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MeRIP sequencing reveals angiogenic properties of vascular endothelial cells

ABSTRACT: We report the application of MeRIP sequencing technology for high-throughput profiling of RNA m6A modifications in wide-type and knock-down METTL3 or WTAP Human Umbilical Vein Endothelial Cells (HUVECs). Both the input samples without immunoprecipitation and the m6A IP samples were used for RNA-seq library generation with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired-end reads. Clean reads of all libraries were aligned to the reference genome (HG19) by Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified by MACS software. Differentially methylated sites were identified by diffReps. And, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and p-value were calculated based on FPKM, differentially expressed mRNA were identified. qRT-PCR validation was performed using SYBR Green assays. Finally, we find that METTL3/WTAP can regulate the expression level of target genes through m6A modification in HUVECs. This study provides a framework for applying MeRIP sequencing profiles to characterize vascular endothelial cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142386 | GEO | 2019/12/20

REPOSITORIES: GEO

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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

2015-02-27 | E-GEOD-56010 | biostudies-arrayexpress

LC-MS/MS analysis of co-immunoprecipitation products of METTL3

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

Profiling of N6-methyladenosine in HepG2 cells: SETD2, METTL3, METTL14 or WTAP knockdown and control cells

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

Binding motif analysis of METTL3，WTAP in METTL3，WTAP overexpressed Human 293T cells

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

Arabidopsis Messenger RNA N6-methyladenosine Demethylation Regulates Plant Development

Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and Î±-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates

2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress

MeRIP-seq of mRNAs in MEFs with or without nutrient starvation

Project description:To investigate the dynamic m6A modification during starvation-induced autophagy, we performed MeRIP-seq in MEFs with or without nutrient deprivation and identified the variations of m6A modification on mRNAs. Briefly, the total polyadenylated mRNAs in MEFs cultured with or without nutrient-deprived medium for 2 hours were isolated using RNAiso plus reagent (Takara) followed Dynabeads mRNA Purification Kit (Invitrogen). The m6A RNA immunoprecipitation was performed using GenSeq m6A RNA IP Kit. NEBNext Ultra II Directional RNA Library Prep Kit (NEB) was used for library generation. Each experiment was conducted in two biological replicates. Methylated sites on peaks were identified by MACS2 peak-calling software (Zhang et al., 2008). Differentially methylated sites were identified by diffReps (Shen et al., 2013) with default setting.

2021-12-30 | GSE158268 | GEO

Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification found in mammalian messenger and non-coding RNAs. The discoveries of functionally significant demethylases that reverse this methylation as well as the recently revealed m6A distributions in mammalian transcriptomes strongly indicate regulatory functions of this modification. Here we report the identification and characterization of the mammalian nuclear RNA N6-adenosine methyltransferase core (RNMTC) complex. Besides METTL3, a methyltransferase which was the only known component of RNMTC in the past, we discovered that a previously uncharacterized methyltransferase, METTL14, exhibits a N6-adenosine methyltransferase activity higher than METTL3. Together with WTAP, the third component that dramatically affects the cellular m6A level, these three proteins form the core complex that orchestrates m6A deposition on mammalian nuclear RNA. Biochemistry assays, imaging experiments, as well as transcriptome-wide analyses of the binding sites and their effects on m6A methylation support methylation function and reveal new insights of RNMTC. PAR-CLIP and m6A-seq in HeLa cells

2013-11-29 | E-GEOD-46705 | biostudies-arrayexpress

m6A-seq of the mouse ESC transcriptome expressing wild-type (R-WT) or non-phosphorylatable (R-3A2A) METTL3/WTAP

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

m6A sequences

Project description:Methylated RNA immunoprecipitation(MeRIP) with specific m6A antibody and used for library construction and the next generation sequencing, to identify m6A modified transcripts in WT or Ythdf1 deficiency colon epithelial cells after DSS treatment

2020-05-06 | GSE149883 | GEO

The Alteration of M6A-tagged Transcript Profiles in The Retina of Rats After Traumatic Optic Neuropathy

Project description:Messager RNA (mRNA) can be modified in a variety of ways, among which the modification of N6-methyladenosine (m6A) is one of the most common ones. Recent studies have found that the m6A modification in mRNA could functionally regulate the splicing, localization, translation and stability of mRNA, which might be closely related to multiple diseases. However, the roles of m6A modification in traumatic optic neuropathy (TON) are unknown. Herein, we detected the expression of m6A-related genes via quantitative real-time PCR (qRT-PCR) and performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) as well as RNA-sequencing to analyze the alteration profiles of m6A modification after TON. The results showed that the expression of m6A-related genes (METTL3, WTAP, FTO and ALKBH5) were all upregulated after TON. In all, 2810 m6A peaks were differentially upregulated and 689 m6A peaks were downregulated. In addition, the hypermethylated and hypomethylated profiles of mRNA transcripts were also identified. To sum up, our study revealed the differentially expressed m6A modification in the early stage of TON, which may provide novel insights into the mechanism and treatment of TON.

2021-01-27 | GSE165520 | GEO

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