TY - JOUR
T1 - An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division
AU - Kittler, Ralf
AU - Putz, Gabriele
AU - Pelletier, Laurence
AU - Poser, Ina
AU - Heninger, Anne Kristin
AU - Drechsel, David
AU - Fischer, Steffi
AU - Konstantinova, Irena
AU - Habermann, Blanca
AU - Grabner, Hannes
AU - Yaspo, Marie Laure
AU - Himmelbauer, Heinz
AU - Korn, Bernd
AU - Neugenbaur, Karla
AU - Pisabarro, Maria Teresa
AU - Buchholz, Frank
N1 - Funding Information:
Acknowledgements We thank M. Miwa and H. Satake for technical assistance. We also thank S. Sugano for donation of the pEF321-T plasmid; K. Ono and K. Tanaka for histological examination of the brain; M. Tamagawa for instruction in electrocardiogram recording; and S. Nishio, N. Tsunekawa and M. Terai for discussions. Amino acid measurements were carried out with the aid of the Center for Analytical Instruments at the National Institute for Basic Biology. This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Funding Information:
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.
PY - 2004/12/23
Y1 - 2004/12/23
N2 - 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 molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. 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 silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs 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.
AB - 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 molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. 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 silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs 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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U2 - 10.1038/nature03159
DO - 10.1038/nature03159
M3 - Article
C2 - 15616564
AN - SCOPUS:19944367565
VL - 432
SP - 1036
EP - 1040
JO - Nature
JF - Nature
SN - 0028-0836
IS - 7020
ER -