Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Mar 21;51(5):2434-2446.
doi: 10.1093/nar/gkad081.

Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis

Affiliations
Free PMC article

Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis

Jue Ju et al. Nucleic Acids Res. .
Free PMC article

Abstract

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

Figures

Figure 1.
Figure 1.
Overall structure of C. elegans METT10-MTD. (A) Schematic diagrams of full-length C. elegans METT10 (CeMETT10-FL), its variant CeMETT10-FLΔL lacking the putative disordered region (residues 386–430), and its N-terminal methyltransferase domain (CeMETT10-MTD: residues 1–314). The MTD is colored cyan, and the C-terminal kinase-associated domains, KA-1a and KA-1b, are magenta and green, respectively. The region between KA-1a and KA-1b is predicted to be disordered. The MTD consists of the N-terminal domain (N) and Rossmann fold (RF). (B) Overall structure of CeMETT10-MTD. Residues 6–187 and 236–309 are modeled in the structure. The extended N-terminal domain and the Rossmann fold are colored magenta and cyan, respectively, and the RNA binding loop (dashed line) between β4 and α4 was disordered and not modeled in the structure. (C) Schematic view of the secondary structure of CeMETT10-MTD. The N-terminal domain and the Rossmann fold are colored as in (B). (D) Stereo view of the structural alignment of Cα atoms between CeMETT10-MTD (cyan) and human METTL16-MTD (hMETTL16-MTD) in complex with SAH (orange, PDB: 6B92) (34).
Figure 2.
Figure 2.
Key residues of CeMETT10- MTD for RNA methylation. (A) The structure of human METTL16-MTD (hMETTL16-MTD) in complex with hMAT2A- hp1 RNA (PDB: 6DU4) (23). hMAT2A-hp1 is shown in green. (B) RNA docking onto the CeMETT10-MTD structure. The structure of hMETTL16-MTD complexed with hMAT2A-hp1 in (A) was superimposed on the structure of CeMETT10-MTD. hMETTL16-MTD was omitted for clarity. hMAT2A-hp1 is colored green. (C) Methylation of sams-hp RNA (see Figure 3B) by METT10-MTD and its variants. The sams-hp RNA (1 μM) was incubated with 0.4 μM METT10-MTD in the presence of 1 mM SAM, at 37°C for 4 min. The methylation of sams-hp RNA by wild-type METT10-MTD was taken as 1.0. The bars in the graphs are the SD of three independent experiments. (D) Model of the hMETTL16 active site with SAH and hMAT2A-hp1. The SAH was modeled into the structure of hMETTL16-MTD in complex with hMAT2A-hp1 by referring to the structure of hMETTL16-MTD in complex with SAH (PDB: 6B92) (34). The methylation site in the RNA (m6A site) is shown in green, and SAH is colored magenta. (E) Superimposition of the active site structures of CeMETT10 (cyan) and hMETTL16 (orange). (F) Methylation of sams-hp RNA by METT10-MTD and its variants, as in (C). The methylation of sams-hp RNA by wild-type METT10-MTD was taken as 1.0. The bars in the graphs are the SD of three independent experiments. (G) Superimposition of the K-loops of CeMETT10-MTD (cyan) and hMETTL16-MTD (orange) in complex with hMAT2A-hp1. SAH was modeled as in (D). (H) Methylation reactions of sams-hp-ls RNA by METT10-MTD and its variants with various concentrations of SAM (0.05, 0.1, 0.2, 0.5, 1.0 and 1.5 mM). The sams-hp RNA (20 μM) was incubated with 0.4 μM METT10-FLΔL or its variants in the presence of various concentrations of SAM, at 37°C for 4 min. The bars in the graphs are the SD of three independent experiments.
Figure 3.
Figure 3.
RNA recognition by C. elegans METT10. (A) Alignments of the nucleotide sequences around the junction between intron 2 and exon 3 of C. elegans sams (-3, -4 and -5) pre-mRNAs and the 3'-UTR hairpin regions of human MAT2A (MAT2A-hp1–6) pre-mRNAs (31). (B) Secondary structures of 3'-UTR hairpin RNA (MAT2A-hp1) of human MAT2A pre-mRNA (left) and C. elegans sams hairpin RNA spanning intron 2 and exon 3, including the 3'-splice site (AG-dinucleotides) (right). The sams RNA also adopts a hairpin structure (sams-hp), as in human MAT2A-hp1. The adenine residues targeted for methylation are circled. The hairpin comprises the loop, transition, and stem regions (23). The loop region is composed of the recognition motif (UACAG: red) and linker (green). (C) Secondary structures of