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.     

Roles of N6‐methyladenosine (m6A) RNA modifications in urological cancers

Epigenetics has long been a hot topic in the field of scientific research. The scope of epigenetics usually includes chromatin remodelling, DNA methylation, histone modifications, non‐coding RNAs and RNA modifications. In recent years, RNA modifications have emerged as important regulators in a variety of physiological processes and in disease progression, especially in human cancers. Among the various RNA modifications, m⁶A is the most common. The function of m⁶A modifications is mainly regulated by 3 types of proteins: m⁶A methyltransferases (writers), m⁶A demethylases (erasers) and m⁶A‐binding proteins (readers). In this review, we focus on RNA m⁶A modification and its relationship with urological cancers, particularly focusing on its roles and potential clinical applications.     

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.     

Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA

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.     

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.     

m6A Regulates Liver Metabolic Disorders and Hepatogenous Diabetes

N⁶-methyladenosine (m⁶A) is one of the most abundant modifications on mRNAs and plays important roles in various biological processes. The formation of m⁶A is catalyzed by a methyltransferase complex (MTC) containing a key factor methyltransferase-like 3 (Mettl3). However, the functions of Mettl3 and m⁶A modification in hepatic lipid and glucose metabolism remain unclear. Here, we showed that both Mettl3 expression and m⁶A level increased in the livers of mice with high fat diet (HFD)-induced metabolic disorders. Overexpression of Mettl3 aggravated HFD-induced liver metabolic disorders and insulin resistance. In contrast, hepatocyte-specific knockout of Mettl3 significantly alleviated HFD-induced metabolic disorders by slowing weight gain, reducing lipid accumulation, and improving insulin sensitivity. Mechanistically, Mettl3 depletion-mediated m⁶A loss caused extended RNA half-lives of metabolism-related genes, which consequently protected mice against HFD-induced metabolic syndrome. Our findings reveal a critical role of Mettl3-mediated m⁶A in HFD-induced metabolic disorders and hepatogenous diabetes.     

A m6A Sensing Method by Its Impact on the Stability of RNA Double Helix

N⁶‐Methyladenosine (m⁶A) is one of the most important RNA modifications in epigenetics. The development of detection method for m⁶A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m⁶A modification and target RNAs without m⁶A modification. We verified the fact that the presence of m⁶A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m⁶A detection methods in the future.     

N~6 -methyl-adenosine (m ~6A) in RNA: An Old Modification with A Novel Epigenetic Function

N6 -methyl-adenosine (m6A) is one of the most common and abundant modifications on RNA molecules present in eukaryotes. However, the biological significance of m6A methylation remains largely unknown. Several independent lines of evidence suggest that the dynamic regulation of m6A may have a profound impact on gene expression regulation. The m6A modification is catalyzed by an unidentified methyltransferase complex containing at least one subunit methyltransferase like 3 (METTL3). m6A modification on messenger RNAs (mRNAs) mainly occurs in the exonic regions and 3’-untranslated region (3’-UTR) as revealed by high-throughput m6A-seq. One significant advance in m6A research is the recent discovery of the first two m6A RNA demethylases fat mass and obesity-associated (FTO) gene and ALKBH5, which catalyze m6A demethylation in an a-ketoglutarate (a-KG)-and Fe2+-dependent manner. Recent studies in model organisms demonstrate that METTL3, FTO and ALKBH5 play important roles in many biological processes, ranging from development and metabolism to fertility. Moreover, perturbation of activities of these enzymes leads to the disturbed expression of thousands of genes at the cellular level, implicating a regulatory role of m6A in RNA metabolism. Given the vital roles of DNA and histone methylations in epigenetic regulation of basic life processes in mammals, the dynamic and reversible chemical m6A modification on RNA may also serve as a novel epigenetic marker of profound biological significances.     

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     

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2′O-methylation, pseudouridylation, N⁶-methyladenosine (m⁶A), and N^6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m⁶A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m⁶A modification, the methyltransferase responsible for the 18S rRNA m⁶A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m⁶A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m⁶A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m⁶A in noncoding RNAs.     

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.     

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.