MeRIP-seq to identify putative Mettl3 targets in the nephron progenitor cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE294483 | GEO | 2025/04/14
REPOSITORIES: GEO
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Project description:MeRIP-seq to identify putative Mettl3 targets in the nephron progenitor cells
| PRJNA1250288 | ENA
Project description:mRNA m6A modification is involved in regulation of immune system. However, its function in antiviral immunity is controversial, and how immune responses regulate m6A modification is unknown. We here found TBK1, a key kinase of antiviral pathways, phosphorylated the core m6A methyltransferase METTL3 at Serine 67. The phosphorylated METTL3 interacted with translational complex and enhanced proteins translation, including IRF3, and facilitated antiviral responses. TBK1 also promoted METTL3 activation and m6A modification, which is required for stabilizing IRF3 mRNA. Type I IFN induction was severely impaired in METTL3 deficient cells. Mettl3flfl-lyz2-Cre mice were significantly more susceptible to IAV-induced lethality than control mice. Consistently, Ythdf1—/— mice cannot control viral infection and showed higher mortality than control mice due to decreased IRF3 expression. Together, we demonstrated that innate signals activated METTL3 via TBK1, and METTL3 and m6A modification secured antiviral immunity by promoting mRNA stability and protein translation.
2020-11-04 | GSE160790 | GEO
Project description:p53 limits the self-renewing ability of a variety of stem cells. Here, contrary to its classical role in restraining cell proliferation, we demonstrate a divergent function of p53 in maintenance of self-renewal of the nephron progenitor population in the embryonic mouse kidney. p53-null nephron progenitor cells (NPC) exhibit progressive loss of the self-renewing progenitor niche in the cap mesenchyme, identified by Cited1 and Six2 expression, and loss of cap integrity. Nephron endowment is regulated by NPC availability and their differentiation to nephrons. Quantitatively, the Six2p53-/- cap has 30% fewer Six2GFP+ cells. While the apoptotic index is unchanged the proliferation index is significantly lower, in accordance with cell cycle analysis data showing less mutant Six2p53-/-;GFP+ cells in S and G2/M phases in comparison to Six2p53+/+;GFP+ cells. The mutant kidneys also show nephron deficit and decreased Fgf8 expression. To investigate the underlying changes in gene expression in the cap mesenchyme that contribute to the Six2p53-/- phenotype, we utilized RNA-Seq for transcriptome comparison. Top biological processes affected by p53 loss are development and morphogenesis, cell adhesion/migration, cell survival and metabolism. Cells from the mutant CM showed increased cellular ROS levels as well as deregulated expression of energy metabolism and mitochondrial genes suggesting metabolic dysfunction. Adhesion defects are visualized by decreased immunostaining of adhesion marker NCAM, and may possibly contribute to the differentiation defect as well. Altogether our data suggest a novel role for p53 in enabling self-renewal of the NPC and preservation of the progenitor niche, and thus regulating nephron endowment. mRNA profiles of wild-type (WT) and conditional p53 knockout (KO) of Six2+ mouse nephron progenitor cells (NPC) at embryonic day 15.5
2015-02-16 | E-GEOD-56253 | biostudies-arrayexpress
Project description:Methyltransferase-like 3 (METTL3) and 14 (METTL14) are core subunits of the methyltransferase complex (MTC) that catalyzes mRNA N6-methyladenosine (m6A) modification. Despite the expanding list of m6A-dependent function of the MTC, m6A independent function of the METTL3 and METTL14 complex remains poorly understood. Here we show that genome-wide redistribution of METTL3 and METTL14 drives senescence-associated secretory phenotype (SASP) in a m6A-independent manner. METTL3 and METTL14 are necessary for SASP. However, SASP is not regulated by m6A mRNA modification. METTL14 is redistributed to the enhancers, while METTL3 is localized to the pre-existing NF-B sites within the promoters of the SASP genes during senescence. METTL3 interacts with NF-B and they are mutually dependent on their associations with the promoters of SASP genes. METTL14 but not METTL3 is necessary for function of SASP gene enhancers. METTL3 and METTL14 are required for both the tumor-promoting and immune surveillance functions of senescent cells mediated by SASP in vivo in mouse models. In summary, our results report a m6A independent function of the METTL3 and METTL14 complex in promoting SASP through regulating transcription by genome-wide redistribution of METTL14 to enhancers and METTL3 to promoters of SASP genes during senescence.
2020-12-10 | GSE141993 | GEO
Project description:N6-methyladenosine (m6A) has been shown to regulate various aspects of mRNA biology. Although m6A has been shown to be co-transcriptionally deposited in mRNA by the chromatin-bound methyltranferase METTL3, the precise role of METTL3 and the resulting m6A methylation remain incompletely understood. Here, we demonstrate that the nascent RNAs generated from enhancers and promoters are co-transcriptionally and dynamically regulated by the chromatin bound METTL3/14 methyltransferase complex, and the demethylase, ALKBH5. Importantly, nascent RNA m6A is recognized by the reader protein hnRNP G, which prevents access of the RNA endonuclease INTS11 and thus protects nascent RNA from inappropriate degradation. Our study thus reveals an important chromatin function of m6A in stabilizing the enhancer- and promoter-derived nascent RNAs, which function to promote transcription activation.
