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SRX15242298: GSM6134111: FBS, MeRIP input, rep3; Mus musculus; RIP-Seq
1 ILLUMINA (Illumina HiSeq 4000) run: 33.7M spots, 1.7G bases, 684.6Mb downloads

External Id: GSM6134111_r1
Submitted by: Ramalho-Santos lab, Lunenfeld-Tanenbaum Research Institute
Study: m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency [MeRIP-seq]
show Abstracthide Abstract
Development is often assumed to be a sequential unfolding of genetic programs towards increased complexity and occurring with a very stereotypical timing. However, embryos across all lineages of metazoans can enter reversible states of developmental pausing in response to adverse environmental conditions. In mammals, pausing manifests as a delayed implantation of the blastocyst, the source of embryonic stem cells (ESCs). Paused pluripotency can be induced in mouse blastocysts and ESCs by inhibition of mTOR, a conserved growth-promoting kinase, and is characterized by a drastic global decrease in biosynthetic activity, including gene transcription. The molecular mechanisms by which this remarkable dormant cellular state is achieved remain largely unknown. Here we show that m6A RNA methylation by Mettl3 is essential for transcriptional dormancy and maintenance of the paused pluripotent state in vitro and in vivo. Mass spectrometry and transcriptome-wide sequencing revealed an increase in m6A in paused ESCs. Knockout of the RNA methyltransferase Mettl3 suppresses transcriptional and proliferation dormancy of ESC specifically in the paused state, and leads to loss of paused blastocysts. Integration of several datasets in vitro and in vivo identified the transcriptional amplifier and oncogene Mycn as a key “anti-pausing factor” regulated by m6A mRNA methylation. Mett3-mediated methylation at a specific site of the Mycn mRNA is essential for its destabilization, which in turns mediates suppression of nascent transcription and proliferation in paused conditions. Our results highlight an intricate feedback between signaling, regulation of RNA stability and nascent transcription during pausing, with Mettl3 as an essential integrator and Mycn as a key downstream anti-pausing factor. These findings shed light on the mechanisms that underlie tuning of global transcriptional output during mammalian developmental pausing, and that may be redeployed in adult stem cells or during cancer dormancy. Overall design: Wildtype mESCs were cultured under control (FBS/LIF) or paused (200nM INK128) conditions for 2 weeks and m6A methylated RNA immunoprecipitation (MeRIP-seq) was performed.
Sample: FBS, MeRIP input, rep3
SAMN28204766 • SRS12975229 • All experiments • All runs
Organism: Mus musculus
Library:
Name: GSM6134111
Instrument: Illumina HiSeq 4000
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: mESCs were spiked with 2% of human cells (HeLa), then poly(A) RNA was extracted using the Magnetic mRNA Isolation Kit (NEB) with two rounds of enrichment. Immunoprecipitation of m6A methylated RNA (MeRIP) was done using the EpiMark® N6-Methyladenosine Enrichment Kit with 4µg spiked poly(A) RNA. MeRIP libraries were constructed from 0.5-1ng of input or IP RNA and prepared using the SMARTR-seq RNA library prep v2 kit (TakaraBio), per manufacturer's recommendations.
Runs: 1 run, 33.7M spots, 1.7G bases, 684.6Mb
Run# of Spots# of BasesSizePublished
SRR1917696933,745,0241.7G684.6Mb2023-05-18

ID:
21721094

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