. For p...