Abstract
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.
Highlights
Background & SummaryCoordination of RNA transcript abundance with cell size has been observed in diverse organisms such as fission yeast, C. elegans, Xenopus, rat, mouse and human – both when comparing organs with differently sized cells[1], and when comparing individual cells in heterogeneous populations[2,3,4]
While the dataset presented here was primarily collected with the purpose of performing a detailed and comprehensive single-cell analysis of transcription rates, and their perturbation in human cells, the dataset contains single-cell-level information about cell and nuclear size and morphology, cell viability, cell-cycle stage, proliferating cell nuclear antigen (PCNA) abundance and localisation, nucleolar morphology, and cell population characteristics – all across perturbations at the genome-wide scale
The dataset is made available on the Image Data Resource (IDR, idr0093) which facilitates user-friendly access to the images and quantitative data derived from the images[11]
Summary
Coordination of RNA transcript abundance with cell size has been observed in diverse organisms such as fission yeast, C. elegans, Xenopus, rat, mouse and human – both when comparing organs with differently sized cells[1], and when comparing individual cells in heterogeneous populations[2,3,4]. To identify regulators of transcriptional scaling to cell size, we have conducted an arrayed image-based genome-wide siRNA screen with single-cell resolution in human HeLa cells. This dataset allows for the detection of perturbations that affect global transcriptional rates, while accounting for cell size and cell cycle stage. While the dataset presented here was primarily collected with the purpose of performing a detailed and comprehensive single-cell analysis of transcription rates, and their perturbation in human cells, the dataset contains single-cell-level information about cell and nuclear size and morphology, cell viability, cell-cycle stage, PCNA abundance and localisation, nucleolar morphology, and cell population characteristics – all across perturbations at the genome-wide scale. The dataset is made available on the Image Data Resource (IDR, idr0093) which facilitates user-friendly access to the images and quantitative data derived from the images[11]
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