M6A-seq of the mouse ESC transcriptome expressing wild-type (R-WT) or non-phosphorylatable (R-3A2A) METTL3/WTAP
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA577117 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10267580.fastq.gz | Fastqsanger.gz | |||
| SRR10267581.fastq.gz | Fastqsanger.gz | |||
| SRR10267582.fastq.gz | Fastqsanger.gz | |||
| SRR10267583.fastq.gz | Fastqsanger.gz | |||
| SRR10267584.fastq.gz | Fastqsanger.gz |
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Project description:We performed immunoprecipitation of m6A-modfiied poly-A tailed RNA in mouse ESCs expressing either wild-type or non-phosphorylatable METTL3/WTAP. By sequencing over 4 billion bases across both Input and Immunoprecipitated (IP) samples, we generate a transcriptome-wide map of m6A methylation enrichment across both cell types. We find that m6A peaks that are differentially methylated between R-WT and R-3A2A mouse ESCs. Several pluripotency transcripts show reduced m6A methylation in R-3A2A cells compared to R-WT. For the CRISPR screen, we transduced a circular RNA GGACU-containing GFP reporter plasmid, followed by transduction of a genome-wide CRISPR sgRNA library. We selected for the top and bottom 5% of GFP expression in HeLa cells and sequenced the sgRNAs.
2020-12-16 | GSE138776 | GEO
Project description:label the cells overexpressed Myc tagged METTL3 and Flag tagged WTAP with 4-SU, the RNA bound by METT3,WTAP can be got by Myc or Flag IP followed by RNA isolation by using the TRIzol (Invitrogen) reagent by following the company manual.the RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 50 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane. discovery of the binding motif of METTL3，WTAP in METTL3，WTAP overexpressed Human 293T cells
2014-01-16 | E-GEOD-50580 | biostudies-arrayexpress
Project description:SETD2 is the specific methyltransferase of H3K36me3, while METTL3, METTL14 and WTAP are the components of m6A methyltransferase complex. To understand the global effect of H3K36me3 on m6A modification, we compared the m6A profiling in SETD2 and METTL3, METTL14 or WTAP knockdown HepG2 cells, and found depletion of H3K36me3 by SETD2 silencing globally reduced m6A in human transcriptome. What’s more, most of the SETD2-dependent hypomethylation sites also responded to knockdown of METTL3, METTL14, or WTAP.
2019-02-21 | GSE110320 | GEO
Project description:N6-methyladenosine (m6A) methylation of mRNA by the methyltransferase complex (MTC), with core components including METTL3-METTL14 heterodimers and Wilms’ tumor 1-associated protein (WTAP), contributes to breast tumorigenesis, but the mechanism of MTC assembly remains elusive. Here, we identify a novel cleaved form METTL3a (residues 239-580 of METTL3), that is highly expressed in breast cancer. Furthermore, we find that both METTL3a and full-length METTL3 are required for MTC assembly, RNA m6A deposition, as well as cancer cell proliferation. Mechanistically, we find that METTL3a is required for METTL3-METTL3 interaction, which is a prerequisite step for recruitment of WTAP in MTC assembly. Analysis of m6A sequencing data shows that depletion of METTL3a globally disrupts m6A methylation, and METTL3a mediates mTOR activation via m6A-mediated suppression of TMEM127 expression. Consequently, we find that METTL3 cleavage is mediated by proteasome in an mTOR-dependent manner, revealing positive regulatory feedback between METTL3a and mTOR signaling. Our findings reveal METTL3a as an important component for MTC assembly, and suggest the METTL3a-mTOR axis as a potential therapeutic target for breast cancer.
2023-08-18 | PXD036899 | Pride
Project description:label the cells overexpressed Myc tagged METTL3 and Flag tagged WTAP with 4-SU, the RNA bound by METT3,WTAP can be got by Myc or Flag IP followed by RNA isolation by using the TRIzol (Invitrogen) reagent by following the company manual.the RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 50 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane.
2014-01-16 | GSE50580 | GEO
Project description:Background: N6-methyladenosine (m6A) RNA modification plays a crucial role in various biological events and is implicated in various metabolic-related diseases. However, its role in MASLD remains unclear. This study aims to investigate the impact of Mettl3 on MASLD through multi-omics analysis, with a focus on exploring its potential mechanisms of action. Methods: MASLD mouse models were established by feeding a high-fat diet for 12 weeks, and Mettl3 stable overexpression AML12 cell models were constructed via lentiviral transfection. Subsequent transcriptomic and proteomic analyses, as well as integrated analysis between different omics datasets, were conducted. Results: Mettl3 expression significantly increased in MASLD mouse models. In the transcriptomic and proteomic analyses, we identified 848 genes with significant inconsistencies between transcriptomic and proteomic datasets. GO/KEGG enrichment terms may involve post-transcriptional modifications, particularly Mettl3-mediated m6A modification. Subsequently, through integrated proteomic analysis of Mettl3-overexpressed AML12 cell models and MASLD mouse models, we selected the top 20 co-upregulated and co-downregulated GO/KEGG terms as the main biological processes influenced by Mettl3 in MASLD. By intersecting with pathways obtained from previous integrated analyses, we identified GO/KEGG terms affected by Mettl3-induced m6A modification. Protein-protein interaction analysis of proteins involved in these pathways highlighted GAPDH, ENO1, and TPI1 as three key hub genes. Conclusion: In MASLD, Mettl3 regulates the glycolytic pathway through m6A modification, influencing the occurrence and development of the disease via the key hub genes GAPDH, ENO1, and TPI1. These findings expand our understanding of MASLD and provide strong evidence for potential therapeutic targets and drug development.
