Unique nascent transcriptomes define naïve, primed and paused embryonic pluripotent states.. Unique nascent transcriptomes define naïve, primed and paused embryonic pluripotent states.

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. Overall design: TT-seq on mESC pluripotent states: SL, 2i, and mTORi. With both label RNA (LRNA) and fragmented total RNA (FRNA).

独特的转录组特征界定了幼稚态、始发态与停滞态的胚胎多能状态。本研究采用校准型瞬时转录测序（calibrated transient transcription sequencing, TT-seq），对不同多能状态下的编码与非编码转录单元（transcription units, TUs）进行从头定义与定量分析。研究发现，在基础态（2i）与停滞态多能性（mTORi）细胞中，RNA合成水平、总RNA总量及周转速率均呈现全局性下降。本研究证实，可通过TT-seq检测的新生RNA与RNA聚合酶II（RNA Polymerase II）富集水平，可靠估算转录延伸速率；同时发现，这两种抑制剂诱导的多能状态中，全转录组层面的转录延伸速率均出现衰减。通过比较延伸速率存在差异的紧密相关转录状态，本研究还揭示了延伸速率与终止通读距离之间的关联。分析结果表明，小鼠胚胎干细胞（embryonic stem cell, ESC）的稳态转录组主要受RNA合成水平调控，而调控不同多能状态的信号通路可直接控制转录的关键参数。实验设计概述：针对小鼠胚胎干细胞多能状态（SL、2i及mTORi）开展TT-seq检测，同时采集标记RNA（label RNA, LRNA）与碎片化总RNA（fragmented total RNA, FRNA）样本。