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 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1036-1040 |
| Number of pages | 5 |
| Journal | Nature |
| Volume | 432 |
| Issue number | 7020 |
| DOIs | |
| State | Published - Dec 23 2004 |
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Cite this
An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. / Kittler, Ralf; Putz, Gabriele; Pelletier, Laurence; Poser, Ina; Heninger, Anne Kristin; Drechsel, David; Fischer, Steffi; Konstantinova, Irena; Habermann, Blanca; Grabner, Hannes; Yaspo, Marie Laure; Himmelbauer, Heinz; Korn, Bernd; Neugenbaur, Karla; Pisabarro, Maria Teresa; Buchholz, Frank.
In: Nature, Vol. 432, No. 7020, 23.12.2004, p. 1036-1040.Research output: Contribution to journal › Article
}
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
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.
UR - http://www.scopus.com/inward/record.url?scp=19944367565&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=19944367565&partnerID=8YFLogxK
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 -