Abstract:tRNAs harbor the most diverse posttranscriptional modifications. The 3-methylcytidine (m3C) is widely distributed at position C32 (m3C32) of eukaryotic tRNAThr and tRNASer species. m3C32 is decorated by the single methyltransferase Trm140 in budding yeasts; however, two (Trm140 and Trm141 in fission yeasts) or three enzymes (METTL2A, METTL2B and METTL6 in mammals) are involved in its biogenesis. The rationale for the existence of multiple m3C32 methyltransferases and their substrate discrimination mechanism is… Show more
“…The two protein molecules bind distinct regions of tRNA Ser (the long variable arm and anticodon loop, respectively), but they do not display steric hindrances. Additionally, both studies 15 , 26 have suggested a tRNA-dependent interaction between hSerRS and hMETTL6; thus, these possible interactions might be insufficient for a stable complex in the absence of tRNA, which awaits further experimental verification. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The assays followed the same procedure as described 26 . The reaction mixes were incubated for 5 min before the reactions were stopped.…”
Section: Methodsmentioning
confidence: 99%
“…These studies demonstrated that METTL6 plays critical role in tumorigenesis and the development, invasion as well as susceptibility to drugs. Recently, we reconstituted the METTL2A and METTL6 methylation systems in vitro and demonstrated that both enzymes need particular prerequisites for the m 3 C32 modification 26 . Specifically, the anticodon loop and the long variable arm of tRNA Ser(GCU) are key determinants for its C32 methylation, which is also critically dependent on the presence of both hMETTL6 and hSerRS 26 .…”
Section: Introductionmentioning
confidence: 99%
“…Recently, we reconstituted the METTL2A and METTL6 methylation systems in vitro and demonstrated that both enzymes need particular prerequisites for the m 3 C32 modification 26 . Specifically, the anticodon loop and the long variable arm of tRNA Ser(GCU) are key determinants for its C32 methylation, which is also critically dependent on the presence of both hMETTL6 and hSerRS 26 . An aminoacylation-defective SerRS mutant is able to stimulate the methylation activity of hMETTL6 to a comparable level of wild-type (WT) SerRS, suggesting that SerRS contributes to the methylation through tRNA binding.…”
In tRNA, the epigenetic m3C modification at position 32 in the anticodon loop is highly conserved in eukaryotes, which maintains the folding and basepairing functions of the anticodon. However, the responsible enzymes METTL2 and METTL6 were identified only in recent years. The loss of human METTL6 (hMETTL6) affects the translational process and proteostasis in cells, while in mESCs cells, it leads to defective pluripotency potential. Despite its important functions, the catalytic mechanism of the C32 methylation by this enzyme is poorly understood. Here we present the 1.9 Å high-resolution crystal structure of hMETTL6 bound by SAH. The key residues interacting with the ligand were identified and their roles were confirmed by ITC. We generated a docking model for the hMETTL6-SAH-CMP ternary complex. Interestingly, the CMP molecule binds into a cavity in a positive patch with the base ring pointing to the inside, suggesting a flipped-base mechanism for methylation. We further generated a model for the quaternary complex with tRNASer as a component, which reasonably explained the biochemical behaviors of hMETTL6. Taken together, our crystallographic and biochemical studies provide important insight into the molecular recognition mechanism by METTL6 and may aid in the METTL-based rational drug design in the future.
