An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.
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.
Kittler
Ralf
R
Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
Putz
Gabriele
G
Pelletier
Laurence
L
Poser
Ina
I
Heninger
Anne-Kristin
AK
Drechsel
David
D
Fischer
Steffi
S
Konstantinova
Irena
I
Habermann
Bianca
B
Grabner
Hannes
H
Yaspo
Marie-Laure
ML
Himmelbauer
Heinz
H
Korn
Bernd
B
Neugebauer
Karla
K
Pisabarro
Maria Teresa
MT
Buchholz
Frank
F
D002455:Cell Division / Q000235:genetics*; D049109:Cell Proliferation; D002470:Cell Survival; D048749:Cytokinesis / Q000235:genetics; D004722:Endoribonucleases / Q000378:metabolism*; D015723:Gene Library; D020043:Genes, Essential / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D018715:Microscopy, Video; D008938:Mitosis / Q000235:genetics; D008969:Molecular Sequence Data; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000502:physiology
D034741:RNA, Small Interfering; D004722:Endoribonucleases