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 pathways 1 , 2 , 3 . In mammalian cells, such screens have been hampered by a lack of suitable t...

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Vydané v:	Nature Ročník 428; číslo 6981; s. 431 - 437
Hlavní autori:	Berns, Katrien, Hijmans, E. Marielle, Mullenders, Jasper, Brummelkamp, Thijn R., Velds, Arno, Heimerikx, Mike, Kerkhoven, Ron M., Madiredjo, Mandy, Nijkamp, Wouter, Weigelt, Britta, Agami, Reuven, Ge, Wei, Cavet, Guy, Linsley, Peter S., Beijersbergen, Roderick L., Bernards, René
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	London Nature Publishing Group UK 25.03.2004 Nature Publishing Nature Publishing Group
Predmet:	Biological and medical sciences Cell cycle Cell Division Cell Line, Tumor Cloning, Molecular Cyclin-Dependent Kinase Inhibitor p16 Cyclin-Dependent Kinase Inhibitor p21 Cyclins - genetics Cyclins - metabolism Down-Regulation Fibroblasts Fundamental and applied biological sciences. Psychology Gene expression Gene Library Genes. Genome Genetic Vectors - genetics Genetics Humanities and Social Sciences Humans letter Mammals Medical screening Molecular and cellular biology Molecular genetics multidisciplinary Reproducibility of Results Retroviridae - genetics Ribonucleic acid RNA RNA Interference RNA, Small Interfering - genetics RNA, Small Interfering - metabolism Science Science (multidisciplinary) shRNA Tumor Suppressor Protein p14ARF - metabolism Tumor Suppressor Protein p53 - genetics Tumor Suppressor Protein p53 - metabolism
ISSN:	0028-0836, 1476-4687, 1476-4687
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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 pathways 1 , 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 expression 4 , 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 p19 ARF -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.
AbstractList	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 pathways 1 , 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 expression 4 , 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 p19 ARF -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. 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 pathways. 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 expression. 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. [PUBLICATION 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 pathways. 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 expression. 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. 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 pathways. 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 expression. 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.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 pathways. 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 expression. 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. 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 pathways. 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 expression. 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 p19 super(ARF)-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.
Audience	Academic
Author	Berns, Katrien Mullenders, Jasper Agami, Reuven Beijersbergen, Roderick L. Madiredjo, Mandy Cavet, Guy Ge, Wei Hijmans, E. Marielle Weigelt, Britta Heimerikx, Mike Bernards, René Velds, Arno Kerkhoven, Ron M. Nijkamp, Wouter Linsley, Peter S. Brummelkamp, Thijn R.
Author_xml	– sequence: 1 givenname: Katrien surname: Berns fullname: Berns, Katrien organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 2 givenname: E. Marielle surname: Hijmans fullname: Hijmans, E. Marielle organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 3 givenname: Jasper surname: Mullenders fullname: Mullenders, Jasper organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 4 givenname: Thijn R. surname: Brummelkamp fullname: Brummelkamp, Thijn R. organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 5 givenname: Arno surname: Velds fullname: Velds, Arno organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 6 givenname: Mike surname: Heimerikx fullname: Heimerikx, Mike organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 7 givenname: Ron M. surname: Kerkhoven fullname: Kerkhoven, Ron M. organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 8 givenname: Mandy surname: Madiredjo fullname: Madiredjo, Mandy organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 9 givenname: Wouter surname: Nijkamp fullname: Nijkamp, Wouter organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 10 givenname: Britta surname: Weigelt fullname: Weigelt, Britta organization: Division of Experimental Therapy, The Netherlands Cancer Institute – sequence: 11 givenname: Reuven surname: Agami fullname: Agami, Reuven organization: Division of Tumor Biology, The Netherlands Cancer Institute – sequence: 12 givenname: Wei surname: Ge fullname: Ge, Wei organization: Rosetta Inpharmatics, Inc – sequence: 13 givenname: Guy surname: Cavet fullname: Cavet, Guy organization: Rosetta Inpharmatics, Inc – sequence: 14 givenname: Peter S. surname: Linsley fullname: Linsley, Peter S. organization: Rosetta Inpharmatics, Inc – sequence: 15 givenname: Roderick L. surname: Beijersbergen fullname: Beijersbergen, Roderick L. email: r.beijersbergen@nki.nl organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute – sequence: 16 givenname: René surname: Bernards fullname: Bernards, René email: r.bernards@nki.nl organization: Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute
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Snippet	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... 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...
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SubjectTerms	Biological and medical sciences Cell cycle Cell Division Cell Line, Tumor Cloning, Molecular Cyclin-Dependent Kinase Inhibitor p16 Cyclin-Dependent Kinase Inhibitor p21 Cyclins - genetics Cyclins - metabolism Down-Regulation Fibroblasts Fundamental and applied biological sciences. Psychology Gene expression Gene Library Genes. Genome Genetic Vectors - genetics Genetics Humanities and Social Sciences Humans letter Mammals Medical screening Molecular and cellular biology Molecular genetics multidisciplinary Reproducibility of Results Retroviridae - genetics Ribonucleic acid RNA RNA Interference RNA, Small Interfering - genetics RNA, Small Interfering - metabolism Science Science (multidisciplinary) shRNA Tumor Suppressor Protein p14ARF - metabolism Tumor Suppressor Protein p53 - genetics Tumor Suppressor Protein p53 - metabolism
Title	A large-scale RNAi screen in human cells identifies new components of the p53 pathway
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