genome-wide rnai analysis of growth and viability in drosophila cells
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Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells. Boutros et al. Overview. Aim : functional analysis of predicted genes after whole genome sequencing Application : RNAi screen* Quatitative assay of cell number** Results : - PowerPoint PPT PresentationTRANSCRIPT
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Genome-Wide RNAi Analysis of Growth and Viability in
Drosophila CellsBoutros et al.
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Overview
• Aim: – functional analysis of predicted genes after whole genome sequencing
• Application: – RNAi screen*– Quatitative assay of cell number**
• Results: – characterize the function of 91% of predicted Drosophila genes in
cell growth and viability*– Identify genes of known and uncharacterized functions
demonstrate the role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival **
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Drosophila
• Model organism • Studies
– Development– Cell Biology– Population genetics– Signal transduction– Gene regulation and function
• Conserved pathways with important roles from flies to humans
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RNA interference (RNAi)
• Use for: – Idetification of gene
function and respective protein
• Drosophila cells– dsRNA treatment lead to:
• Depletion of corresponding transcript
• Loss-of-function phenotypic analysis
• Faster and effective way to turn off genes.– Development of new drugs
capable of turning off disease causing genes
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• 21,306 primers pairs used for:– Amplification of gene-specific fragments used for synthesis of dsRNAs
• dsRNA library targets all genes in the Drosophila genome
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Experimental approach for genome-wide RNAi screens.
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Quantitative Assay
• Reduction of signal to dying cells
• RNAi of the D. melanogaster apoptosis 1 inhibitor D-IAP1
• Left: Luciferous activity indicate ATP levels correlated with the number of Drosophila cells
• Right: Treatment of green fluorescencent protein (gfp) dsRNA targeting D-IAP1 induced time-dependent decrease in cell viability
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Flourescence Microscopy
• Cells after 3 days RNAi
• Treatment of D-IAP1 RNAi compared to control dsRNAs
• More dying cells with D-IAP1 by the ratio of SYTOX green-labeled nuclei vs. Hoeschst 33342-labeled nuclei.
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Genome-wide RNAi screen
• 5 days dsRNA treatment • Two embryotic hemocyte (blood cell) lines (Kc187 and S2R+)• 77,880 RNAi experiments
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Reproducibility
• Screens reproducible
• Two independent RNAi screen
• Phenotypes with similar z scores
• Correlation coefficient=0.86
• Z score- severity or rank of specific RNAi phenotypes
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Quantitative RNAi phenotypes of genes
• Gray bars- averaged RNAi phenotypes of 72 genes encoding all annotated ribosomal proteins tested
• White bars- gfp dsRNA are the negative controls
• Black bars- D-IAP1 dsRNA are the more severe phenotypes. Used as the positive control
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Frequency of RNAi Phenotypes
• Reduced cell number – Z greater of equal to 3– Threshold shows severity of z
score• Defects
– cell growth– cell survival
• 20% of identified genes has associated mutant alleles with Drosophila– Roles in:
• Cell growth• Cell cycle • Anti-apoptotic cell survival
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Frequency of Functional Groups
• Distribution of most abundant domain predicted gene functions differed with the quatitative severity of the RNAi phenotypes
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Classification of Quantitative RNAi phenotypes
• Duplicate screens per cell types
• Identification of:
– Serpent, srp
– CG11700-ubiquitin-like gene• Protein degradation
– CG15455- AML1-like Transcription factor
• Acute myeloid leukemia gene
– Oncogene
– Encodes transcriptional factors
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Classification of Quantitative RNAi Phenotypes Cont’d
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Flow Cytometry Analysis
• Scans single cells flowing past excitation sources in a liquid medium
• Measure fluorescence intensity produced by fluorescent-labeled antibiodies and ligands that bind specific cell-associated molecules
• Propidium Iodide stained DNA after 3 days RNAi
• Decrease cell size and DNA content
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Proportion of Apoptotic Cells (TUNEL)
•Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL)
•Terminal Transerase labeled DNA breaks
• Severe RNAi phenotypes distinguished with dsRNA treatment
• 95% treated with dsRNa to CG11700 or DIAP1
• 20% treated with dsRNA to CG15455
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Epistasis Analysis
• Pan-caspase inbitor (z-VAD-fmk) reverted cell death in response to
•RNAi of D-IAP1 and CG11700
•CG15455 and other transcription factor at a lesser extent
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Results
• Proteome comparison– Percentage of ortholog found for the genes with RNAi viability
phenotypes was High
– 50 genes had homology to human disease genes
– 10 genes implicated in blood-cell leukemia (AML-1)
– Genes with antiapopototic functions (FOXOA1 AND MLK)
• CG11700 may act in the same pathway as D-IAP1
– Directly preventing Nc caspase-activated apoptotic cell death.
• CG15455 and set of TFs may regulate complex responses for cell fate, proliferation, and/or cell survivial