“…The two protein molecules bind distinct regions of tRNA Ser (the long variable arm and anticodon loop, respectively), but they do not display steric hindrances. Additionally, both studies 15 , 26 have suggested a tRNA-dependent interaction between hSerRS and hMETTL6; thus, these possible interactions might be insufficient for a stable complex in the absence of tRNA, which awaits further experimental verification. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The assays followed the same procedure as described 26 . The reaction mixes were incubated for 5 min before the reactions were stopped.…”
Section: Methodsmentioning
confidence: 99%
“…These studies demonstrated that METTL6 plays critical role in tumorigenesis and the development, invasion as well as susceptibility to drugs. Recently, we reconstituted the METTL2A and METTL6 methylation systems in vitro and demonstrated that both enzymes need particular prerequisites for the m 3 C32 modification 26 . Specifically, the anticodon loop and the long variable arm of tRNA Ser(GCU) are key determinants for its C32 methylation, which is also critically dependent on the presence of both hMETTL6 and hSerRS 26 .…”
Section: Introductionmentioning
confidence: 99%
“…Recently, we reconstituted the METTL2A and METTL6 methylation systems in vitro and demonstrated that both enzymes need particular prerequisites for the m 3 C32 modification 26 . Specifically, the anticodon loop and the long variable arm of tRNA Ser(GCU) are key determinants for its C32 methylation, which is also critically dependent on the presence of both hMETTL6 and hSerRS 26 . An aminoacylation-defective SerRS mutant is able to stimulate the methylation activity of hMETTL6 to a comparable level of wild-type (WT) SerRS, suggesting that SerRS contributes to the methylation through tRNA binding.…”
In tRNA, the epigenetic m3C modification at position 32 in the anticodon loop is highly conserved in eukaryotes, which maintains the folding and basepairing functions of the anticodon. However, the responsible enzymes METTL2 and METTL6 were identified only in recent years. The loss of human METTL6 (hMETTL6) affects the translational process and proteostasis in cells, while in mESCs cells, it leads to defective pluripotency potential. Despite its important functions, the catalytic mechanism of the C32 methylation by this enzyme is poorly understood. Here we present the 1.9 Å high-resolution crystal structure of hMETTL6 bound by SAH. The key residues interacting with the ligand were identified and their roles were confirmed by ITC. We generated a docking model for the hMETTL6-SAH-CMP ternary complex. Interestingly, the CMP molecule binds into a cavity in a positive patch with the base ring pointing to the inside, suggesting a flipped-base mechanism for methylation. We further generated a model for the quaternary complex with tRNASer as a component, which reasonably explained the biochemical behaviors of hMETTL6. Taken together, our crystallographic and biochemical studies provide important insight into the molecular recognition mechanism by METTL6 and may aid in the METTL-based rational drug design in the future.
“…In Saccharomyces cerevisiae (S. cerevisiae ), the m 3 C methyltransferase Trm140 mediates modification of both cytoplasmic tRNA Thr/Ser whereas in Schizosaccharomyces pombe (S. pombe ) two alternative enzymes, Trm140 and Trm141, install m 3 C 32 modifications in cytoplasmic tRNAs Thr and tRNAs Ser respectively 30 , 37 . The mammalian homologues of the S. pombe proteins, METTL2 (with paralogues A and B in humans) and METTL6 are likewise responsible for installing these modifications into the human tRNAs Thr and tRNA Ser 38 – 41 . The m 3 C 32 modification is also present in mammalian tRNA Arg(CCU) and tRNA Arg(UCU) , and METTL2A/B, together with the cofactor protein DALRD3, has recently been identified as the methyltransferase responsible for introducing these modifications 40 .…”
Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs.
BackgroundN6‐methyladenosine (m6A) modification is a widespread and reversible epigenetic alteration in eukaryotic mRNA, playing a pivotal role in various biological functions. Its significance in female reproductive development and associated diseases has recently become a focal point of research.ObjectiveThis review aims to consolidate current knowledge of the role of m6A modification in female reproductive tissues, emphasizing its regulatory dynamics, functional significance, and implications in reproductive health and disease.MethodsA comprehensive analysis of recent studies focusing on m6A modification in ovarian development, oocyte maturation, embryo development, and the pathogenesis of reproductive diseases.Resultsm6A modification exhibits dynamic regulation in female reproductive tissues, influencing key developmental stages and processes. It plays critical roles in ovarian development, oocyte maturation, and embryo development, underpinning essential aspects of reproductive health. m6A modification is intricately involved in the pathogenesis of several reproductive diseases, including polycystic ovary syndrome (PCOS), premature ovarian failure (POF), and endometriosis, offering insights into potential molecular mechanisms and therapeutic targets.ConclusionThe review highlights the crucial role of m6A modification in female reproductive development and related diseases. It underscores the need for further research to explore innovative diagnostic and therapeutic strategies for reproductive disorders, leveraging the insights gained from understanding m6A modification's impact on reproductive health.
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