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Identification of pathways regulating cell size and cell-cycle progression by RNAi

Nature volume 439, pages 1009–1013 (2006)Cite this article

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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Figure 1: Cell-cycle regulation by protein phosphorylation in S2 cells.
Figure 2: The DGC RNAi library screen.
Figure 3: Classification of genes identified in the DGC screen.

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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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Author notes

  1. Mikael Björklund and Minna Taipale: *These authors contributed equally to this work

Authors and Affiliations

  1. Molecular and Cancer Biology Program, University of Helsinki, PO Box 63 , Haartmaninkatu 8, FI-00014, Finland

    Mikael Björklund, Minna Taipale, Markku Varjosalo & Jussi Taipale

  2. High Throughput Center, Biomedicum Helsinki, University of Helsinki, PO Box 63, Haartmaninkatu 8, FI-00014, Finland

    Juhani Lahdenperä & Jussi Taipale

  3. Department of Molecular Medicine, National Public Health Institute, FI-00251, Helsinki, Finland

    Mikael Björklund, Minna Taipale, Markku Varjosalo, Juha Saharinen & Jussi Taipale

  4. Biomedicum Bioinformatics Unit, Biomedicum, FI-00251, Helsinki, Finland

    Juha Saharinen

Authors
  1. Mikael Björklund
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Correspondence to Minna Taipale or Jussi Taipale.

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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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