Unique nascent transcriptomes define naïve, primed and paused embryonic pluripotent states.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA707057 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13866843_1.fastq.gz | Fastqsanger.gz | |||
| SRR13866843_2.fastq.gz | Fastqsanger.gz | |||
| SRR13866844_1.fastq.gz | Fastqsanger.gz | |||
| SRR13866844_2.fastq.gz | Fastqsanger.gz | |||
| SRR13866845_1.fastq.gz | Fastqsanger.gz |
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Project description:Unique transcriptomes define naïve, primed and paused embryonic pluripotent states. Here we perform calibrated transient transcription sequencing (TT-seq) to de novo define and quantify coding and non-coding transcription units (TUs) in different pluripotent states. We observe a global reduction of RNA synthesis, total RNA amount and turnover rates in ground state (2i) and paused pluripotency (mTORi). We demonstrate that elongation speed can be reliably estimated from TT-seq nascent RNA and RNA Polymerase II occupancy levels, and observe a transcriptome-wide attenuation of elongation speeds in these two inhibitor-induced states. Comparing closely related transcriptional states with different elongation speeds, we also discover a relationship between elongation speed and termination read-through distance. Our analysis suggests that steady-state transcriptomes in mouse ESC cells are controlled predominantly on the level of RNA synthesis and signaling pathways governing different pluripotent states directly control key parameters of transcription.
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Project description:RNAP II is frequently paused near gene promoters in mammals and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes. SR ChIP-seq, Pol II ChIP-seq, and Gro-seq
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Project description:RNAP II is frequently paused near gene promoters in mammals and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes.
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Project description:Translational control plays a central role in regulation of gene expression and can lead to significant divergence between mRNA- and protein-abundance. The translational landscape of early mammalian development and its impact on cellular proteome, however, remains largely un-explored. Here we used genome-wide approaches combined with time-course analysis to measure the mRNA-abundance, mRNA-translation rate and protein expression during the transition of naïve into primed embryonic stem cells (ESCs). We found that the ground state ESCs cultured with GSK3- and MEK-inhibitors and LIF (2iL) display higher ribosome density on a selective set of mRNAs. These mRNAs show reduced translation during the exit from ground state pluripotency and transition to serum/LIF (SL) culture or upon commitment to primed epiblast-like stem cells (EpiLSCs). Strikingly, integrative analysis with cellular proteome indicate a strong translational buffering of this set of mRNAs in 2iL-ESCs leading to stable protein expression levels. Our data reveal that the global alteration of cellular proteome is largely accompanied by transcriptional rewiring. Furthermore, we identified a set of genes (including UHRF1 and KRAS) that undergo selective post-translational regulation during the transition of naïve into primed pluripotency and linked the observed changes to upstream GSK- and MEK/MAPK-signaling pathways using single inhibitor treated ESCs. Thus, we provide a comprehensive and detailed overview of the global changes in gene expression during the transition of naïve to primed pluripotency and dissect the relative contributions of RNA-transcription, translation and regulation of protein stability in controlling protein abundance.
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Project description: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.
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