M?A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins.
ABSTRACT: The control of gene expression is a multi-layered process occurring at the level of DNA, RNA, and proteins. With the emergence of highly sensitive techniques, new aspects of RNA regulation have been uncovered leading to the emerging field of epitranscriptomics dealing with RNA modifications. Among those post-transcriptional modifications, N6-methyladenosine (m6A) is the most prevalent in messenger RNAs (mRNAs). This mark can either prevent or stimulate the formation of RNA-protein complexes, thereby influencing mRNA-related mechanisms and cellular processes. This review focuses on proteins containing a YTH domain (for YT521-B Homology), a small building block, that selectively detects the m6A nucleotide embedded within a consensus motif. Thereby, it contributes to the recruitment of various effectors involved in the control of mRNA fates through adjacent regions present in the different YTH-containing proteins.
Project description: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.
Project description:More than 100 types of chemical modifications in RNA have been well documented. Recently, several modifications, such as N6-methyladenosine (m6A), have been detected in mRNA, opening the window into the realm of epitranscriptomics. The m6A modification is the most abundant modification in mRNA and non-coding RNA (ncRNA). At the molecular level, m6A affects almost all aspects of mRNA metabolism, including splicing, translation, and stability, as well as microRNA (miRNA) maturation, playing essential roles in a range of cellular processes. The m6A modification is regulated by three classes of proteins generally referred to as the "writer" (adenosine methyltransferase), "eraser" (m6A demethylating enzyme), and "reader" (m6A-binding protein). The m6A modification is reversibly installed and removed by writers and erasers, respectively. Readers, which are members of the YT521-B homology (YTH) family proteins, selectively bind to RNA and affect its fate in an m6A-dependent manner. In this review, we summarize the structures of the functional proteins that modulate the m6A modification, and provide our insights into the m6A-mediated gene regulation.
Project description: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 alpha-helix/beta-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.
Project description:BACKGROUND:N6-Methyladenosine (m6A) is the most widespread RNA modification that plays roles in the regulation of genes and genome stability. YT521-B homology (YTH) domain-containing RNA-binding proteins are important RNA binding proteins that affect the fate of m6A-containing RNA by binding m6A. Little is known about the YTH genes in common wheat (Triticum aestivum L.), one of the most important crops for humans. RESULTS:A total of 39 TaYTH genes were identified in common wheat, which are comprised of 13 homologous triads, and could be mapped in 18 out of the 21 chromosomes. A phylogenetic analysis revealed that the TaYTHs could be divided into two groups: YTHDF (TaDF) and YTHDC (TaDC). The TaYTHs in the same group share similar motif distributions and domain organizations, which indicates functional similarity between the closely related TaYTHs. The TaDF proteins share only one domain, which is the YTH domain. In contrast, the TaDCs possess three C3H1-type zinc finger repeats at their N-termini in addition to their central YTH domain. In TaDFs, the predicated aromatic cage pocket that binds the methylysine residue of m6A is composed of tryptophan, tryptophan, and tryptophan (WWW). In contrast, the aromatic cage pocket in the TaDCs is composed of tryptophan, tryptophan, and tyrosine (WWY). In addition to the general aspartic acid or asparagine residue used to form a hydrogen bond with N1 of m6A, histidine might be utilized in some TaDFb proteins. An analysis of the expression using both online RNA-Seq data and quantitative real-time PCR verification revealed that the TaDFa and TaDFb genes are highly expressed in various tissues/organs compared with that of TaDFcs and TaDCs. In addition, the expression of the TaYTH genes is changed in response to various abiotic stresses. CONCLUSIONS:In this study, we identified 39 TaYTH genes from common wheat. The phylogenetic structure, chromosome distribution, and patterns of expression of these genes and their protein structures were analyzed. Our results provide a foundation for the functional analysis of TaYTHs in the future.
Project description:N(6)A 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 N(6)-methylated adenosine reader domain and report its solution structure in complex with a N(6)-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m(6)A recognition. These findings establish a molecular function for YTH domains as m(6)A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m(6)A recognition.
