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.

Molecular Simulations Matching Denaturation Experiments for N6-Methyladenosine

“…Post-transcriptionally modified nucleotides are crucial for RNA function. , Methylation of adenine in the N6 position (m 6 A) is the most prevalent chemical modification in mRNAs and has been observed in both coding and noncoding RNAs. − m 6 A can finely regulate the interaction of RNA with specific proteins known as m 6 A readers. In addition, similarly to other chemical modifications, it can directly affect RNA stability and structural dynamics (see, e.g., refs − ). Specifically, m 6 A has been suggested to weaken Watson–Crick pairings due to the incompatibility of its most stable conformation ( syn ) with duplex formation − (see also Figure ).…”

METTL3 drives telomere targeting of TERRA lncRNA through m6A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma

“…Xist is associated with adaptor protein RBM15 which recruits the METTL3/METTL14 complex to deposit m 6 A on Xist RNA. The reader protein YTHDC1 has been shown to recognize m 6 A in the UCG tetra-loop of Repeat A in the Xist RNA and to be required for Xist mediated gene silencing (3,15). The presence of m 6 A modification is also detected in non-canonical GGAAU motif in HSATIII lncRNA and has been shown to be critical for the sequestration of splicing factors in nuclear stress bodies (16).…”

Relevance of RNA N6-Methyladenosine Regulators for Pulmonary Fibrosis: Implications for Chronic Hypersensitivity Pneumonitis and Idiopathic Pulmonary Fibrosis

“…METTL14, METTL16, WTAP, VIRMA, ZC3H13, CBLL1, RBM15, and RBM15B are also included in the complex of m6A “writers” ( Meyer and Jaffrey, 2017 ; Wen et al, 2018 ; Gu et al, 2022 ; Shen et al, 2022 ; Su et al, 2022 ). Accordingly, YT521-B homology (YTH) domain family is the main “readers” ( Jones et al, 2022 ) and “erasers” consisting of FTO and ALKBH5 ( Kaur et al, 2022 ). Recent studies have focused on the importance of m6A modification in tumors ( Jiang et al, 2022 ; Ma et al, 2022 ; Xiong et al, 2022 ; Yadav et al, 2022 ) and non-neoplastic diseases like asthma and pulmonary hypertension ( Teng et al, 2021 ; Zeng et al, 2021 ; Zhou et al, 2021 ).…”