Quantification of subcellular mRNA kinetics in mouse embryonic stem cells (flavopiridol)

In this work, we quantified mRNA flow rates between subcellular compartments in mouse embryonic stem cells. Combining metabolic RNA labeling, biochemical cell fractionation and RNA sequencing with mathematical modeling we were able to determine the kinetic rates of nuclear pre-, nuclear mature, cytosolic and membrane mRNA derived from more than 9000 protein-coding genes. For more than 5000 genes, we additionally estimated the transcript elongation rate. For most of the transcripts, mature nuclear half-lives are the longest, suggesting nuclear retention to be the rate-limiting step in the mRNA life cycle. Genes encoding transcription factors and immediate early genes possess fast kinetic rates, specifically a short nuclear half-life. Differentially localized mRNAs exhibit distinct combinations of rate constants, suggesting modular control within subcellular compartments. We show that membrane stability is high for membrane-localized mRNA and that cytosolic stability is high for cytosol-localized mRNA. Genes encoding target signals, such as signal peptides or transmembrane domains, have low cytosolic and high membrane half-lives with only slight differences between target signals. Nuclear-encoded mitochondrial proteins show long nuclear mature half-lives and otherwise similar features of cytoplasmic kinetics that do not resemble co-translational targeting to the mitochondria. Overall design: To obtain global insights into the nucleocytoplasmic kinetics of mRNA we conducted a time-resolved SLAM-seq experiment with subcellular fractionation in mouse embryonic stem cells (mESCs). To experimentally validate our metabolic labeling-derived subcellular flow rates with an orthogonal method, we inhibited transcription using flavopiridol followed by subcellular fractionation and RNA sequencing, resulting in a second set of subcellular rates (this repository). For the SLAM-seq dataset, please go to GSE252199. Three independently passaged biological replicates of mESCs (~3.5 × 107 cells per replicate) were cultured in “2i + LIF” ES medium supplemented with 1µM flavopiridol to block the transcription. Cells were harvested at 0, 30, 60, 120 and 180 min after addition of flavopiridol followed by cell fractionation as described above. Obtained fractions were mixed with Trizol LS and RNA was isolated following the manufacturer instructions. One microgram of total RNA was used as an input for TruSeq Stranded mRNA Library Prep Kit according to the instructions of the manufacturer. The multiplexed libraries were sequenced using HiSeq 4000 for pair-end 75 cycles. Reads were aligned with STAR (v2.7.6) and counted with RSEM (v1.3.3). Library sizes were normalized using genes with known half-lives greater than 14 hours (Herzog et al, 2017). Samples of each time series were further normalized to the corresponding t=0 time point. We fitted a simple exponential decay model per compartment for roughly 10,000 genes, giving the aggregated subcellular turnover rates, in contrast to the rates of transition from one compartment to the next.