Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells

Genome-Wide RNAi Analysis of Growth and Viability in

Drosophila CellsBoutros et al.

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 **

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

• 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.

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

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.

Genome-wide RNAi screen

• 5 days dsRNA treatment • Two embryotic hemocyte (blood cell) lines (Kc187 and S2R+)• 77,880 RNAi experiments

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

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

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

Frequency of Functional Groups

• Distribution of most abundant domain predicted gene functions differed with the quatitative severity of the RNAi phenotypes

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

Classification of Quantitative RNAi Phenotypes Cont’d

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

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

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

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