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: –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

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 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 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 –CG 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