Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1

Abstract

The A-repeat region of the lncRNA Xist is critical for X inactivation and harbors several N6-methyladenosine (m6A) modifications. How the m6A modification affects the conformation of the conserved AUCG tetraloop hairpin of the A-repeats and how it can be recognized by the YTHDC1 reader protein is unknown. Here, we report the NMR solution structure of the (m6A)UCG hairpin, which reveals that the m6A base extends 5' stacking of the A-form helical stem, resembling the unmethylated AUCG tetraloop. A crystal structure of YTHDC1 bound to the (m6A)UCG tetraloop shows that the (m6A)UC nucleotides are recognized by the YTH domain of YTHDC1 in a single-stranded conformation. The m6A base inserts into the aromatic cage and the U and C bases interact with a flanking charged surface region, resembling the recognition of single-stranded m6A RNA ligands. Notably, NMR and fluorescence quenching experiments show that the binding requires local unfolding of the upper stem region of the (m6A)UCG hairpin. Our data show that m6A can be readily accommodated in hairpin loop regions, but recognition by YTH readers requires local unfolding of flanking stem regions. This suggests how m6A modifications may regulate lncRNA function by modulating RNA structure.

Graphical Abstract

Recognition of a m6A modification in an Xist A-repeat RNA hairpin loop leads to local unfolding. This may modulate tertiary RNA contacts or interactions with RNA-binding proteins.

Figure 1.

Structure of the (m⁶A)UCG tetraloop in Xist A-repeats. (A) Schematic overview of the reader (RBM15) and writer proteins (METTL3 and WTAP) that facilitate m⁶A modification of adenosines located within the tetraloop of the Xist AUCG tetraloop hairpin. YTHDC1 recognizes and binds m⁶A-modified Xist RNA. (B) Overview of the non-methylated AUCG tetraloop structure (PDB ID: 2Y95). (C) Schematic of base orientation and sugar puckering of the (m⁶A)UCG tetraloop. (D) NOEs between the m⁶A H2 and methyl (H9*) protons, and inter-nucleotide NOEs between m⁶A H2 and methyl (H9*) protons to ribose protons of C8 and G9, as observed in the ¹H–¹H NOESY spectra at 150 ms mixing time. (E) Zoomed view of the (m⁶A)UCG tetraloop nucleotides in the lowest energy structure of the NMR ensemble of the (m⁶A)UCG tetraloop hairpin. The methyl group of m⁶A is marked with a blue asterisk. (F) Zoomed view of the (m⁶A)UCG tetraloop residues highlighting inter-residue NOEs between the m⁶A H2 and methyl (H9*) protons.

Figure 2.

Binding of m⁶A modified RNAs by the YTH domain. Isothermal titration calorimetry experiments for binding of the YTH domain (A) to 5′-CC(m⁶A)UCG-3′ RNA, (B) to the m⁶A-modified Xist A-repeat tetraloop hairpin and (C) to a variant of the m⁶A tetraloop with a destabilized closing base pair (CG to UG). (D, E) Zoomed-in regions of ¹H–¹⁵N HSQC spectra overlays of the YTH domain free (black), (D) in complex with m⁶A-modified 5′-CC(m⁶A)UCG-3' hexameric oligo (red: 1:1), and (E) when bound to the m⁶A modified Xist A-repeat tetraloop hairpin (blue: 1:1).

Figure 3.

Structure of YTH/(m⁶A)UCG tetraloop complex. (A) Crystal structure of YTH bound to the (m⁶A)UCG tetraloop of a Xist A-repeat. (B) The aromatic cage that recognizes the m⁶A is formed by W428, W377 and L439 side chains. (C) Hydrogen bonds (dashed lines) formed between S378, N367 and N363 mediate specific contacts to the m⁶A base. (D) A positively charged surface area interacts with the backbone of (m⁶A)UCG tetraloop and (E) mediates electrostatic interactions with the RNA (dashed lines). (F) π/cation stacking interaction between R475 and the U7 base.

Figure 4.

YTH domain binding requires opening of the closing base pair of the (m⁶A)UCG tetraloop stem. (A) ¹H 1D and ¹H–¹H NOESY spectra showing broadening imino proton resonances upon addition of YTH domain protein (black: apo RNA, blue: 0.25:1, green: 0.5:1 and red: 1:1). The G10 imino proton resonance (as indicated by the dashed line) disappears even in the presence of 0.25 molar ratio of YTH protein. (B) Schematic representation of the (m⁶A)UCG tetraloop hairpin with a fluorophore conjugated to the 5′ end and a quencher to the 3′ end. When the two probes are separated, there is emission of fluorescence intensity. (C) Bar plot showing the effect the YTH domain has on unwinding of the lower stem of the (m⁶A)UCG tetraloop hairpin at increasing concentration of protein. The RNA concentration was held constant at 400 nM. (D) Model illustrating the effects of m⁶A modification and YTHDC1 binding on the upper region of the Xist (m⁶A)UCG stem–loop. The conformational changes induced in the RNA upon YTH binding might modulate interactions with RNA-binding proteins or tertiary contacts involving the hairpin RNA.