Protein Blotting Guide

Abstract

To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non‐perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half‐lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress‐responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress‐induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA‐derived mRNA synthesis rates identified 16 stress‐specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.Visual OverviewSynopsisNascent transcriptome analysis reveals dynamics of mRNA synthesis and decay in yeast.The first step in the expression of the genome is the synthesis of messenger‐RNA (mRNA). In all cells, the regulation of mRNA levels in response to changing environmental conditions is a fundamental process. Classical methods to study such changes in mRNA levels, however, fail to unravel whether such changes are due to changes in mRNA synthesis (transcription) or changes in mRNA decay, which both contribute to setting mRNA levels. Therefore, the regulation of mRNA stability and turnover is poorly understood, and new methods for a quantitative analysis of mRNA synthesis and decay are urgenlty sought.In this study, we describe a novel method termed dynamic transcriptome analysis (DTA), which can be used to determine synthesis and decay rates of mRNAs on a genome‐wide level in yeast and other eukaryotic cells. We applied DTA to the model organism Saccharomyces cerevisiae and analyzed the dynamics of the transcriptome under standard growth conditions as well as under osmotic stress conditions. DTA relies on a combination of biochemistry, high‐throughput data acquisition, and computational biology. It uses metabolic labeling of newly synthesised RNA with the nucleoside analogon 4‐thiouridine (4sU), purification of labeled, newly synthesized RNA, and subsequent microarray hybridization. An improved mathematical model enables synthesis and decay rates of esentially all mRNAs in the cell to be determined with accuracy.In this study, we found that under normal growth conditions the synthesis rates for most mRNAs are low and that the decay rates are not correlated with synthesis. Addition of salt to the culture, however, induced three phases of changes in mRNA synthesis and decay. During the initial shock phase, there is a global repression of synthesis and a reduction of decay of most mRNAs. The subsequent induction phase involves strongly increased synthesis of stress mRNAs, which are also destabilized. Finally, the recovery phase restores decay rates, but leaves synthesis rates altered, apparently to allow for cellular growth under the new conditions.DTA shows a higher sensitivity and better temporal resolution than classical methods such as transcriptomics. Also, DTA is non‐perturbing and allows for an unbiased monitoring of genomic regulatory systems in living cells. Previously used methods are invasive and likely alter cellular physiology and thereby mRNA dynamics. DTA has a high potential to become a standard technique in molecular biology that may replace standard transcriptomics to study gene regulatory systems. In the future, DTA may be used to study dynamic changes in cellular mRNA metabolism induced by chemical inhibitors or defined mutations or changes in the environment.Rates of mRNA synthesis and decay can be measured on a genome‐wide scale in yeast by dynamic transcriptome analysis (DTA), which combines non‐perturbing metabolic RNA labeling with dynamic kinetic modeling.DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half‐lives range around a median of 11 min.DTA realistically monitors the cellular response to osmotic stress with higher sensitivity and temporal resolution than transcriptomics, and can be used to follow changes in RNA metabolism in gene regulatory systems.