A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation

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.

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. 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. 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.