Abstract
Many high-throughput loss-of-function analyses of the eukaryotic cell cycle have relied on the unicellular yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. In multicellular organisms, however, additional control mechanisms regulate the cell cycle to specify the size of the organism and its constituent organs1. To identify such genes, here we analysed the effect of the loss of function of 70% of Drosophila genes (including 90% of genes conserved in human) on cell-cycle progression of S2 cells using flow cytometry. To address redundancy, we also targeted genes involved in protein phosphorylation simultaneously with their homologues. We identify genes that control cell size, cytokinesis, cell death and/or apoptosis, and the G1 and G2/M phases of the cell cycle. Classification of the genes into pathways by unsupervised hierarchical clustering on the basis of these phenotypes shows that, in addition to classical regulatory mechanisms such as Myc/Max, Cyclin/Cdk and E2F, cell-cycle progression in S2 cells is controlled by vesicular and nuclear transport proteins, COP9 signalosome activity and four extracellular-signal-regulated pathways (Wnt, p38βMAPK, FRAP/TOR and JAK/STAT). In addition, by simultaneously analysing several phenotypes, we identify a translational regulator, eIF-3p66, that specifically affects the Cyclin/Cdk pathway activity.
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Acknowledgements
We thank the Berkeley Drosophila Genome Project, L. Lum and P. A. Beachy for constructs and reagents; T. Mäkelä, M. Laiho, M. Bonke and P. Ojala for critical review of the manuscript, and R. Medema for discussion. This work was supported by the Centre of Excellence in Translational Genome-Scale Biology of the Academy of Finland, Biocentrum Helsinki, University of Helsinki, Sigrid Jusélius Foundation, Finnish Cultural Foundation, Maud Kuistila Foundation and Finnish Cancer Research Organizations.
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Supplementary information
Supplementary Methods
This file contains additional methods used in the study.
Supplementary Table 1
This file contains the identified components of pathways regulating cell cycle.
Supplementary Tables
This file contains Supplementary Tables 2–13.
Supplementary Figure Legends
This file contains text to accompany Supplementary Figures 1–12.
Supplementary Figure 1
An overview of the flow cytometry screen.
Supplementary Figure 2
Supplementary Figure 2 nature04469-s06.eps Micrographs from cells treated with dsRNAs targeting cell cycle-related genes.
Supplementary Figure 3
The flow cytometry data from kinase/phosphatase screen with standard errors from individual samples.
Supplementary Figure 4
The a) kinase and b) phosphatase dendrograms.
Supplementary Figure 5
Compares our data with the data of Bettencourt-Dias et al.
Supplementary Figure 6
The G1 and G2 phenotype of the samples in DGC screen.
Supplementary Figure 7
The correlation between cell size at G1 and G2 phases.
Supplementary Figure 8
This figure shows a) the apoptosis/cell death scores in DGC screen, b) representative micrographs from cells treated with dsRNA causing cell death, c). over-4N DNA content of treated cells from the DGC screen identifying cytokinesis and DNA replication defects, d) representative micrographs from cells treated with dsRNA causing over-4N DNA content.
Supplementary Figure S9
This figure shows the comparison of microscopy and MTS assay with flow cytometry.
Supplementary Figure 10
Analysis of yeast-two hybrid and genetic interactions in various RNAi screens.
Supplementary Figure 11
This file depicts western blots showing the effectiveness of RNAi.
Supplementary Figure S12
This file contains analysis of 'hit-rate' in selected protein complexes/pathways.
Supplementary Figure 13
Unsupervised hierarchical clustering of all the genes identified in DGC screen.
Supplementary Figure 14
This figure displays the expression pattern of the identified genes during Drosophila life cycle. Cyclin E, E2f and eIF-3p66 are indicated with an arrow.
Supplementary Figure 15
This figure shows the pathways regulating cell cycle in S2 cells.
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Björklund, M., Taipale, M., Varjosalo, M. et al. Identification of pathways regulating cell size and cell-cycle progression by RNAi. Nature 439, 1009–1013 (2006). https://doi.org/10.1038/nature04469
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DOI: https://doi.org/10.1038/nature04469
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