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, 10 (2), 93-5

A METTL3-METTL14 Complex Mediates Mammalian Nuclear RNA N6-adenosine Methylation

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A METTL3-METTL14 Complex Mediates Mammalian Nuclear RNA N6-adenosine Methylation

Jianzhao Liu et al. Nat Chem Biol.

Abstract

N(6)-methyladenosine (m(6)A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m(6)A RNA methylation. Together with METTL3, the only previously known m(6)A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-METTL14 that functions in cellular m(6)A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. METTL3, METTL14, and WTAP affect the cellular m6A level in polyadenylated RNA with METTL3 and METTL14 forming a stable complex
(a) LC-MS/MS quantification of the m6A/A ratio in polyadenylated RNA isolated from HeLa and 293FT with the control and single knockdown of METTL3, METTL14, or WTAP. Both groups of data were assessed using student’s t-test with P value < 1e-6 (calculated between control and specific knockdown sample). Error bars indicate mean ± s.d. (n = 10 for HeLa, five biological replicates × two technical replicates, and n = 8 for 293FT, four biological replicates × two technical replicates). (b) Gel filtration traces of individual Flag-tagged METTL3, METTL14, and WTAP, co-expressed Flag-METTL14/His6-METTL3 as well as mixed Flag-METTL14/Flag-METTL3/Flag-WTAP with equal molar amount. All proteins were expressed in insect cells and purified by Flag-IP. Markers: 669 kDa (thyroglobulin, bovine), 200 kDa (β-amylase from sweet potato), and 66 kDa (bovine serum albumin). (c) Coomassie staining of two-dimensional native/SDS PAGE of the Flag-IP product from insect cells co-expressing Flag-METTL14/His6-METTL3. The band of ~219 kDa corresponds to the METTL3-14 heterodimer, while the band of ~504 kDa represents dimer of dimer. Full images of gels are presented in Supplementary Fig. 15.
Figure 2
Figure 2. In vitro methylation activity of METTL3, METTL14, and WTAP
The in vitro RNA N6-adenosine methylation activities of Flag-tagged METTL3, METTL14, or WTAP as well as the combination of METTL3 and METTL14 were tested using different RNA probes (numbered 1–6) with/without the consensus sequence of GGACU in the stem and/or loop, and with/without a stem-loop secondary structure in the presence of isotope-labeled cofactor d3-SAM (S-(5′-adenosyl)-L-methionine-d3). The methylation yields were calculated based on the molar ratio of d3-m6A to specific probe, measured by LC-MS/MS. Error bars indicate mean ± s.d., n = 4 (two biological replicates × two technical replicates).
Figure 3
Figure 3. RNA N6-adenosine methylation by the METTL3-14 complex isolated from HeLa cell nuclear extract and identification of their RNA-binding sites
(a) Left panel: Methylation activity tests of HeLa cell nuclear extracts, which were separated step-by-step according to the scheme shown in Supplementary Fig. 6a. Right panel: Methylation activity of gel filtration products of NE-A2. The corresponding western blotting of each fraction was shown at the bottom of each panel. Error bars indicate mean ± s.d., n = 2 (two biological replicates). Full images of gels are presented in Supplementary Fig. 16. (b) Consensus motifs identified within 4SU-PAR-CLIP binding sites of METTL3 (P = 1e-93), METTL14 (P = 1e-47), and METTL3/METTL14 overlay (P = 1e-79). (c) A schematic illustration for the reversible methylation of N6-adenosine in RNA. METTL3 and METTL14 form a heterodimeric methyltransferase complex within the cell nuclei that performs the RNA methylation function. WTAP interacts with the METTL3-14 complex to affect the m6A deposition. The demethylase removes the m6A mark, demonstrating a reversible process.

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