Flexible Binding of m⁶A Reader Protein YTHDC1 to Its Preferred RNA Motif

Abstract

N6-methyladenosine (m 6 A) is the most prevalent chemical modification in human mRNAs. Its recognition by reader proteins enables many cellular functions, including splicing and translation of mRNAs. However, the binding mechanisms of m 6 A-containing RNAs to their readers are still elusive due to the unclear roles of m 6 A-flanking ribonucleotides. Here, we use a model system, YTHDC1 with its RNA motif 5'-G-2G-1(m 6 A)C+1U+2-3', to investigate the binding mechanisms by atomistic simulations, X-ray crystallography, and isothermal titration calorimetry. The experimental data and simulation results show that m 6 A is captured by an aromatic cage of YTHDC1 and the 3' terminus nucleotides are stabilized by cation-π-π interactions, while the 5' terminus remains flexible. Moreover, simulations of unbound RNA motifs reveal that the methyl group of m 6 A and the 5' terminus shift the conformational preferences of the oligoribonucleotides to the bound-like conformation, thereby facilitating the association process. The proposed binding mechanisms may help in the discovery of chemical probes against m 6 A reader proteins.

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

“…Consistent with this, a recent analysis of m 6 A/YTH domain interactions suggests that residues located upstream, i.e. 5′, of the m 6 A residue do not form stable interactions with the protein and remain flexible ( 34 ). Strikingly, our NMR and fluorescence data demonstrate that the lower region of the stem remains base paired, and thus, the RNA hairpin is not completely unfolded.…”

“…Overall, the structure of the YTH/(m 6 A)UCG complex tetraloop structure resembles the recognition of m 6 A seen in other reported structures of YTH domains with single-stranded RNA ligands. A structural alignment of the YTH/(m 6 A)UCG complex and the YTH/(m 6 A)CU RNA in PDB 6RT4 ( 34 ) shows that the two YTH domain structures are nearly identical, with a root mean square deviation (RMSD) of 0.146 Å for heavy atoms ( Supplementary Figure S5C ). This indicates a conserved mode of m 6 A recognition in a single-stranded RNA context and implies that the YTH binding requires at least a local conformational change of the (m 6 A)UCG tetraloop, which avoids steric clashes of the remaining helical stem region with the YTH domain ( Supplementary Figure S5D ).…”

“…However, the fact that the m 6 A base is not readily accessible in the (m 6 A)UCG tetraloop structure raises the question of how the YTH domain can induce conformational changes in the hairpin RNA to enable recognition of the m 6 A. It has been suggested that the interaction formed between the two nucleotides immediately 3′ of m 6 A nucleotides (in our case U7 and C8) with the charged pocket may facilitate the recognition of m 6 A by the YTH domain through a two-step mechanism ( 34 , 59 ). Following a zipper-like mechanism of binding, the charged pocket on the surface of the YTH domain may first interact with the sugar-phosphate backbone of the two nucleotides 3′ of the m 6 A base, thus guiding the m 6 A base into the aromatic pocket.…”

Epigenetics: Roles and therapeutic implications of non-coding RNA modifications in human cancers

“…The major m6A reading elements cover the family of protein supplemented by the YT521-B homology (YTH) domain (e.g., the cytoplasmic YTHDF3, YTHDF2, YTHDF1, and YTHDC2 and the nuclear Homo sapiens YTH domain containing 1 [YTHDC1]). 27 , 28 The rest of the RNA-binding proteins, e.g., nuclear factor κB (NF-κB)-related protein (NKAP) and heterogeneous nuclear ribonucleoprotein (HNRNP) family (HNRNPG, HNRNPC, and HNRNPA2B1), impact the fates of RNAs and the functions exhibited by cells via the particular identification of m6A ( Figure 1 ). 29 Alarcón et al.…”

Selectively Disrupting m6A-Dependent Protein–RNA Interactions with Fragments

“…Therefore, the m 6 A modification machinery has recently attracted broad attention from the drug discovery community. , Importantly, the molecular recognition and physicochemical properties of RNA change upon its posttranscriptional covalent modifications. In the case of m 6 A modification, the base becomes more hydrophobic . The corresponding reader proteins (i.e., proteins that specifically recognize the modification to elicit cellular response to it) feature a binding pocket [Figure A] that is more hydrophobic than the interfaces that bind unmodified RNA .…”