Deficiency in a Glutamine-Specific Methyltransferase for Release Factor Causes Mouse Embryonic Lethality

Biological methylation is a fundamental enzymatic reaction for a variety of substrates in multiple cellular processes. Mammalian N6amt1 was thought to be a homologue of bacterial N⁶-adenine DNA methyltransferases, but its substrate specificity and physiological importance remain elusive. Here, we demonstrate that N6amt1 functions as a protein methyltransferase for the translation termination factor eRF1 in mammalian cells both in vitro and in vivo. Mass spectrometry analysis indicated that about 70% of the endogenous eRF1 is methylated at the glutamine residue of the conserved GGQ motif. To address the physiological significance of eRF1 methylation, we disrupted the N6amt1 gene in the mouse. Loss of N6amt1 led to early embryonic lethality. The postimplantation development of mutant embryos was impaired, resulting in degeneration around embryonic day 6.5. This is in contrast to what occurs in Escherichia coli and Saccharomyces cerevisiae, which can survive without the N6amt1 homologues. Thus, N6amt1 is the first glutamine-specific protein methyltransferase characterized in vivo in mammals and methylation of eRF1 by N6amt1 might be essential for the viability of early embryos.Nucleic acids, proteins, carbohydrates, and lipids, as well as a body of small molecules, are subject to methylation in a wide variety of biological contexts (3). The majority of methylation reactions are catalyzed by S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases). These enzymes ubiquitously exist in species from all three domains of life.Methylation of DNA occurs on one of two bases: cytosine or adenine (19). In prokaryotes, adenine methylation is as widespread as cytosine methylation. In contrast, eukaryotic genomes are devoid of adenine methylation or this type of methylation is too rare to be detected (23, 26). Intriguingly, two putative N⁶-adenine DNA MTases, named N6amt1 and N6amt2, are encoded in the mouse and human genomes. In addition to the conserved AdoMet-binding signature motif GXGXG and other sequence elements, they possess the NPPY motif characteristic of the N⁶-adenine or N⁴-cytosine DNA MTases in bacteria (6, 14). N6amt1 was thus proposed as an AdoMet-dependent DNA MTase, although no evidence had been provided that N6amt1 could methylate DNA (23).No functional clue for N6amt1 existed until two groups independently identified Escherichia coli HemK, distantly related to N6amt1, as a protein MTase for polypeptide release factors RF1 and RF2 (8, 17). The HemK gene was initially discovered in a genetic screen for heme biosynthesis mutants (18), although subsequent studies revealed no direct involvement in heme metabolism. The presence of an NPPY motif, thought to be restricted to members of the adenine and cytosine amino methyltransferases, led to the suggestion that HemK could be an AdoMet-dependent DNA MTase (2). However, a series of genetic and biochemical experiments finally revealed that HemK methylates the side-chain amide group of a glutamine residue in the universally conserved tripeptide motif GGQ of the two release factors in E. coli (8, 17). Methylation of the release factors ensures efficient translation termination and release of newly synthesized peptide from the ribosome (16). Similarly, the yeast HemK homologue, YDR140w (Mtq2p), was confirmed to methylate the eukaryotic release factor eRF1 on a corresponding glutamine residue (9, 22). More recently, the human homologue N6amt1 (HemK2) was reported to methylate release factor 1 (eRF1) in vitro (5).We initially sought to characterize the function of N6amt1 as a potential DNA adenine MTase. Interestingly, the human N6amt1 gene is located on chromosome 21q21.3, a critical region for Down syndrome (1, 20). In this study, we report the identification of murine N6amt1 as a glutamine-specific MTase of eRF1 both in vitro and in vivo. Mammalian eRF1, the only mammalian release factor, is indeed methylated at the glutamine residue of the GGQ motif. Inactivation of the N6amt1 gene by targeted disruption led to embryonic lethality in the mouse. These data confirm that N6amt1 functions as a protein MTase in mammals and indicate that modulation of the eRF1 activity by N6amt1-mediated glutamine methylation might be essential for embryo viability.