N6A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N6-methylated adenosine reader domain and report its solution structure in complex with a N6-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m6A recognition. These findings establish a molecular function for YTH domains as m6A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m6A recognition.

The YTH (YT521-B homology) domain was identified by sequence comparison and is found in 174 different proteins expressed in eukaryotes. It is characterized by 14 invariant residues within an ␣-helix/␤-sheet structure. Here we show that the YTH domain is a novel RNA binding domain that binds to a short, degenerated, single-stranded RNA sequence motif. The presence of the binding motif in alternative exons is necessary for YT521-B to directly influence splice site selection in vivo. Array analyses demonstrate that YT521-B predominantly regulates vertebrate-specific exons. An NMR titration experiment identified the binding surface for single-stranded RNA on the YTH domain. Structural analyses indicate that the YTH domain is related to the pseudouridine synthase and archaeosine transglycosylase (PUA) domain. Our data show that the YTH domain conveys RNA binding ability to a new class of proteins that are found in all eukaryotic organisms.The binding of proteins to RNA is a fundamental aspect of biology that interferes with most aspects of gene expression and cellular functions. The presence of various binding motifs defines the group of RNA binding proteins (1). Commonly found RNA binding domains include the RNA recognition motif (RRM), 3 the double-stranded RNA binding domain, the Piwi Argonaut and Zwille domain, and the heterogeneous nuclear ribonucleoprotein K homology domain. The most prominent RNA binding domain is the RRM that is found in ϳ2% of human proteins (2). The RRM is composed of two consensus sequences RNP2 and RNP1 that contain aromatic residues important for RNA binding. In other RNA binding motifs, such as the PUA (pseudouridine synthase and archaeosine transglycosylase) and OB-fold (oligonucleotide/oligosaccaride binding fold), the RNA interacts with the ␤-sheets that form pseudobarrels (3). The general composition of the PUA domain is reminiscent of the OB-fold, a nucleic acid binding motif that displays only a low degree of sequence similarity between its members. The OB-fold consists of two three-stranded antiparallel ␤-sheets, where strand 1 is shared by both sheets. The individual ␤-sheets can be separated by protein parts of different length, which makes the identification based on primary structure difficult (4). The ␤-sheets in the PUA and OB-folds form a ligand binding surface that can bind to nucleic acids through aromatic stacking, hydrogen bonding, as well as polar and hydrophobic interactions. The so-far unexplained RNA binding activities of proteins such as apontic (5) demonstrate that not all RNA binding domains have been described.One of the potentially new RNA binding domains is the YTH (YT521 homology) domain. The YTH domain is highly conserved during evolution and was identified by comparing all known protein sequences with the splicing factor YT521-B (6). The domain is found only in eukaryotes and is abundant in plants. The YTH domain can be between 100 and 150 amino acids in size and is characterized by 14 invariant and 19 highly conserved residues. It is predicted to contain ...

Partitioning of chromatids during mitosis requires that chromosome compaction and spindle length scale appropriately with each other. However, it is not clear whether chromosome condensation and spindle elongation are linked. Here, we find that yeast cells could cope with a 45% increase in the length of their longest chromosome arm by increasing its condensation. The spindle midzone, aurora/Ipl1 activity, and Ser10 of histone H3 mediated this response. Thus, the anaphase spindle may function as a ruler to adapt the condensation of chromatids, promoting their segregation regardless of chromosome or spindle length.

The YTH domain of YTHDC1 belongs to a class of protein "readers", recognizing the N6methyladenosine (m 6 A) chemical modification in mRNA. Static ensemble-averaged structures revealed details of N6-methyl recognition via a conserved aromatic cage. Here, we performed molecular dynamics (MD) simulations along with nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) to examine how dynamics and solvent interactions contribute to the m 6 A recognition and negative selectivity towards unmethylated substrate. The structured water molecules surrounding the bound RNA and the methylated substrate's ability to exclude bulk water molecules contribute to the YTH domain's preference for m 6 A. Intrusions of bulk water deep into the binding pocket disrupt binding of unmethylated adenosine. The YTHDC1's preference for the 5′-Gm 6 A-3′ motif is partially facilitated by a network of water-mediated interactions between the 2-amino group of the guanosine and residues in the m 6 A binding pocket. The 5′-Im 6 A-3′ (where I is inosine) motif can be recognized too but disruption of the water network lowers affinity. The D479A mutant also disrupts the water network and destabilizes m 6 A binding. Our interdisciplinary study of YTHDC1 protein/RNA complex reveals an unusual physical mechanism by which solvent interactions contributes towards m 6 A recognition.