2022-02-25 | GSE196564 | GEO
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:All the three enzymes, METTL3, METTL14, and METTL16, belong to methyltransferase like (METTL) family and possess the ability to deposit N6-methyladenosine (m6A) in mRNA. Via immunofluorescence staining and wertern blotting, we discovered either METTL3 or METTL14 mainly localize in nuclear, but METTL16 localizes in both cytosol and nuclei. To compare the m6A methyltransferase activities of the three m6A 'writers' and better understand their differences, MeRIP-seq (m6A-seq) was conducted with poly(A) RNAs isolaed from HEK293T cells with METTL3, 14 and 16 knockout.
2021-10-28 | GSE182607 | GEO
Project description:Methyltransferase-like 3 (METTL3) is the best known m6A methyltransferase of the N6-adenosine-methyltransferase complex that functions in the reversible epi-transcriptome modulation of m6A modification. Besides acting as a m6A methyltransferase, METTL3 also regulates mRNA translation as well as other biological processes either through m6A “reader”-mediated downstream effect or directly binding to m6A sites, but the underlying mechanism and biological significance of these cytoplasmic events remain undefined. Here, we demonstrated that METTL3 inhibition significantly delayed gastric tumorigenesis in patient-derived xenograft model. Intriguingly, global METTL3-binding profiling revealed METTL3 occupied numerous oncogenic mRNAs without m6A signals, indicating a novel mechanism independent of m6A machinery. Furthermore, METTL3 was observed to translocate from nucleus to cytoplasm during gastric carcinogenesis, and its cytoplasm-to-nucleus ratio was correlated with cancer progression. In addition, the nuclear retention of METTL3 nearly abolished its tumor-promoting activity. Mechanistically, METTL3 interacted with PABPC1 to promote RNA looping, thus facilitating the translation of multiple oncogenic mRNAs. Taken together, our findings indicate an unexpected role of METTL3 as an important translational regulator independent of either m6A-“writer” or -“reader” activities and suggest METTL3 as a therapeutic target in gastric cancer.
2022-05-16 | GSE163310 | GEO
Project description:Purpose: To find m6A modified genes which were affected in Mettl3-KO (Mettl3fl/fl; Lgr5-EGFP-IRES-CreERT2; Villin-CreERT2) Intestinal stem cells, we used a method combining RNA-protein immunoprecipitation with high-throughput sequencing to achieve RNA methylation, exactly m6A, analysis at the full transcriptome level. Methods: Intestinal stem cells (Lgr5-high cells) were sorted by flow cytometry in WT and Mettl3-KO intestinal epithelium at 4 dpt. mRNA was purified using Dynabeads mRNA Purification kit (Invitrogen, 61006). Then, mRNA was fragmented into ~100-nucleotide-long fragments by RNA Fragmentation Reagents (Ambion, AM8740) at 94 ℃ for 45s. After that, mRNA were immunoprecipitation with m6A antibody (NEB, NO.E1610S) to enrich the m6A-marked mRNA followed by RNA extraction by RNA clean&concentrator-5 (ZYMO RESEARCH, 1013). The cDNA libraries were generated using SMARTER Stranded Total RNA-seq kit v2. In each sample, reads were aligned to the mouse genome in Ensembl (release 95) with HISAT2. m6A peaks in each sample were identified using exomePeak (v2.17.0) R/Bioconductor package with default parameters. Conclusions: We found that 2074 transcripts showed a reduced m6A modification in Mettl3-KO Lgr5-high ISCs at 4 dpt. The most of targets contained the common m6A motif “RRACH (GGACU)” in the protein-coding region and 3’UTR, especially enriched near the stop codon.
2023-04-13 | GSE186914 | GEO
Project description:N6-methyladenosine (m6A) methyltransferase METTL3 mainly mediates mRNA m6A methylation and plays important roles in various biological processes. Here we report a chromatin-based role for METTL3 in heterochromatin integrity regulation in mouse embryonic stem cells (mESCs). We show that in mESCs METTL3 predominantly localizes to one of the most active endogenous retroviruses (ERVs) families, the intracisternal A-type particles (IAPs). We further show that METTL3 knockout (KO) impairs deposition of multiple heterochromatin marks on METTL3-targeted IAPs, and upregulates IAP transcription without affecting the stability of the resulting RNAs. De-repression can be rescued by wildtype but not catalytically inactive METTL3, suggesting that METTL3-mediated methylation is important for heterochromatin silencing. Mechanistically, we demonstrate that METTL3 physically interacts with the H3K9 tri-methyltransferase SETDB1 and its regulator TRIM28, and reciprocally promote each other’s localization to IAPs. Importantly, we show that m6A catalytical activity of METTL3 is necessary for its own recruitment to chromatin but not its interactions with SETDB1 and TRIM28. Taken together, our findings demonstrate that RNA m6A modification mediated by METTL3 is important for IAP heterochromatin integrity in mESCs.
2020-12-09 | GSE154138 | GEO