Department of Molecular Life Sciences, University of Zurich, Zürich, Switzerland.
Institute of Neuropathology, University of Zurich, Zurich, Switzerland.
Sci Data. 2021 Jun 28;8(1):162. doi: 10.1038/s41597-021-00944-5.
Coordination of RNA abundance and production rate with cell size has been observed in diverse organisms and cell populations. However, how cells achieve such 'scaling' of transcription with size is unknown. Here we describe a genome-wide siRNA screen to identify regulators of global RNA production rates in HeLa cells. We quantify the single-cell RNA production rate using metabolic pulse-labelling of RNA and subsequent high-content imaging. Our quantitative, single-cell measurements of DNA, nascent RNA, proliferating cell nuclear antigen (PCNA), and total protein, as well as cell morphology and population-context, capture a detailed cellular phenotype. This allows us to account for changes in cell size and cell-cycle distribution (G1/S/G2) in perturbation conditions, which indirectly affect global RNA production. We also take advantage of the subcellular information to distinguish between nascent RNA localised in the nucleolus and nucleoplasm, to approximate ribosomal and non-ribosomal RNA contributions to perturbation phenotypes. Perturbations uncovered through this screen provide a resource for exploring the mechanisms of regulation of global RNA metabolism and its coordination with cellular states.
在不同的生物和细胞群体中，已经观察到 RNA 丰度和产生率与细胞大小的协调。然而，细胞如何实现这种与大小相关的转录“缩放”仍然未知。在这里，我们描述了一个全基因组 siRNA 筛选，以鉴定 HeLa 细胞中全局 RNA 产生率的调节剂。我们使用代谢脉冲标记 RNA 并随后进行高内涵成像来定量单个细胞的 RNA 产生率。我们对 DNA、新生 RNA、增殖细胞核抗原 (PCNA) 和总蛋白的单细胞测量，以及细胞形态和群体背景，捕获了详细的细胞表型。这使我们能够在扰动条件下解释细胞大小和细胞周期分布（G1/S/G2）的变化，这些变化间接影响全局 RNA 产生。我们还利用亚细胞信息来区分定位于核仁与核质中的新生 RNA，以近似核糖体和非核糖体 RNA 对扰动表型的贡献。通过这种筛选发现的扰动为探索全局 RNA 代谢的调节机制及其与细胞状态的协调提供了资源。