C. elegans sams-hp RNA and its variants used for the methylation assays in (D). (D) Methylation of sams-hp RNA and its variants by METT10-FLΔL. The sams-hp RNA (1 μM) and its variants were each incubated with 0.4 μM METT10-FLΔL in the presence of 1 mM SAM, at 37°C for 4 min. The methylation of wild-type sams-hp RNA by METT10-FLΔL was taken as 1.0. The bars in the graph are the SD of three independent experiments. (E) G8–A22 base pairing in the transition region near the stem of hMAT2A-hp (left) and the possible U9–C22 base pairing at the corresponding position in the sams-hp (right). Possible hydrogen bonds between the sugar edge of U9 and the 4-NH2 group of C22 are depicted by dashed lines.
Figure 4.
Figure 4.
Predicted structure of the C-terminal region of CeMETT10. (A) Structure of the C-terminal KA-1 domain of human METTL16 (PDB: 6M1U) (35). The disordered loop (dashed line) is inserted between KA-1a (magenta) and KA-1b (green). (B) Model structures of the C-terminal regions of CeMETT10 and hMETTL16 homologs from invertebrates [Caenorhabditis elegans (MET16_CAEEL), Octopus vulgaris (A0A6P7TIF1_OCTVU), Tetranychus urticae (T1JVZ0_TETUR), Actinia tenebrosa (A0A6P8ITS0_ACTTE), and Amphimedon queenslandica (A0A1 × 7U5N0_AMPQE)], and a fission yeast [Schizosaccharomyces pombe (MTL16_SCHPO)] generated by Alphafold2 (44,45). The disordered region between KA-1a (magenta) and KA-1b (green), predicted by Alphafold2 in each structure, is depicted by dashed lines (Supplementary Figures 2, 6). The arginine-rich region, RRR, is colored blue. (C) Alignments of the amino acid sequences of the C-terminal regions of hMETTL16 and CeMETT10. The secondary structural elements (α-helices and β-strands) for hMETTL16 and CeMETT10 are shown in parallel above and below the alignment, respectively.
Figure 5.
Figure 5.
The C-terminal KA-1 of CeMETT10 facilitates m6A modification of RNAs. (A) Alignment of amino acid sequences around the arginine-rich region (RRR) in the C-terminal KA-1 domains from various organisms (Supplementary Figures 2 and 3), including invertebrates (C. elegans, O. vulgaris, T. urticae, A. queenslandica and A. tenebosa), a fission yeast (S. pombe), and vertebrates (H. sapiens, G. gallus, C. picta, X. laevis and D. rerio). (B) Schematic diagram of CeMETT10-FL and its variants used in the methylation assays in (D) and (E). MTD, KA-1a and KA-1b are colored cyan, magenta, and green, respectively. RRR (arginine-rich region: RARKRAK) is colored blue, and its mutant is red (RRR/7E). (C) Secondary structures of sams-hp-ls and U6 snRNA. (D, E) Steady-state kinetics of the methylation of sams-hp-ls (D) and U6 snRNA (E) by METT10-FLΔL, -MTD, and -FLΔL_RRR/7E. Various concentrations of RNA [0–8 μM for sams-hp-ls (D) and 1–4 μM for U6 snRNA (E)] were incubated with 0.4 μM METT10- FLΔL, -MTD or -FLΔL _RRR/7E in the presence of 1 mM SAM, at 37°C for 4 min, and the initial reaction velocities were calculated. The bars in the graph are the SD of three independent experiments. (F) Gel-shifts of sams-hp-ls by wild-type METT10-FLΔL (0–1.0 μM), -MTD (0–100 μM) or -FLΔL _RRR/7E (0–20 μM). The graphs on the left in (F) and (G) are magnified views of the graphs on the right at the lower protein concentration ranges. (G) Gel-shifts of U6 snRNA by wild-type METT10-FLΔL (0–1.0 μM), -MTD (0–100 μM) or -FLΔL _RRR/7E (0–20 μM). The bars in the graphs (F, G) are the SD of three independent experiments.

Similar articles

References

    1. Pan T. N6-methyl-adenosine modification in messenger and long non-coding RNA. Trends. Biochem. Sci. 2013; 38:204–209. - PMC - PubMed
    1. Dominissini D. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012; 485:201–206. - PubMed
    1. Meyer K.D., Saletore Y., Zumbo P., Elemento O., Mason C.E., Jaffrey S.R.. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 2012; 149:1635–1646. - PMC - PubMed
    1. Yue Y., Liu J., He C.. RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation. Genes Dev. 2015; 29:1343–1355. - PMC - PubMed
    1. Wang X., Lu Z., Gomez A., Hon G.C., Yue Y., Han D., Fu Y., Parisien M., Dai Q., Jia G.et al. .. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014; 505:117–120. - PMC - PubMed

Publication types