Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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


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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others


  1. Lum, L. et al. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045 (2003)

    Article  ADS  CAS  Google Scholar 

  2. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003)

    Article  ADS  CAS  Google Scholar 

  3. Ashrafi, K. et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268–272 (2003)

    Article  ADS  CAS  Google Scholar 

  4. Brummelkamp, T. R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002)

    Article  ADS  CAS  Google Scholar 

  5. Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J. & Conklin, D. S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002)

    Article  CAS  Google Scholar 

  6. Hannon, G. J. RNA interference. Nature 418, 244–251 (2002)

    Article  ADS  CAS  Google Scholar 

  7. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001)

    Article  ADS  CAS  Google Scholar 

  8. Sherr, C. J. The ink4a/ARF network in tumour suppression. Nature Rev. Mol. Cell Biol. 2, 731–737 (2001)

    Article  ADS  CAS  Google Scholar 

  9. Brummelkamp, T., Bernards, R. & Agami, R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243–247 (2002)

    Article  CAS  Google Scholar 

  10. Brummelkamp, T. R., Nijman, S. M., Dirac, A. M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003)

    Article  ADS  CAS  Google Scholar 

  11. Semizarov, D. et al. Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl Acad. Sci. USA 100, 6347–6352 (2003)

    Article  ADS  CAS  Google Scholar 

  12. Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21, 635–637 (2003)

    Article  CAS  Google Scholar 

  13. Kamijo, T. et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91, 649–659 (1997)

    Article  CAS  Google Scholar 

  14. Lakin, N. D. & Jackson, S. P. Regulation of p53 in response to DNA damage. Oncogene 18, 7644–7655 (1999)

    Article  CAS  Google Scholar 

  15. El-Deiry, W. S. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993)

    Article  CAS  Google Scholar 

  16. Brugarolas, J. et al. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377, 552–557 (1995)

    Article  ADS  CAS  Google Scholar 

  17. Waldman, T., Kinzler, K. W. & Vogelstein, B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res. 55, 5187–5190 (1995)

    CAS  PubMed  Google Scholar 

  18. Brown, J. P., Wei, W. & Sedivy, J. M. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277, 831–834 (1997)

    Article  CAS  Google Scholar 

  19. Miyashita, T. & Reed, J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293–299 (1995)

    Article  CAS  Google Scholar 

  20. Barak, Y., Juven, T., Haffner, R. & Oren, M. mdm2 expression is induced by wild type p53 activity. EMBO J. 12, 461–468 (1993)

    Article  CAS  Google Scholar 

  21. Kubbutat, M. H., Jones, S. N. & Vousden, K. H. Regulation of p53 stability by Mdm2. Nature 387, 299–303 (1997)

    Article  ADS  CAS  Google Scholar 

  22. Brummelkamp, T. R. & Bernards, R. New tools for functional mammalian cancer genetics. Nature Rev. Cancer 3, 781–789 (2003)

    Article  CAS  Google Scholar 

  23. Shoemaker, D. D., Lashkari, D. A., Morris, D., Mittmann, M. & Davis, R. W. Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy. Nature Genet. 14, 450–456 (1996)

    Article  CAS  Google Scholar 

  24. Brummelkamp, T. R. et al. TBX-3, the gene mutated in Ulnar-Mammary Syndrome, is a negative regulator of p19ARF and inhibits senescence. J. Biol. Chem. 277, 6567–6572 (2002)

    Article  CAS  Google Scholar 

  25. Kanda, T., Sullivan, K. F. & Wahl, G. M. Histone–GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377–385 (1998)

    Article  CAS  Google Scholar 

  26. Wang, A. H. et al. HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor. Mol. Cell. Biol. 19, 7816–7827 (1999)

    Article  CAS  Google Scholar 

  27. Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003)

    Article  CAS  Google Scholar 

  28. Heetebrij, R. J. et al. Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays. Chembiochem 4, 573–583 (2003)

    Article  CAS  Google Scholar 

  29. Trettel, F. et al. Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum. Mol. Genet. 9, 2799–2809 (2000)

    Article  CAS  Google Scholar 

  30. Dirac, A. M. & Bernards, R. Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53. J. Biol. Chem. 278, 11731–11734 (2003)

    Article  CAS  Google Scholar 

Download references


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).

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Roderick L. Beijersbergen or René Bernards.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing