title: An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.
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.
authors.affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Kittler
authors.forename: Gabriele
authors.initials: G
authors.lastname: Putz
authors.forename: Laurence
authors.initials: L
authors.lastname: Pelletier
authors.forename: Ina
authors.initials: I
authors.lastname: Poser
authors.forename: Anne-Kristin
authors.initials: AK
authors.lastname: Heninger
authors.forename: David
authors.initials: D
authors.lastname: Drechsel
authors.forename: Steffi
authors.initials: S
authors.lastname: Fischer
authors.forename: Irena
authors.initials: I
authors.lastname: Konstantinova
authors.forename: Bianca
authors.initials: B
authors.lastname: Habermann
authors.forename: Hannes
authors.initials: H
authors.lastname: Grabner
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Yaspo
authors.forename: Heinz
authors.initials: H
authors.lastname: Himmelbauer
authors.forename: Bernd
authors.initials: B
authors.lastname: Korn
authors.forename: Karla
authors.initials: K
authors.lastname: Neugebauer
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Pisabarro
authors.forename: Frank
authors.initials: F
authors.lastname: Buchholz
mesh_terms: 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
chemical_list: D034741:RNA, Small Interfering; D004722:Endoribonucleases