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...