Structural Basis for the Discriminative Recognition of N6-Methyladenosine RNA by the Human YT521-B Homology Domain Family of Proteins

N⁶-Methyladenosine (m⁶A) is the most abundant internal modification in RNA and is specifically recognized by YT521-B homology (YTH) domain-containing proteins. Recently we reported that YTHDC1 prefers guanosine and disfavors adenosine at the position preceding the m⁶A nucleotide in RNA and preferentially binds to the GG(m⁶A)C sequence. Now we systematically characterized the binding affinities of the YTH domains of three other human proteins and yeast YTH domain protein Pho92 and determined the crystal structures of the YTH domains of human YTHDF1 and yeast Pho92 in complex with a 5-mer m⁶A RNA, respectively. Our binding and structural data revealed that the YTH domain used a conserved aromatic cage to recognize m⁶A. Nevertheless, none of these YTH domains, except YTHDC1, display sequence selectivity at the position preceding the m⁶A modification. Structural comparison of these different YTH domains revealed that among those, only YTHDC1 harbors a distinctly selective binding pocket for the nucleotide preceding the m⁶A nucleotide.     

YTH Domain: A Family of N6-methyladenosine (m6A) Readers

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N⁶-methyladenosine (m⁶A) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, m⁶A can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the m⁶A modification is reversible and dynamic. Moreover, m⁶A is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the m⁶A recognition by YTH domain-containing proteins, which would shed new light on m⁶A-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.     

Downregulation of m6A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

Lung cancer is one of the most common types of carcinoma worldwide. Cigarette smoking is considered the leading cause of lung cancer. Aberrant expression of several YT521-B homology (YTH) family proteins has been reported to be closely associated with multiple cancer types. The present study aims to evaluate the function and regulatory mechanisms of the N6-methyladenosine (m⁶A) reader protein YTH domain containing 2 (YTHDC2) by in vitro, in vivo and bioinformatics analyses. The results revealed that YTHDC2 was reduced in lung cancer and cigarette smoke-exposed cells. Notably, bioinformatics and tissue arrays analysis demonstrated that decreased YTHDC2 was highly associated with smoking history, pathological stage, invasion depth, lymph node metastasis and poor outcomes. The in vivo and in vitro studies revealed that YTHDC2 overexpression inhibited the proliferation and migration of lung cancer cells as well as tumor growth in nude mice. Furthermore, YTHDC2 decreased expression was modulated by copy number deletion in lung cancer. Importantly, the cylindromatosis (CYLD)/NF-κB pathways were confirmed as the downstream signaling of YTHDC2, and this axis was mediated by m⁶A modification. The present results indicated that smoking-related downregulation of YTHDC2 was associated with enhanced proliferation and migration in lung cancer cells, and appeared to be regulated by DNA copy number variation. Importantly, YTHDC2 functions as a tumor suppressor through the CYLD/NF-κB signaling pathway, which is mediated by m⁶A modification.     

YTH: a new domain in nuclear proteins

A novel 100-150-residue domain has been identified in the human splicing factor YT521-B and its Drosophila and yeast homologues. Homology searches show that the domain is typical for the eukaryotes and is particularly abundant in plants. It is predicted to adopt a mixed alpha-helix-beta-sheet fold and to bind to RNA. We propose the name YTH (for YT521-B homology) for the domain.     

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

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.     

A novel RNA-binding mode of the YTH domain reveals the mechanism for recognition of determinant of selective removal by Mmi1

The YTH domain-containing protein Mmi1, together with other factors, constitutes the machinery used to selectively remove meiosis-specific mRNA during the vegetative growth of fission yeast. Mmi1 directs meiotic mRNAs to the nuclear exosome for degradation by recognizing their DSR (determinant of selective removal) motif. Here, we present the crystal structure of the Mmi1 YTH domain in the apo state and in complex with a DSR motif, demonstrating that the Mmi1 YTH domain selectively recognizes the DSR motif. Intriguingly, Mmi1 also contains a potential m⁶A (N⁶-methyladenine)-binding pocket, but its binding of the DSR motif is dependent on a long groove opposite the m⁶A pocket. The DSR-binding mode is distinct from the m⁶A RNA-binding mode utilized by other YTH domains. Furthermore, the m⁶A pocket cannot bind m⁶A RNA. Our structural and biochemical experiments uncover the mechanism of the YTH domain in binding the DSR motif and help to elucidate the function of Mmi1.     

Linking the YTH domain to cancer: the importance of YTH family proteins in epigenetics

N6-methyladenosine (m6A), the most prevalent and reversible modification of mRNA in mammalian cells, has recently been extensively studied in epigenetic regulation. YTH family proteins, whose YTH domain can recognize and bind m6A-containing RNA, are the main “readers” of m6A modification. YTH family proteins perform different functions to determine the metabolic fate of m6A-modified RNA. The crystal structure of the YTH domain has been completely resolved, highlighting the important roles of several conserved residues of the YTH domain in the specific recognition of m6A-modified RNAs. Upstream and downstream targets have been successively revealed in different cancer types and the role of YTH family proteins has been emphasized in m6A research. This review describes the regulation of RNAs by YTH family proteins, the structural features of the YTH domain, and the connections of YTH family proteins with human cancers.Subject terms: Cancer, Epigenetics     

A dynamic reversible RNA N6-methyladenosine modification: current status and perspectives

N⁶-methyladenosine (m⁶A), as the most abundant RNA epigenetic modifications, has been shown to play critical roles in various biological functions. Research about enzymes that can catalyze and remove m⁶A have revealed its comprehensive roles in messenger RNA (mRNA) metabolism and other physiological processes. The “readers” including YTH domain-containing proteins, hnRNPC, hnRNPG, hnRNPA2B1, IGF2BP1, IGF2BP2, and IGF2BP3, which can affect the fates of mRNA in an m⁶A-dependent manner. In this review, we focus on recent advances in the research of the m⁶A modifications, especially about the latest functions of its writers, erasers, readers in RNA metabolism, cancer, and lipid metabolism. In the end, we provide insights into the underlying molecular mechanisms of m⁶A modifications.     

m6A甲基转移酶家族Fiona/METT-10/METTL16的功能

RNA碱基上的化学修饰在其功能的精准调节中发挥关键作用,其中m⁶A是自然界中最普遍的RNA修饰之一,且该修饰在调控RNA稳定性、pre-mRNA剪接、翻译等方面具有重要功能。在真核生物中,m⁶A修饰主要由两种甲基转移酶完成,其在哺乳动物中分别命名为METTL3和METTL16。与METTL3相似,METTL16的底物多种多样,包括pre-mRNA、rRNA、snRNA和lncRNA等,因此似乎难以用一种分子机理解释METTL16对不同RNA底物进行m⁶A修饰的功能。此外,METTL16还在翻译调控中发挥重要作用,但此过程不依赖其甲基转移酶活性,这进一步增加了高度保守的METTL16的功能复杂性。本综述总结了METTL16及其同源蛋白质的结构域、甲基化底物以及它们的潜在功能,着重阐述了在不同物种中关于METTL16研究结果的矛盾之处,并推测METTL16调控S-腺苷基甲硫氨酸(SAM)代谢的功能是趋同进化的一个潜在案例。     

Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

N⁶-Methyladenosine (m⁶A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m⁶A in single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC₅₀, ∼488 μm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m⁶A binding pocket of ALKBH5 and the key residues involved in m⁶A recognition using mutagenesis and ITC binding experiments.