Gummesson Bertil, Shah Shiraz Ali, Borum Alexander Skov, Fessler Mathias, Mitarai Namiko, Sørensen Michael Askvad, Svenningsen Sine Lo
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark.
Front Genet. 2020 Mar 5;11:144. doi: 10.3389/fgene.2020.00144. eCollection 2020.
cells respond to a period of famine by globally reorganizing their gene expression. The changes are known as the stringent response, which is orchestrated by the alarmone ppGpp that binds directly to RNA polymerase. The resulting changes in gene expression are particularly well studied in the case of amino acid starvation. We used deep RNA sequencing in combination with spike-in cells to measure global changes in the transcriptome after valine-induced isoleucine starvation of a standard K12 strain. Owing to the whole-cell spike-in method that eliminates variations in RNA extraction efficiency between samples, we show that ribosomal RNA levels are reduced during isoleucine starvation and we quantify how the change in cellular RNA content affects estimates of gene regulation. Specifically, we show that standard data normalization relying on sample sequencing depth underestimates the number of down-regulated genes in the stringent response and overestimates the number of up-regulated genes by approximately 40%. The whole-cell spike-in method also made it possible to quantify how rapidly the pool of total messenger RNA (mRNA) decreases upon amino acid starvation. A principal component analysis showed that the first two components together described 69% of the variability of the data, underlining that large and highly coordinated regulons are at play in the stringent response. The induction of starvation by sudden addition of high valine concentrations provoked prominent regulatory responses outside of the expected ppGpp, RpoS, and Lrp regulons. This underlines the notion that with the high resolution possible in deep RNA sequencing analysis, any different starvation method (e.g., nitrogen-deprivation, removal of an amino acid from an auxotroph strain, or valine addition to K12 strains) will produce measurable variations in the stress response produced by the cells to cope with the specific treatment.
细胞通过全面重组基因表达来应对一段时期的饥荒。这些变化被称为严谨反应，由直接结合RNA聚合酶的警报素ppGpp精心调控。在氨基酸饥饿的情况下，基因表达的相应变化得到了特别深入的研究。我们使用深度RNA测序结合掺入细胞来测量标准K12菌株在缬氨酸诱导异亮氨酸饥饿后转录组的全局变化。由于全细胞掺入方法消除了样品间RNA提取效率的差异，我们发现异亮氨酸饥饿期间核糖体RNA水平降低，并且我们量化了细胞RNA含量的变化如何影响基因调控的估计。具体而言，我们表明，依赖于样品测序深度的标准数据归一化低估了严谨反应中下调基因的数量，并高估了上调基因的数量约40%。全细胞掺入方法还使得量化氨基酸饥饿时总信使RNA（mRNA）池减少的速度成为可能。主成分分析表明，前两个成分共同描述了69%的数据变异性，强调了大型且高度协调的调控子在严谨反应中发挥作用。突然添加高浓度缬氨酸诱导饥饿引发了预期的ppGpp、RpoS和Lrp调控子之外的显著调控反应。这强调了这样一种观念，即由于深度RNA测序分析具有高分辨率，任何不同的饥饿方法（例如，氮剥夺、从营养缺陷型菌株中去除一种氨基酸或向K12菌株中添加缬氨酸）都会在细胞为应对特定处理而产生的应激反应中产生可测量的变化。
