Adenine Methylation Enhances the Conformational Flexibility of an RNA Hairpin Tetraloop

Abstract

The N⁶-methyladenosine modification is one of the most abundant post-transcriptional modifications in ribonucleic acid (RNA) molecules. Using molecular dynamics simulations and alchemical free-energy calculations, we studied the structural and energetic implications of incorporating this modification in an adenine mononucleotide and an RNA hairpin structure. At the mononucleotide level, we found that the syn configuration is more favorable than the anti configuration by 2.05 ± 0.15 kcal/mol. The unfavorable effect of methylation was due to the steric overlap between the methyl group and a nitrogen atom in the purine ring. We then probed the effect of methylation in an RNA hairpin structure containing an AUCG tetraloop, which is recognized by a "reader" protein (YTHDC1) to promote transcriptional silencing of long noncoding RNAs. While methylation had no significant conformational effect on the hairpin stem, the methylated tetraloop showed enhanced conformational flexibility compared to the unmethylated tetraloop. The increased flexibility was associated with the outward flipping of two bases (A6 and U7) which formed stacking interactions with each other and with the C8 and G9 bases in the tetraloop, leading to a conformation similar to that in the RNA/reader protein complex. Therefore, methylation-induced conformational flexibility likely facilitates RNA recognition by the reader protein.

Figure 1

Two configurations of the methylated adenine (m6A) mononucleotide. (A) Structures of m⁶A in syn and anti configurations, (B) distances between the N7 atom (center of mass) and the methyl group (center of mass) during the alchemical transformation of the syn or anti configuration methylated adenine into an unmethylated adenine nucleotide, and (C) snapshots from an alchemical transformation simulation of anti m⁶A highlighting the steric overlap of the methyl group and the N7 atom (red arrow). In all panels, the methyl group in the anti configuration is highlighted in dark green color. See also Figures S4, S5.

Figure 2

Secondary and tertiary structures of RNA hairpin in methylated and unmethylated forms. (A) Secondary structure, (B) methylated NMR structure (PDB code: 7POF) with the methyl group highlighted in red color, and (C) unmethylated NMR structure (PDB code: 2Y95). Key nucleotides are uniquely colored and labeled.

Figure 3

RMSD and occupancy analyses. (A) Histograms (with error bars) of mean RMSD values computed with respect to the initial structure based on MD simulations of the unmethylated (lighter shades) and methylated (darker shades) hairpin including the entire tetraloop motif (labeled TL), the individual tetraloop nucleotides (labeled A6/A6*, U7, C8, G9), and the entire stem motif (labeled stem). The experimental structures of the RNA tetraloop in (B) methylated and (C) unmethylated states, highlighting initial distances between residues involved in stacking and hydrogen-bonding interactions for which the occupancy values (shown as heat maps in panels B and C) are computed based on 5 independent MD simulations of each hairpin state. Nucleotides are uniquely colored and labeled.

Figure 4

Base flipping in the RNA hairpin tetraloop. (A) Traces of the base flipping angle (φ) from a representative MD simulation for each of the unmethylated (light green) and methylated A6 (dark green) states and (B) snapshots showing conformational rearrangements in methylated A6 and other neighboring tetraloop nucleotides, corresponding to various values of φ labeled with ① through ⑤ in panel A. The gray rectangle in panel A marks the angle range (0° ≤ φ ≤ 60°) for the inward flipped state. Nucleotides are uniquely colored and labeled. See also Figures S7, S8.

Figure 5

(A) RMSD distribution for the tetraloop nucleotides (A6*, U7, C8, and G9), computed based on data from an MD simulation of the methylated hairpin, relative to the protein-bound conformation of the tetraloop observed in the crystal structure (PDB code: 7PO6). (B) Structural comparison of tetraloop conformations in the crystal structure (left) and from an MD simulation (right). See also Figures S11, S12.