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Genome-scale RNAi profiling of cell division in human tissue culture cells

Abstract

Cell division is fundamental for all organisms. Here we report a genome-scale RNA-mediated interference screen in HeLa cells designed to identify human genes that are important for cell division. We have used a library of endoribonuclease-prepared short interfering RNAs for gene silencing and have used DNA content analysis to identify genes that induced cell cycle arrest or altered ploidy on silencing. Validation and secondary assays were performed to generate a nine-parameter loss-of-function phenoprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by ascribing novel functions to genes in mitosis and cytokinesis. In particular, we identify two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin.

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Figure 1: Screening strategy.
Figure 2: High-throughput detection and reproducibility of cell cycle parameters by DNA content analysis.
Figure 3: Phenotypic profiling of cell cycle defects.
Figure 4: Profile analysis of cell division defects.
Figure 5: Mitotic phenotype of CASC5 depletion.
Figure 6: Characterization of LIN54 function in cell division.

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Acknowledgements

We thank S. Payne for support with laser scanning cytometry; E. Krausz for support with automated transfection; D. Pinchev for help with the CASC5 experiments; E. Tanaka, F. Stewart and W. Zachariae for critical reading of an earlier version of the manuscript; P. Goodwin and C. Brown for development of the DeltaVision/CellWorX screening platform; and all members of the Buchholz laboratory and our colleagues in Mitocheck and SMP-RNAi for discussions. This study was funded by the Max Planck Society, the Bundesministerium für Bildung und Forschung grant SMP-RNAi under the framework of NGFN-2 (01GR0402) and the EU-FP6 grant Mitocheck (LSHG-CT-2004-503464). During the course of this work R.K. and L.P. were supported by postdoctoral fellowships of the Human Frontier Science Program, and L.P. was supported by the Samuel Lunenfeld Research Institute.

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Contributions

R.K and L.P. contributed equally to this work. R.K., L.P. and F.B. performed project planning, experimental work, data analysis and wrote the manuscript. A.-K.H., M.S., M.T., L.M., I.P., S.L., H.G., K.K., J.W., V.S., C.R., W.B., A.L.J. and B.H. performed experimental work and data analysis. A.A.H. performed data analysis.

Corresponding author

Correspondence to Frank Buchholz.

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Kittler, R., Pelletier, L., Heninger, AK. et al. Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat Cell Biol 9, 1401–1412 (2007). https://doi.org/10.1038/ncb1659

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