Abstract
The relative ease of mouse Embryonic Stem Cells (mESCs) culture and the potential of these cells to differentiate into any of the three primary germ layers: ectoderm, endoderm and mesoderm (pluripotency), makes them an ideal and frequently used ex vivo system to dissect how gene expression changes impact cell state and differentiation. These efforts are further supported by the large number of constitutive and inducible mESC mutants established with the aim of assessing the contributions of different pathways and genes to cell homeostasis and gene regulation. Gene product abundance is controlled by the modulation of the rates of RNA synthesis, processing, and degradation. The ability to determine the relative contribution of these different RNA metabolic rates to gene expression control using standard RNA-sequencing approaches, which only capture steady state abundance of transcripts, is limited. In contrast, metabolic labeling of RNA with 4-thiouridine (4sU) coupled with RNA-sequencing, allows simultaneous and reproducible inference of transcriptome wide synthesis, processing, and degradation rates. Here we describe, a detailed protocol for 4sU metabolic labeling in mESCs that requires short 4sU labeling times at low concentration and minimally impacts cellular homeostasis. This approach presents a versatile method for in-depth characterization of the gene regulatory strategies governing gene steady state abundance in mESC.
Highlights
Gene expression control is central to ensure appropriate responses to intrinsic and extrinsic cellular stimuli and cellular homeostasis
We conclude that 15 min of incubation with 200 μM 4sU is sufficient to label a sizable RNA fraction in mouse Embryonic Stem Cells (mESCs)
As previously reported (Marzi et al, 2016), following normalization, we find that 4sUrel.−enr is inversely correlated (R2 = 0.80) (Figure 2C) with previously published stabilities obtained using transcriptional inhibition in mESC (Sharova et al, 2009)
Summary
Gene expression control is central to ensure appropriate responses to intrinsic and extrinsic cellular stimuli and cellular homeostasis. The steady state abundance of RNA transcripts is controlled by the rates at which the gene is transcribed (transcription), processed (processing), and degraded (degradation). Understanding how these three RNA metabolic rates change in response to different cellular cues, is paramount for in-depth characterization of how gene expression regulation, in health and disease, contributes to the maintenance or changes in cell state during development and in adulthood
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