Project description:To profile the landscape of methylation N6 adenosine (m6A) RNA regulators in colonic adenocarcinoma (COAD) and to explore potential diagnostic and prognostic biomarkers, we assessed the differential expression patterns of m6A RNA methylation regulators between 418 COAD patients and 41 controls based on profiling from The Cancer Genome Atlas (TCGA) database. We plotted the receiver operating characteristic (ROC) curves and calculated the area under the curve (AUC) values to estimate the discrimination ability. The relationship between the expression of m6A RNA methylation regulators and clinicopathological characteristics was explored. Kaplan-Meier plotter, log-rank test, and Cox regression were used and a nomogram was created to explore the prognostic significance of m6A-related genes in overall survival at the mRNA level. Pathway analysis was performed by gene set enrichment analysis (GSEA) using TCGA dataset, and a coexpression network was built based on the STRING database. We observed that YTHDF1, METTL3, and KIAA1429 were significantly upregulated, while YTHDF3, YTHDC2, METTL14, and ALKBH5 were significantly downregulated in COAD samples compared to normal samples. YTHDF1 had the highest diagnostic value. Low expression of YTHDF3 predicted a poor survival rate in COAD patients. YTHDC2 was related to sex and showed a downward trend as clinical stage increased. Our results indicate that the YT521-B homology (YTH) domain family ("readers"), especially YTHDF1, YTHDF3, and YTHDC2, might play a significant role in the detection, progression, and prognosis of COAD, indicating that they are promising cancer biomarkers.
Project description: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.
Project description:BACKGROUND:Plasmodium falciparum exhibits high translational plasticity during its development in RBCs, yet the regulation at the post-transcriptional level is not well understood. The N6-methyl adenosine (m6A) is an important epigenetic modification primarily present on mRNA that controls the levels of transcripts and efficiency of translation in eukaryotes. Recently, the dynamics of m6A on mRNAs at all three developmental stages of P. falciparum in RBCs have been profiled; however, the proteins that regulate the m6A containing mRNAs in the parasites are unknown. RESULTS:Using sequence analysis, we computationally identified that the P. falciparum genome encodes two putative YTH (YT521-B Homology) domain-containing proteins, which could potentially bind to m6A containing mRNA. We developed a modified methylated RNA immunoprecipitation (MeRIP) assay using PfYTH2 and find that it binds selectively to m6A containing transcripts. The PfYTH2 has a conserved aromatic amino acid cage that forms the methyl-binding pocket. Through site-directed mutagenesis experiments and molecular dynamics simulations, we show that F98 residue is important for m6A binding on mRNA. Fluorescence depolarization assay confirmed that PfYTH2 binds to methylated RNA oligos with high affinity. Further, MeRIP sequencing data revealed that PfYTH2 has more permissive sequence specificity on target m6A containing mRNA than other known eukaryotic YTH proteins. Taken together, here we identify and characterize PfYTH2 as the major protein that could regulate m6A containing transcripts in P. falciparum. CONCLUSION:Plasmodium spp. lost the canonical m6A-specific demethylases in their genomes, however, the YTH domain-containing proteins seem to be retained. This study presents a possibility that the YTH proteins are involved in post-transcriptional control in P. falciparum, and might orchestrate the translation of mRNA in various developmental stages of P. falciparum. This is perhaps the first characterization of the methyl-reading function of YTH protein in any parasites.
Project description:N6-methyladenosine (m6A) is a well-known post-transcriptional modification that is the most common type of methylation in eukaryotic mRNAs. The regulation of m6A is dynamic and reversible, which is erected by m6A methyltransferases ("writers") and removed by m6A demethylases ("erasers"). Notably, the effects on targeted mRNAs resulted by m6A predominantly depend on the functions of different m6A-binding proteins ("readers") including YT521-B homology (YTH) domain family, heterogeneous nuclear ribonucleoproteins (HNRNPs), and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs). Indeed, m6A readers not only participate in multiple procedures of RNA metabolism, but also are involved in a variety of biological processes. In this review, we summarized the specific functions and underlying mechanisms of m6A-binding proteins in tumorigenesis, hematopoiesis, virus replication, immune response, and adipogenesis.
Project description:N1-methyladenosine (m1A) is an important post-transcriptional modification in RNA; however, the exact biological role of m1A remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of m1A in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to m1A in RNA. We also found that Trp432 in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N6-methyladenosine (m6A), is required for its recognition of m1A. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and m1A sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to m1A in cells. Together, our results uncovered YTH domain-containing proteins as readers for m1A in RNA and provided new insight into the functions of m1A in RNA biology.