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M6A modification segregates the stemness program from proliferation in ISCs [MeRIP-seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE186914 | GEO | 2023/04/13

REPOSITORIES: GEO

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m6A modification segregates the stemness program from proliferation in ISCs [RNA-seq]

Project description:Purpose: One goal of this Bulk RNA-seq is to compare genome-wide transcriptome profiles in WT and Mettl3-KO mice intestinal stem cells at indicated time point.The other goal is to compare expression of stemness genes in WT, Mettl3-KO, Mettl3 KO and AKF-OE or AKFP-OE organoids. Methods: For mice, intestinal crypts were isolated and digested with Tryple, Lgr5-high or Lgr5-low cells were sorted by flow cytometry. For organoids, organoids were digested with Tryple and alive cells were sorted by flow cytometry. Total RNA was extracted with RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Bulk RNA-seq was performed using the Illumina Hiseq X. RNA-seq reads were aligned to the mouse genome available in Ensembl (release 95) with HISAT2. Conclusions: Mettl3 deletion results in reduced expression of most of the stem cell signature genes in Mettl3-KO ISCs, including Lgr5, Olfm4, Ascl2, and Smoc2, but increased gene expression related to cell death, p53, MAPK, YAP and regeneration pathway. AKF and AKFP-overexpression could rescue the expression level of some ISC signature genes compared with Mettl3 detion organoids.

2023-04-13 | GSE186912 | GEO

Mettl3-m6A-YTHDF1 axis promotion of mitochondrial dysfunction in metabolic dysfunction–associated steatotic liver disease (MASLD)

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2024-09-20 | PXD047819 | Pride

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Project description:Purpose: Determine the differential m6A methylation pattern of Mettl3 between wildtype, Pkd1F/RC-KO and Pkd1F/RC-Mettl3-DKO mouse kidneys. Methods: m6A RNA IP using the Magna MeRIP™ m6A Kit (EMD Millipore, catalog #17-10499) was performed on P18 wildtype, Pkd1F/RC-KO and Pkd1F/RC-Mettl3-DKO mouse kidney samples. Two biological replicate samples from each genotype were sequenced. Each sample was a pool of 6 kidneys of the same genotype. The input samples were also sequenced to obtain mRNA profiles. Results: 133 mRNAs were found to be differentially hypermethylated in the Pkd1-KO kidneys compared to the control kidneys and differentially hypomethylated in the Pkd1-Mettl3-DKO kidneys compared to the Pkd1-KO kidneys. Two key pathogenic mRNAs namely, c-Myc and Avpr2 were identified and validated as Mettl3 targets. The mRNA transcript levels were unchanged between Pkd1-KO and Pkd1-Mettl3-DKO kidneys. Conclusion: Mettl3/m6A promotes translation of pathogenic mRNAs to mediate cystogenesis in mouse models of ADPKD.

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m6A RNA Methylation is Critical for Adequate Exit from Naïve Pluripotency and Execution of Mammalian Development (Ribo-seq)

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2014-11-02 | E-GEOD-61996 | biostudies-arrayexpress

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2020-02-29 | E-MTAB-6501 | biostudies-arrayexpress

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Project description:In this study we identify Mettl3, an m6A RNA modification writer, as a critical regulator for terminating naM-CM-/ve pluripotency and a positive maintainer of primed pluripotency in vitro and in vivo. Remarkably, Mettl3 knockout pre-implantation epiblasts and naM-CM-/ve ES cells, entirely lack m6A on coding mRNAs and are viable. Yet, they fail to adequately terminate the naM-CM-/ve pluripotent state, and subsequently undergo aberrant priming and early lineage commitment at the post-implantation stage. A comprehensive functional and genomic analysis involving profiling of m6A, RNA transcription and translation in Mettl3 wild-type and knockout pluripotent and differentiated cells, identified m6A as a critical determinant that destabilizes secondary naM-CM-/ve specific pluripotency genes Esrrb, Klf4 and Nanog, and restrains their transcript stability and translation efficiency. In summary, our findings provide for the first time evidence for a critical role for an mRNA epigenetic modification in early mammalian development in vivo, and identify a mechanism that functionally regulates mouse naM-CM-/ve and primed pluripotency in an opposing manner. 3' polyA RNA-sequencing (equivalent to Digital Gene Expression) measured in mouse Embryonic Stem Cells (ESCs) and mouse Embriod bodies (EBs) 0,4 & 8 hours after treatment with Actinomycin which halts transcription. Measured in both WT and Mettl3-KO cells.

2014-11-02 | E-GEOD-61994 | biostudies-arrayexpress

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m6A RNA Methylation is Critical for Adequate Exit from Naïve Pluripotency and Execution of Mammalian Development (RNA-Seq)

Project description:In this study we identify Mettl3, an m6A RNA modification writer, as a critical regulator for terminating naïve pluripotency and a positive maintainer of primed pluripotency in vitro and in vivo. Remarkably, Mettl3 knockout pre-implantation epiblasts and naïve ES cells, entirely lack m6A on coding mRNAs and are viable. Yet, they fail to adequately terminate the naïve pluripotent state, and subsequently undergo aberrant priming and early lineage commitment at the post-implantation stage. A comprehensive functional and genomic analysis involving profiling of m6A, RNA transcription and translation in Mettl3 wild-type and knockout pluripotent and differentiated cells, identified m6A as a critical determinant that destabilizes secondary naïve specific pluripotency genes Esrrb, Klf4 and Nanog, and restrains their transcript stability and translation efficiency. In summary, our findings provide for the first time evidence for a critical role for an mRNA epigenetic modification in early mammalian development in vivo, and identify a mechanism that functionally regulates mouse naïve and primed pluripotency in an opposing manner. polyA RNA-seq was measured in mouse embryonic stem cells (ESCs) and embroid bodies (EBs), each in WT and in Mettl3-KO cell lines. RNA-seq was measured also from WT mouse embronic fibroblasts (MEF). 3 biological replicates are available from ESCs and 2 from EBs. Replicate C in ESCs was measured alongside protein levels (SILAC) and was used for the analysis of that assay.

2014-11-02 | E-GEOD-61997 | biostudies-arrayexpress

Widespread N6-methyladenosine-dependent RNA Structural Switches Regulate RNA-Protein Interactions

Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling. Measure the m6A methylated hnRNP C binding sites transcriptome-wide by PARCLIP-MeRIP; measure the differential hnRNP C occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPC, METTL3 and METTL14 knockdown

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