2024-09-20 | PXD050565 | Pride
Project description:The N6-methyladenosine (m6A) mRNA modification and the mitochondrial respiratory chain (MRC) hold paramount importance in the advancement of MASLD. This study thoroughly investigates the relationship and impact of m6A mRNA modification and mitochondrial function in the progression of MASLD. Here we report that the mRNA and protein levels of mitochondrial respiratory chain (MRC) subunits showed inconsistent trends in vivo experiments. Abnormal m6A modification and mitochondrial dysfunction in MASLD were attributed to the upregulation of methyltransferase like 3 (Mettl3) and the downregulation of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) induced by high-fat foods. Mettl3 promoted the MRC's function. However, knockout of the reader protein YTHDF1, which plays a crucial role in the m6A modification process, counteracted the effect of Mettl3 and suppressed MRC. In MASLD, damage to the MRC may be regulated by the Mettl3-m6A-YTHDF1 complex axis, especially by the role of YTHDF1. Our research has offered a novel perspective on the involvement of m6A mRNA methylation in the pathogenesis of MASLD.
2024-09-20 | PXD047819 | Pride
Project description:Methyltransferase-like 3 (METTL3) is the predominant catalytic enzyme which forms a stable complex with METTL14 and WTAP to promote m6A methylation in the nucleus. Recently, accumulating evidence has shown that METTL3 can express in the cytoplasm but the function of cytoplasmic METTL3 is not fully understood. Here, we demonstrated that METTL3 inhibition significantly delayed tumorigenesis in gastric cancer both in and ex vivo. Surprisingly, our data revealed that METTL3 could not only facilitate cancer progression via mRNA m6A modification, but also bind to numerous non-m6A-modified mRNAs, suggesting an unexpected role of METTL3 that was independent of m6A modification. Mechanistically, cytoplasm-anchored METTL3 interacted with poly(A) binding protein cytoplasmic 1 (PABPC1) to stabilize its association with cap-binding complex eIF4F, which preferentially promoted the translation of epigenetic factors without m6A modification. Clinical investigation using both xenograft models and patient samples showed that cytoplasmic distributed METTL3 was highly correlated with gastric cancer progression, and this finding could be expanded in prostate cancer patients. We therefore propose that cytoplasmic METTL3 enhances the translation of both m6A and non-m6A-modified epigenetic mRNAs thus serves as an oncogenic driver in cancer progression, and METTL3 subcellular distribution can assist diagnose and predict prognosis for cancer patients.
2022-05-16 | GSE191170 | GEO
Project description:WTAP is an essential component of the RNA N-6-methyladenosine (m6A) modification complexes that guides METLL3-METLL14 heteroduplexes to target RNAs in the nucleus of mammalian cells. Through mining the genotype-tissue expression (GTEx) datasets, we initially found that TTC22 expression was highly correlated with WTAP and FTO in many normal human tissues. Our experimental results indicate that TTC22 could directly capture RNA binding protein RPL4, induce the binding between RPL4 and WTAP mRNA in the cytoplasm, which increased the m6A level, induced alterative splicing, enhanced the stability and translation efficiency of WTAP mRNA, and consequently upregulated the level of total m6A RNA. These results indicate that WTAP mRNA is a m6A target and there is a positive feedback loop between total m6A and WTAP expression. YTHDF1 was found to be an essential m6A WTAP mRNA binding protein. Downregulation of RPL4, WTAP, or YTHDF1 expression could reverse TTC22-enhanced total m6A RNA level. m6A-specific antibody immunoprecipitated RNA-sequencing (meRIP-seq) demonstrated that TTC22 caused dramatic expression changes of genes related to metabolic pathways, ribosome biogenesis, and RNA spliceosome. Furthermore, we also found that TTC22 upregulated the expression of epithelial-mesenchymal transition (EMT)-related gene SNAI1 via m6A, and promoted metastasis of colon cancer in vitro and in mice. In conclusion, our study illustrates that WTAP mRNA is a m6A target using YTHDF1 as the binding protein. TTC22 could upregulate the levels of WTAP expression and total m6A RNA through the PRL4 binding. The m6A-mediated upregulation of SNAI1 expression may contribute to TTC22-enhanced colon cancer metastasis.
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