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A large-scale RNAi screen in human cells identifies new components of the p53 pathway

Nature volume 428, pages 431–437 (2004)Cite this article

Abstract

RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signalling pathways1,2,3. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression4,5. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.

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Figure 1: RNAi library screen in conditionally immortalized cells.
Figure 2: Validation of new p53 pathway components.
Figure 3: Downregulation of p21 transcription.
Figure 4: Knockdown of p21cip1 is sufficient to bypass a p53-dependent arrest.
Figure 5: siRNA bar-code screens.

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Acknowledgements

We thank S. Friend and J. Downward for their support of this project, M. Voorhoeve, Z. Wu, X.-j. Yang, H. Yntema and Kreatech Biotechnology for reagents, the NKI microarray facility group for assistance, A. Dirac and S. Nijman for technical help, and members of the Bernards laboratory for discussions. This work was supported by grants from the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO), Cancer Research UK (CRUK), the Centre for Biomedical Genetics (CBG), the Dutch Cancer Society (KWF) and Utrecht University (ABC cluster).

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  1. Katrien Berns and E. Marielle Hijmans: These authors contributed equally to this work

Authors and Affiliations

  1. Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands

    Katrien Berns, E. Marielle Hijmans, Jasper Mullenders, Thijn R. Brummelkamp, Arno Velds, Mike Heimerikx, Ron M. Kerkhoven, Mandy Madiredjo, Wouter Nijkamp, Roderick L. Beijersbergen & René Bernards

  2. Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands

    Britta Weigelt

  3. Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands

    Reuven Agami

  4. Rosetta Inpharmatics, Inc., 12040 115th Avenue NE, Kirkland, Washington, 98034, USA

    Wei Ge, Guy Cavet & Peter S. Linsley

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Corresponding authors

Correspondence to Roderick L. Beijersbergen or René Bernards.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Schematic representation of the 59 nt oligonucleotides. (PPT 21 kb)

Supplementary Figure 2

Knockdown of endogenous gene expression. (PPT 27 kb)

Supplementary Table 1

Genes in the NKI RNAi library. (XLS 1082 kb)

Supplementary Table 2

Sequence information of identified shRNA vectors. (PDF 14 kb)

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Berns, K., Hijmans, E., Mullenders, J. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004). https://doi.org/10.1038/nature02371

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