Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

Abstract

Methylation of the N6 position of selected internal adenines (m 6 A) in mRNAs and noncoding RNAs is widespread in eukaryotes, and the YTH domain in a collection of proteins recognizes this modification. We report the crystal structure of the splicing factor YT521-B homology (YTH) domain of Zygosaccharomyces rouxii MRB1 in complex with a heptaribonucleotide with an m 6 A residue in the center. The m 6 A modification is recognized by an aromatic cage, being sandwiched between a Trp and Tyr residue and with the methyl group pointed toward another Trp residue. Mutations of YTH domain residues in the RNA binding site can abolish the formation of the complex, confirming the structural observations. These residues are conserved in the human YTH proteins that also bind m 6 A RNA, suggesting a conserved mode of recognition. Overall, our structural and biochemical studies have defined the molecular basis for how the YTH domain functions as a reader of methylated adenines.T he methylation of the N6 position of selected internal adenines (m 6 A) modification is widespread in eukaryotic mRNAs and noncoding RNAs (1-3), reviewed in refs. 4-6. Recent studies have linked this modification to the regulation of alternative splicing (2), RNA processing, and mRNA degradation (7). Although the exact cellular functions of this modification are still not completely understood, m 6 A has been linked to the regulation of the circadian clock (8) and m 6 A levels are highest during yeast meiosis (1). The m 6 A methyl group can be removed by the dioxygenases FTO (9) and ALKBH5 (10, 11), suggesting that m 6 A is a reversible modification on the RNA.A consensus sequence G(m 6 A)C has been identified for this modification based on transcriptome-wide mapping (1, 2), which is consistent with that identified from earlier biochemical studies (4-6). Several YTH domain (12) family members, YTHDF1, YTHDF2, and YTHDF3 in humans (2, 7) and methylated RNAbinding protein 1 (MRB1) in yeast (1), have been shown to bind RNAs with m 6 A modification, and the binding consensus for the YTH domain of YTHDF2 is also G(m 6 A)C (7), consistent with that found by transcriptome-wide mapping.The YTH domain contains ∼160 residues and is found in yeast, plants, and animals ( Fig. S1) (12, 13). The domain is located at the C-terminal end of yeast MRB1 and human YTHDF1-3 (Fig. 1A), and its sequence is well conserved among these proteins (Fig. 1B and Fig. S1). The N-terminal regions of these proteins are poorly conserved, although that of YTHDF2 mediates its function in regulating mRNA localization and degradation (7). Yeast MRB1 regulates phosphate metabolism by destabilizing the mRNA of a transcription factor of the pathway and, hence, it is also known as Pho92 (14), although direct evidence of MRB1 regulating m 6 A-containing mRNAs in yeast cells is lacking.The structures of the YTH domains of two related proteins, human YTH domain containing protein 1 [YTHDC1; Protein Data Bank (PDB) ID code 2YUD] and YTHDC2 (2YU6), have been reported. Human YTHDC1 has 29% sequence i...

Crystal structure of the YTH domain of YTHDF2 reveals mechanism for recognition of N6-methyladenosine

“…Notably, residues W432 and W486 are highly conserved among the YTHDF family members from yeast to human ( Figure 1A), further supporting their important role in mediating specific recognition of m 6 A-RNA. When our manuscript was under revision, two crystal structures of m 6 A-RNA in complex with YTH domains from YTHDC1 [14] and Zygosaccharomyces rouxii MRB1 (ZrMRB1) [15] were reported. Structural comparison shows that residues W432 and W486 of YTH YTHDF2 adopt a similar conformation to that of m 6 A-binding residues in the two complex structures (Supplementary information, Figure S1H), suggesting a conserved mechanism for m 6 A-RNA recognition by YTH domains.…”

Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast

“…8B). YTH domain proteins have attracted great attention recently as "readers" of the N6-methyladenine (m 6 A) mark in eukaryal mRNA Luo and Tong 2014;Xu et al 2014Xu et al , 2015. m 6 A-reading YTH proteins recognize the RNA sequence 5 ′ -GG(m 6 A)C, which bears no resemblance to the fission yeast DSR sequence that attracts Mmi1.…”

“…The turnover of these lncRNAs by the exosome is aided by Mmi1 (Ard et al 2014;Shah et al 2014), a YTH domaincontaining protein that recognizes determinants of select removal (DSRs) in the target RNA (Harigaya et al 2006;Yamashita et al 2012). YTH domain proteins from other eukaryal taxa are specific "readers" of the modified nucleoside N6-methyladenosine (m 6 A) in RNA Luo and Tong 2014;Xu et al 2014Xu et al , 2015, but it is not known whether or how inscription of an m 6 A mark might affect phosphate homeostasis.…”

Structure of the YTH domain of human YTHDF2 in complex with an m6A mononucleotide reveals an aromatic cage for m6A recognition

“…As the G nucleotide at the −1 position relative to the m 6 A site adopts different conformations in the two recently reported structures [11,12], further studies are needed to illustrate the structural basis of preferential recognition of the conserved G(m 6 A)C motif by YTH-YTHDF2 [7].…”

“…The third aromatic cage residue (Trp491 in YTHDF2) is replaced by Tyr237 in PHO92, the only known YTH domain-containing protein in Saccharomyces cerevisiae (S. cerevisiae) [9], and a leucine residue in YTHDC1/2 which have been reported to bind to a degenerate unmethylated RNA sequence [10] (Figure 1E). During the preparation of this manuscript, structures of the YTH domains of human YTHDC1 [11] and ZrMRB1 (the ortholog of S. cerevisiae PHO92 in Zygosaccharomyces rouxii) [12] were reported. The three YTH domains show high structural similarity with all pairwise backbone RMSD values below 1 Å and possess a similar pocket for m 6 A binding despite the variance in the third aromatic cage residue (Supplementary information, Figure S1C and S1D).…”