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
Project description:To further understand the relationship between the level of gene expression and protein translation in human placenta, we focused on N6-methyladenosine: m6A, which is a methylation modification of mRNA acting as a post-transcriptional regulation which has drawn attention in recent years. It is said that m6A affects the fate of mRNA. In addition, it is thought that m6A affects gene expression in the placenta of preeclampsia which is a representative disease of perinatal period and fetal growth abnormality, and the placentas of children who were small for date (SFD) or heavy for date (HFD) were studied in addition to appropriate for date (AFD) preganancies. The SFD placentas contained half of the cases with PE. HEK293T cell was used to confirm the experimental system. PolyA RNA was purified from each tissue and cell, and libraries were prepared from immunoprecipitated (IP) samples using anti-m6A antibody and input samples, and sequenced with Hiseq 1500/2500 and RNAseq was carried out.
Project description:We report an epitranscriptome-wide mapping of m6A-modified circRNAs (m6A-circRNA) in oral squamous cell carcinoma (OSCC). Utilizing the data of m6A-circRNAs epitranscriptomic microarray analysis, we found that m6A-circRNAs exhibited their particular modification style in OSCC. anti-m6A antibody Synaptic Systems, cat. No. 202003)
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 (“m6A levels”), 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 3’ untranslated regions (3’-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.
Project description:N6-methyl-adenosine (m6A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m6A by mapping the m6A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m6A modification, including transcripts encoding core pluripotency transcription factors. m6A is enriched over 3M-bM-^@M-^Y untranslated regions at defined sequence motifs, and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m6A methylases, led to m6A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESCM-bM-^@M-^Ys exit from self-renewal towards differentiation into several lineages in vitro and in vivo. Thus, m6A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages. Examing m6A modification differences in two different cell types
Project description:Glutathione peroxidase 8 (GPX8) is a key regulator of redox homeostasis. Whether its antioxidant activity participates in the regulation of m6A modification is a crucial issue, which has important application value in cancer treatment. MeRIP-seq was used to explore the characteristics of transcriptome-wide m6A modification in GPX8-deficient oral cancer cells in this study. Oxidative stress caused by lack of GPX8 resulted in 1,279 hyper- and 2,287 hypo-methylated m6A peaks and 2,036 differentially expressed genes in GPX8-KO cells. Twenty-eight differentially expressed genes were related to the cell response to oxidative stress, and half of them changed their m6A modification. In GPX8-KO cells, m6A regulators IGF2BP2 and IGF2BP3 were upregulated, while FTO, RBM15, VIRMA, ZC3H13 and YTHDC2 were downregulated. After H2O2 treatment, the expression changes of RBM15, IGF2BP2 and IGF2BP3 were further enhanced. These data indicated that GPX8-mediated redox homeostasis regulated m6A modification, thereby affecting the expression and function of downstream genes. This study highlights the possible significance of GPX8 and the corresponding m6A regulatory or regulated genes as novel targets for antioxidant intervention in cancer therapy
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) has been one of the most abundant and well-known modifications in mRNA since its discovery in 1970s. Recent studies have demonstrated that m6A gets involved in various biological processes such as alternative splicing and RNA degradation, playing an important role in all kinds of diseases. To better understand the role of m6A, transcriptome-wide m6A profiling data is indispensable. In these years, the Oxford Nanopore Technology Direct RNA Sequencing (DRS) platform has shown promise in RNA modification detection based on current disruptions measured in transcripts. However, decoding current intensity data into modification profiles remains a challenging task. Here, we introduce m6A Transcriptome-wide Mapper (m6ATM), a novel Python-based computational pipeline that applies deep neural networks to predict m6A sites at single-base resolution using DRS data. The m6ATM model architecture incorporates a WaveNet encoder and a dual-stream multiple instance learning model to extract features from specific target sites and characterize the m6A epitranscriptome. For validation, m6ATM achieved an accuracy of 80 to 98% across in-vitro transcription datasets containing varying m6A modification ratios and outperformed other tools in benchmarking with human cell-line data. Moreover, we demonstrated the versatility of m6ATM in providing reliable stoichiometric information and used it to pinpoint PEG10 as a potential m6A target transcript in liver cancer cells. In conclusion, we showed that m6ATM is a high-performance m6A detection tool and our results paved the way for epitranscriptomic precision medicine.
Project description:N6-methyladenosine (m6A) has been one of the most abundant and well-known modifications in mRNA since its discovery in 1970s. Recent studies have demonstrated that m6A gets involved in various biological processes such as alternative splicing and RNA degradation, playing an important role in all kinds of diseases. To better understand the role of m6A, transcriptome-wide m6A profiling data is indispensable. In these years, the Oxford Nanopore Technology Direct RNA Sequencing (DRS) platform has shown promise in RNA modification detection based on current disruptions measured in transcripts. However, decoding current intensity data into modification profiles remains a challenging task. Here, we introduce m6A Transcriptome-wide Mapper (m6ATM), a novel Python-based computational pipeline that applies deep neural networks to predict m6A sites at single-base resolution using DRS data. The m6ATM model architecture incorporates a WaveNet encoder and a dual-stream multiple instance learning model to extract features from specific target sites and characterize the m6A epitranscriptome. For validation, m6ATM achieved an accuracy of 80 to 98% across in-vitro transcription datasets containing varying m6A modification ratios and outperformed other tools in benchmarking with human cell-line data. Moreover, we demonstrated the versatility of m6ATM in providing reliable stoichiometric information and used it to pinpoint PEG10 as a potential m6A target transcript in liver cancer cells. In conclusion, we showed that m6ATM is a high-performance m6A detection tool and our results paved the way for epitranscriptomic precision medicine.