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

Abstract

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 N 6 -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.

Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates

“…Here, we identify several novel protein substrates of HEMK2 in vitro and in vivo, and our data expand the range of proteins potentially subjected to glutamine methylation significantly. The strong phenotype of HEMK2 deletion in mice (17) and the important biological role of the novel HEMK2 substrates described here may suggest that the biological function of HEMK2 is not restricted to its role in translational termination mediated by eRF1. Hence, the search for the biological role of HEMK2 must be continued beyond eRF1 methylation.…”

“…Cloning of Proteins-A bacterial expression pRSF-Duet1 vector that encodes the His-tagged murine HEMK2 (N6AMT1) and untagged TRM112, a pRSET vector encoding human eRF1, and mammalian expression constructs of HA-tagged HEMK2 and Myc-tagged TRM112 were kindly provided by Dr. G. L. Xu (17). The coding sequences of other putative substrate protein domains (see Table 2) were amplified from cDNA prepared from HEK293 cells and cloned into the pGEX-6P-2 vector as GST fusion proteins.…”

“…This led to the suggestion that HemK was itself an AdoMet-dependent DNA MTase (15), and its classification in data bases as "probable MTase modifying N6-adenine or N4-cytidine in DNA" including the renaming of many entries to N6AMT. However, no evidence could be found that HemK is able to methylate DNA (16,17). Later it was found that E. coli HemK methylates a glutamine residue in the ribosomal release factors (RF1 and RF2) at the universally conserved tripeptide GGQ motif (18,19), indicating that the alignment based assignment of HemK as an AdoMet dependent MTase was correct although the predicted substrate was wrong.…”

“…In addition to that, Trm112 binding masks a hydrophobic region of Mtq2, and this prevents Mtq2 homodimer formation and enhances the expression of the enzyme in soluble form (27). In mice, HEMK2 knock-out heavily impaired postimplantation development of mutant embryos, and it led to early embryonic lethality indicating that HEMK2 is an essential protein in mammals (17). However, the mechanistic basis of the interaction of HEMK2 with its eRF1 substrate is not fully understood.…”

“…The HemK enzyme has two homologs in many eukaryotes, HEMK1 in mitochondria and HEMK2 in the cytosol. The human HEMK2, also called N6AMT1, and its yeast homolog called YDR140w or Mtq2p were shown to methylate the glutamine residue in the GGQ motif of the eukaryotic release factor eRF1 (17,20,21). The human HEMK1 (HMPrmc) and its yeast homolog Mtq1p methylate the corresponding site in the mitochondrial release factor and regulate the mitochondrial translational activity (22).…”