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Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets

Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets. Ulrike Eggert, Amy Kiger, Constance Richter, Zachary Perlman, Norbert Perrimon, Tomothy Mitchison, Christine Field Pat Ekpanyaskun Mar 3, 2005. Small molecule inhibitors

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Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets

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  1. Parallel Chemical Genetic and Genome-Wide RNAi Screens Identify Cytokinesis Inhibitors and Targets Ulrike Eggert, Amy Kiger, Constance Richter, Zachary Perlman, Norbert Perrimon, Tomothy Mitchison, Christine Field Pat Ekpanyaskun Mar 3, 2005

  2. Small molecule inhibitors directly inhibit protein fx. search by phenotypic screening -> i.d. targets / pathway binding of irrelevant prot. To study protein function

  3. RNA interference • RNAi • dsRNA targets destruction of RNA • inhibit prot. fx in a genome-wide basis

  4. Mitosis • daughter cells are separated by constriction of a cleavage furrow

  5. This paper Goal • Complete list of prot. required for cytokinesis in Drosophila cells Approach • Genome-wide RNAi screening & small molecule inhibitors

  6. dsRNA screening - >90% annotated genome dsRNAs - incubate ~4 d Small molecule screening - 51,000 inhibitors 2 d Screening Immunofluorescence • dsRNAs of 214 genes • 1 new gene (Borr/CG4454) • 50 inhibitors • (commercial lib, bioactive, nat • prod. extract)

  7. Predicted Functional Annotations of 214 Genes Associated with RNAi Binucleate Phenotypes

  8. 24% 32% 44% 6% 20% 74% Penetrance of binucleate phenotype 40% 28% Inhibitor RNAi 29% 51%

  9. 24% 40% 8% 28% 12% 29% 51% Phenotypic classes binucleate w large, diffuse DNA Binucleate Inhibitor RNAi Low cell count Low cell count Binucleate with MT extension

  10. RNAi inhibitors Two phenotypes correlate in both methods • dsRNA targeting Act5COR Cytochalasin D

  11. Two phenotypes correlate in both methods • Binucleate with diffuse DNA • Chromosomal passenger protein that co-localize with Aurora B throughout mitosis & cytokinesis

  12. Aurora B • Chromosomal passenger prot • Localize on centromeres (prophase-metaphase) • Relocalize to spindle midzone and cortex (anaphase) • Disruption causes loss of phosphorylation on histone H3 and failure in cytokinesis Andrews et al 2003

  13. Two phenotypes correlate in both methods • Chromosomal passenger protein that co-localize with Aurora B throughout mitosis & cytokinesis

  14. Borr/CG4454 co-localizes with Aurora B

  15. Borr is required for INCENP localizationAurora B RNAi and Binucleine 2 shares same phenotype and affect localization of INCENP Cells treated to aurora B dsRNA, borr/CG4454 dsRNA or Binucleine 2 results in an absence of H3

  16. Summary • i.d. new prot. involved in cytokinesis and new small molecule that inhibits it • 214 prot. important for cytokinesis in Drosophila • Borr/CG4454 is a chromosomal passenger prot that co-localize with Aurora B • Depletion had a profound effect on cytokinesis • Binucleine 2 as a cancer drug ?

  17. Message • Integration of data from different tools to answer biological questions • Subjective to visual inspection Comment

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