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M6A-seq on chromatin-assoicated RNA (ChrMeRIP-seq) in mouse embryonic stem cell [ChrMeRIP-seq]

ABSTRACT: N6-methyladenosines (m6A) are stoichiometrically deposited on exons of nearly one third of RNA Pol II transcriptome, mainly catalysed by METTL3/14 complex. However, neither the intronic methylation pattern and its functional relevance nor the immediate response upon m6A loss have been fully understood. Here we applied MeRIP-seq on mESC nascent transcriptome and thus revealed that approximately 6-10% of m6A peaks occurred at intronic regions, preferentially the conserved and alternative exon/intron part of longer introns, proximately to 5’-splice sites. These intronic m6A deposition correlates with both Rbm15 binding and H3K36me3. Moreover, coupled 4sU-seq with METTL3 dTAG system in mESC, we found that m6A mediates alternative exon/intron inclusive in nascent transcriptome. Intriguingly, m6A self-regulation including writers and readers are early response and partially contributed by alternative splicing changes. Collectively, our study presents a unified model that m6A mediates alternative splicing, and dTAG METTL3 opens an avenue to interrogate the direct response of functional m6A disruption.

ORGANISM(S): Mus musculus

PROVIDER: GSE154706 | GEO | 2020/09/15

REPOSITORIES: GEO

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N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]

Project description:N6-methyladenosines (m6A) are stoichiometrically deposited on exons of nearly one third of RNA Pol II transcriptome, mainly catalysed by METTL3/14 complex. However, neither the intronic methylation pattern and its functional relevance nor the immediate response upon m6A loss have been fully understood. Here we applied MeRIP-seq on mESC nascent transcriptome and thus revealed that approximately 6-10% of m6A peaks occurred at intronic regions, preferentially the conserved and alternative exon/intron part of longer introns, proximately to 5’-splice sites. These intronic m6A deposition correlates with both Rbm15 binding and H3K36me3. Moreover, coupled 4sU-seq with METTL3 dTAG system in mESC, we found that m6A mediates alternative exon/intron inclusive in nascent transcriptome. Intriguingly, m6A self-regulation including writers and readers are early response and partially contributed by alternative splicing changes. Collectively, our study presents a unified model that m6A mediates alternative splicing, and dTAG METTL3 opens an avenue to interrogate the direct response of functional m6A disruption.

2020-09-15 | GSE154705 | GEO

m6A RNA modification controls cell fate transition in mouse embryonic stem cells (gene expression analysis)

Project description:Here we determine the map of RNA methylation (m6A) in mouse embryonic stem cells, and embryoid bodies. Gene expression analysis: To establish the impact of METTL3 loss of function on gene expression in mESC. RNA hybridized to GeneChip Mouse Exon 1.0 ST Array

2014-10-16 | E-GEOD-52680 | biostudies-arrayexpress

N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome

Project description:N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome

| PRJNA647274 | ENA

N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]

Project description:N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]

| PRJNA647276 | ENA

RNA methylation destabilizes developmental regulators in murine embryonic stem cells (MoGene-2)

Project description:Recent methylome studies have located N6-methyladenosine (m6A) RNA modification on thousands of mammalian transcripts. However, its functional mechanism remains unclear. In this study, we examined the role of m6A methylation in mouse embryonic stem cells. To gain an understanding of dynamic changes in cells depleted with (proposed) methyltranferases at molecular level, we examined the time-series gene expression pattern in mESC lines using microarray analysis. After 0, 4 and 8 h incubation with scramble, shRNA for Mettl3 or Mettl14, mESC cells were collected and RNAs were isolated for microarray analysis using Affymetrix Genechip Mouse Gene 2.0 ST array. After 0 h incubation with scramble, shRNA for Mettl3 or Mettl14, mESC cells were collected and RNAs were isolated for microarray analysis using Affymetrix Genechip Mouse Gene 2.0 ST array.

2014-01-06 | E-GEOD-46879 | biostudies-arrayexpress

Depletion of m6A methylation by conditional Mettl3 knockout in Ascl1-induced neurons

Project description:We have perturbed RNA methylation machinery and investigated the change in subcellular localization of mRNA in Ascl1-induced neurons (iNeurons). Mutant iNeuron line harbouring tagged endogenous Mettl3 was generated which allowed for the selective and targeted depletion of Mettl3 protein with the addition of dTAG. Cells were treated with dTAG for 40 hours then separated into compartments (neurites and soma) and then sequenced in parallel with samples which received no dTAG treatment.

2023-07-12 | E-MTAB-9643 | biostudies-arrayexpress

m6A-seq profiling in Mettl3 Ctrl and dTAG13 mESCs [m6Aseq]

Project description:dTAG-13 treated METTL3_FKBP12(F36V) is sufficient to functionally deplete m6A methylation, that catalysed by METTL3/14 complex.

2020-09-15 | GSE154707 | GEO

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

Regulation of pre-mRNA splicing by m6A through the low-complexity protein hnRNPG and co-transcriptional pausing

Project description:Co-transcriptional processing of nascent transcripts is coupled with transcription through the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). The mRNA modification N6-methyladenosine (m6A) occurs co-transcriptionally and impacts pre-mRNA processing. How alternative splicing of pre-mRNA is co-transcriptionally regulated in an m6A-dependent manner is not well understood. Furthermore, splicing regulation through RNAPII pausing has been frequently suggested but the underlying control mechanism remains unclear. Here, we show that the m6A reader protein hnRNPG directly binds to the phosphorylated CTD of RNAPII using Arg-Gly-Gly (RGG) motifs in its low-complexity region. This RGG-phospho-CTD interaction and nascent RNA binding by hnRNPG enables its co-transcriptional association with RNAPII, resulting in transcriptome-wide regulation of alternative splicing and transcript abundance. m6A sites near exon splice sites in nascent mRNA further modulate hnRNPG binding and exon splicing. Exon inclusion is associated with RNAPII pausing downstream of the m6A site. Our results reveal an integrated nuclear mechanism of m6A-mediated gene regulation, in which an m6A reader protein regulates gene expression by using RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA, while the interplay between hnRNPG binding and RNAPII pausing modulates alternative splicing regulation.

2019-09-05 | GSE114311 | GEO

Dynamic Control of Chromatin-associated m6A Methylation Regulates Nascent RNAs Stability [ChIP-seq Dm]

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 | GSE186853 | GEO

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