Project description:The aim of the study was to uncover the mechanism behind m6A methylation on E. coli mRNA, and to elude to its functional roles by characterising it and asessing its affects under stress. We carried out m6A sequencing under different conditions.
Project description:The aim of the study was to uncover the mechanism behind m6A methylation on E. coli mRNA, and to elude to its functional roles by characterising it and asessing its affects under stress. We carried out m6A sequencing to meet these aims, and conclude that there is no enzymatic mechanism acting directly on the mRNA responsible for m6A installation, and that the modification is most likely non-functional, and appears randomly throughout the transcriptome.
Project description:m6A is a ubiquitous RNA modification in eukaryotes. Transcriptome-wide m6A patterns in Arabidopsis have been assayed recently. However, m6A differential patterns among organs have not been well characterized. The goal of the study is to comprehensively analyze m6A patterns of numerous types of RNAs, the relationship between transcript level and m6A methylation extent, and m6A differential patterns among organs in Arabidopsis. In total, 18 libraries were sequneced. For the 3 organs: leaf, flower and root, each organ has mRNA-Seq, m6A-Seq and Input sequenced. And each sequence has 2 replicats.
Project description:N6-methyladenosine (m6A) is one of the most abundant modifications in eukaryotic RNA. Recent mapping of m6A methylomes in mammals, yeast, and plants as well as characterization of m6A methyltransferases, demethylases, and binding proteins have revealed regulatory functions of this dynamic RNA modification. In bacteria, although m6A is present in ribosomal RNA (rRNA), its occurrence in messenger RNA (mRNA) still remains elusive. Here, we used liquid chromatography-mass spectrometry (LC-MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved distinct m6A pattern that is significantly different from that in eukaryotes. Most m6A peaks are located inside open reading frames (ORF), and carry a unique consensus motif (GCCAU). Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified transcripts are associated with respiration, amino acids metabolism, stress response, and small RNAs genes, suggesting potential regulatory roles of m6A in these pathways. m6A profiling in E.coli and P.aeruginosa mRNA
Project description:N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNA of all higher eukaryotes. Here we present that m6A is selectively recognized by human YTH domain family (YTHDF2) protein to regulate mRNA degradation. By using crosslinking and immunoprecipitation, we have identified over 4000 substrate RNA of YTHDF2 with conserved core motif of G(m6A)C. We further estabilshed the role of YTHDF2 in RNA metabolism by a combination of ribosome profiling, RNA sequencing, m6A level quantification and cell-based imaging: the C-terminal domain of YTHDF2 selectively binds to m6A of mRNA and the N-terminal domain is responsive for localizing mRNA from translatable pool to processing body where mRNA decay occurs. PAR-CLIP and RIP was used to identify YTHDF2 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF2 siRNA knock-down
Project description:Glioblastoma Multiforme (GBM) is one of the most malignant tumors, characterized by high treatment costs and poor outcomes, thus urgently necessitating the development of new therapeutic approaches. Recent studies have found that intermittent fasting (IF) not only can suppress GBM among various tumors but also can reconstruct the gut microbiome and alter metabolism. However, the mechanism by which IF inhibits GBM is not yet fully understood. Using animal models, I discovered that IF can effectively suppress GBM and alter the gut microbiome.Furthermore, microbial metabolites such as methionine, m6A, and the m6A-targeted oncogene TGFB2 were also downregulated. Therefore, we hypothesize that IF, by reconstructing the gut microbiome, reduces the levels of microbial metabolite methionine, subsequently decreasing m6A in GBM, leading to the downregulation of the m6A-targeted oncogene TGFB2, thereby inhibiting the development of GBM.
Project description:The detection of low-abundance DNA N6-methyladenine (DNA-m6A) remains challenging, limiting our understanding of this novel base in eukaryotes. To address this, we introduce an approach for systematically validating the selectivity and sensitivity of antibody-based DNA-m6A methods, revealing most commercial antibodies as poorly selective towards DNA-m6A. Finally, we validate selective anti-DNA-m6A antibodies with sensitivity <2 ppm, and expose distinct pathways mediating endogenous DNA-m6A in C. reinhardtii, A. thaliana, and D. melanogaster.
Project description:N6-methyladenosine (m6A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m6A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m6A, especially gene- and cell-type-specific m6A mRNA modifications. We also show that microRNAs (miRNAs) regulate m6A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m6A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m6A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m6A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m6A formation of mRNAs and provide a foundation for future functional studies of m6A modification in cell reprogramming. m6A-seq in ESC, iPSC, NSC and sertoli cells.
Project description:N6-methyladenosine (m6A) is a widespread reversible chemical modification of RNAs, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (âm6A levelsâ), or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of non-stoichiometric m6A levels with cell type specificity. At the level of isoform characterization, we discovered widespread differences in use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3â untranslated regions (3â-UTRs), while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications. m6A-LAIC-seq of H1-ESC and GM12878 cell lines, each cell line has two replicates