Recognition of G-quadruplex RNA by a crucial RNA methyltransferase component, METTL14

Abstract

N6-methyladenosine (m6A) is an important epitranscriptomic chemical modification that is mainly catalyzed by the METTL3/METTL14 RNA methyltransferase heterodimer. Although m6A is found at the consensus sequence of 5'-DRACH-3' in various transcripts, the mechanism by which METTL3/METTL14 determines its target is unclear. This study aimed to clarify the RNA binding property of METTL3/METTL14. We found that the methyltransferase heterodimer itself has a binding preference for RNA G-quadruplex (rG4) structures, which are non-canonical four-stranded structures formed by G-rich sequences, via the METTL14 RGG repeats. Additionally, the methyltransferase heterodimer selectively methylated adenosines close to the rG4 sequences. These results suggest a possible process for direct recruitment of METTL3/METTL14 to specific methylation sites, especially near the G4-forming regions. This study is the first to report the RNA binding preference of the m6A writer complex for the rG4 structure and provides insights into the role of rG4 in epitranscriptomic regulation.

Graphical Abstract

The RNA methyltransferase METTL3/METTL14 heterodimer was shown to preferentially bind to RNA G-quadruplex (rG4) structures and methylate adenosines close to rG4, providing insights into the role of rG4 in epitranscriptomic regulation.

Figure 1.

The RNA methyltransferase construct and RNAs used in this study. (A) The schematic representation of G4 RNA binding by the m6A writer complex. (B) The schematic representation of MTD3/MTD14-RGG. (C) The oligonucleotide sequences of G4 RNA1 and Non-G4 RNA1 are shown. G-quadruplex-forming guanines are shown in bold. Methylation consensus sequences are underlined. (D) The circular dichroism spectra of G4 RNA1 (left) and Non-G4 RNA1 (right) in 10 mM potassium phosphate buffer (pH 7.5), 10 mM potassium phosphate buffer (pH 7.5) with 100 mM KCl, 10 mM lithium phosphate buffer (pH 7.5) or 50 mM Tris-HCl (pH 7.5) (No-salt) buffer.

Figure 2.

Specific interactions between the G-quadruplex structure of G4 RNA1 and MTD3/MTD14-RGG. (A and B) Representative results of the electrophoretic mobility shift assay (EMSA) of MTD3/MTD14-RGG and G4 RNA1 (upper), and Non-G4 RNA1 (lower) in 10 mM potassium phosphate (K⁺) buffer (A) or 10 mM lithium phosphate (Li⁺) buffer (B) in the presence of yeast tRNA. The concentrations of MTD3/MTD14-RGG are 0, 3, 8, 19, 48, 120 and 300 nM. (C) Analysis of EMSA data. Fractions of protein-bound RNA are plotted against MTD3/MTD14-RGG concentration.

Figure 3.

Formation of rG4 structure in free and MTD3/MTD14-RGG (MTase)-bound G4 RNA1. (A) CD spectra of 2 μM G4 RNA1 in 10 mM KP buffer ([a]; solid line) and 10 mM LiP buffer (long dashed line), 2 μM G4 RNA1 and 1 μM MTD3/MTD14-RGG mixture ([b]; dashed line), 2 μM G4 RNA1 and 2 μM MTD3/MTD14-RGG mixture ([c]; dash-dotted line), 1 μM MTD3/MTD14-RGG only ([d]; dotted line), and 2 μM MTD3/MTD14-RGG only ([e]; dotted line) in 10 mM KP buffer. (B) Difference in the CD spectra between [b] and [d] (dashed line) and between [c] and [e] (dotted line), in which CD spectra of protein only (1 or 2 μM; [d] or [e]) were subtracted from mixture of protein (1 or 2 µM) and G4 RNA1 ([b] or [c]). For a reference, the CD spectrum of 2 μM G4 RNA1 ([a]; solid line) was shown in both figures.

Figure 4.

Preferential methylation of G4-forming oligonucleotide RNA by MTD3/MTD14-RGG. The methylation rates of G4 RNA1–4, and Non-G4 RNA3 (5′- UGAGUGUUAGGGACAUGAGUGUGAGUG-3′) by 100 nM MTD3/MTD14-RGG for 60 min in non-competitive conditions are shown on the left side (border color). The methylateion percentages of G4 RNA1–4 and Non-G4 RNA3 in the mixture of one of the G4 RNAs and Non-G4 RNA3 in the presence of HeLa total RNA (competitive condition) are shown on the right side; *, P< 0.01; n.s., P > 0.05.