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An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

Nature volume 432, pages 1036–1040 (2004)Cite this article

Abstract

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules1,2,3,4. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions5,6,7,8. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing9,10,11. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs12 from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

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Figure 1: Library generation and screening strategy.
Figure 2: Cell division phenotypes visualized by videomicroscopy.
Figure 3: Spindle and cytokinesis defects observed for RNAi phenotypes.
Figure 4: Efficiency and specificity of esiRNA.

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Acknowledgements

We thank F. Stewart, I. Baines, T. Hyman and M. Slabicki for critical reading and comments on the manuscript. We thank K. Weis for helpful discussions. We are grateful to M. Boutros for providing protein accession numbers and sequences of the cell viability screen in Drosophila cells. This study was supported by the Max Planck Society and by the EU-FP6 grant Mitocheck. L.P. is supported by a postdoctoral fellowship from the HFSP.

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Author notes

  1. Maria Teresa Pisabarro

    Present address: TU Dresden, Biotechnologisches Zentrum, Tatzberg 47-51, D-01307, Dresden, Germany

Authors and Affiliations

  1. Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307, Dresden, Germany

    Ralf Kittler, Gabriele Putz, Laurence Pelletier, Ina Poser, Anne-Kristin Heninger, David Drechsel, Steffi Fischer, Irena Konstantinova, Hannes Grabner, Karla Neugebauer, Maria Teresa Pisabarro & Frank Buchholz

  2. Scionics Computer Innovation, GmbH, Pfotenhauerstrasse 110, D-01307, Dresden, Germany

    Bianca Habermann

  3. Max Planck Institute for Molecular Genetics, Ihnestrasse 73, D-14195, Berlin-Dahlem, Germany

    Marie-Laure Yaspo & Heinz Himmelbauer

  4. RZPD-Ressourcenzentrum für Genomforschung, Im Neuenheimer Feld 506, D-69120, Heidelberg, Germany

    Bernd Korn

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Supplementary information

Supplementary Information

Supplementary Data, Supplementary Methods, Supplementary Tables 1–3, Supplementary Figure Legends 1–4 and references. (DOC 72 kb)

Supplementary Figure 1

Mitotic indices for 29 genes identified to cause a mitotic arrest. (PDF 349 kb)

Supplementary Figure 2

Nuclear localization and domain structure of KIAA1387. (JPG 169 kb)

Supplementary Figure 3

Effect on cell viability of 26 esiRNAs targeting genes encoding ribosomal proteins. (PDF 287 kb)

Supplementary Figure 4

Sequence analysis of RPS4Y and RPS4X. (PDF 101 kb)

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Kittler, R., Putz, G., Pelletier, L. et al. An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036–1040 (2004). https://doi.org/10.1038/nature03159

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