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Combinatorial RNAi screens for cell growth and viability

Combinatorial RNAi screens for cell growth and viability. Co-RNAi of 84 protein and lipid phosphatases in Drosophila Map Interactions to identify phosphatases are involved in similar processes Scalability: pilot experiments for larger combinatorial screens. Types of interactions. A*B*. wt.

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Combinatorial RNAi screens for cell growth and viability

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  1. Combinatorial RNAi screens for cell growth and viability • Co-RNAi of 84 protein and lipid phosphatases in Drosophila • Map Interactions to identify phosphatases are involved in similar processes • Scalability: pilot experiments for larger combinatorial screens

  2. Types of interactions A*B* wt A* B* A*B* wt A* A B viability positive (synergistic) interaction X B negative (buffering) interaction viability A B* negative (suppressing) interaction viability B A A*B* wt A* B*

  3. Experimental set up Int Pos Neg P1 4 Plates 6 Plates P2 P3 . . . P1 P2 P3 . . . P – Neg P – Pos, P – Int Pi – Pj Pi – Pi 24 Plates

  4. Experimental set up Ready-to-screen assay plates Seed cells (10.000 – 20.000)‏ Transfection of dsRNAs Kc167 SL2 S2R+ 5 days incubation (ensure protein depletion)‏ Readout (single channel)‏

  5. Experimental set up • “all against all” • single knock-downs (PPX-Neg) • double knock-downs (PPX-PPX) • combination knock-downs (PPX-PPY) • possible findings • viability hits • control rescues • PPX_PPY interactions PP3 PP2 PP1 intermediate neg pos PP21 ………………………………………………………. PP1 PP2 PP3 … … … … … … … … … … … … … PP14 pos neg Plate 1 of 24

  6. Quality assessment PPX-PPY vs PPY-PPX Slope ~1 Outliers Systematic error

  7. Interaction estimates viability hits single effects synergistic interactions antagonistic interactions interactions

  8. Phosphatase-pair specific T-scores

  9. cluster of lipid phosphatases (CG11437, CG11438, wun)‏ interact with JNK signaling Related phosphatases share interaction profiles synergistic antagonistic interactions

  10. Networks based on synthetic interactions Limited number of PP with many interactions, e.g. puc PP1- a96 Expansion to larger network sizes, more complex phenotypes Application to RNAi-drug interactions

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