1. Project 3: Functional validation of ncRNAs Norbert Perrimon and Bernard Mathey-Prevot

2. The meaning of “Functional” 1. “being expressed significantly above the noise” 2. “serving a biological purpose in the context of where it is expressed” Functionally validate (through loss of function or gain of function studies) a subset of ncRNAs identified in the other Aims, using a battery of cell-based assays that we developed. We propose We propose to:

3. Synthesize dsRNAs or ncRNA expression constructs Array reagents into screening plate Validation and hit selection Process cells for screen read-out Use established high-throughput screening assays Data acquisition and storage into database Data Analysis and statistical treatment Meta-analysis of results General experimental flow-chart Plate cells for RNAi or overexpression treatment

4. Transcriptional-Luciferase Reporter Assays Protein modification (phospho-specific antibodies) Microscopy-based assays Plate reader-based assays GFP or antibodies DRSC screening platform for RNAi cell-based assays pros: Fast, numeric data, quantitative cons: Costly, limited in information pros: Feature rich, cheaper cons: Analysis can be challenging (Aerius) (Discovery-1 or Opera confocal) P Cell number 800 nm Fluorescence P-Akt level 700 nm Fluorescence Z-scores

5. Multimode plate reader Luminescence, fluorescence intensity, absorbance, FP, bidirectional stackers Barcode reader FITC, TRITC, CFP, YFP, Cy5 filter sets Li-COR Aerius scanning reader 2 color scanning (far red) for simultaneous detection and quantification (in-cell western) 20-500  m resolution Scans microplates or membranes Plate-reader and plate-reader/scanner

6. Fully automated confocal imaging and autofocus system 4 laser based excitation sources (405, 488, 532, 635 nm) Non-confocal Epi-Fluorescence Imaging Up to 4 independent CCD detectors Compatible with all plate types (24 to 2080 wells) High speed data acquisition (up to 100,000 image sets in 24 h) On board image analysis script library and analysis 20x (dry) and 40x, 60x water- immersion lenses HTS microscope Opera HTS confocal (Evotec)

7. DRSC Database and Website Internally developed LIMS suite to manage the inventory and QC of dsRNA libraries Metadata storage Tools and links for data normalization/analysis and for the processing of experimental data Database DRSC portal to the scientific community Resource for RNAi (protocols, screen data, etc.) Information on how to apply for a screen Tools and links related to RNAi and data mining Website (http://flyrnai.org)

8. 54 ncRNAs (hand-picked by J. Manak) 1 We were able to map 45 to unique sites Synthesized 1 or 2 dsRNA per ncRNA (72 total) Tested dsRNAs in the MAPK assay 2 (A. Friedman) 1 Manak et al, Nature Genet. (2006) 2 Friedman et al, Nature (2006)2 DRSC36511 targets ncRNA 2L16745000 ncRNA 2L16745000 MAPK Assay Measure amount of dp-Erk (relative to total Erk) after EGF stimulation in S2R+ cells treated with dsRNAs for 3 days Look NIH, we can do it…

9. Not so fast says Sue! ncRNA 2L16745000 is in fact the 3’-UTR of CG17912 CG17912 is a confirmed hit in the MAPK screen by Friedman & Perrimon

10. miR-315 is a potent and specific activator of Wg signaling in Drosophila Clone 8 cells Serena Silver, Joshua Hagen, Eric Lai