Drosophila miRNA affinity purification for cell-type and tissue-specific miRNA profiling. Amanda Thomas¹, Weimin Xiao², Cristian Coarfa³, Pei-Jung Lee¹, Esther Jung¹, Gregg Roman^2,4, Preethi Gunaratne², Herman Dierick^1,5. 1) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2) Department of Biology and Biochemistry, University of Houston, Houston, TX; 3) Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX; 4) Biology and Behavior Insitute, University of Houston, Houston, TX; 5) Department of Neuroscience, Baylor College of Medicine, Houston, TX.

   MicroRNA expression and function play a wide variety of roles throughout an organism, although little is known about the specificity of microRNAs (miRNAs) in any specific tissue or cell type. The highly heterogeneous and intermixed neuronal subtypes in the fly brain makes cell type specific miRNA analysis even more pertinent in understanding neuronal function. In miRNA biogenesis, Argonaut proteins associate with the fully mature, 21-23 nt miRNAs. A technique, called miRAP (miRNA affinity purification) has been developed in worms and mice and uses tagged components of the RNA induced silencing complex to co-immunoprecipitate mature miRNAs; however, no such system exists for cell-type specific miRNA profiling in flies. We have generated transgenic lines with random insertions of a UAS-Ago1::GFP fusion gene to examine the miRNA profiles of specific cell-types when combined with specific GAL4 drivers. Expression of UAS-Ago1::GFP with pan-neuronal driver Elav-GAL4 showed expected tissue wide protein localization of the GFP-tagged Ago1 in the cyptoplasm. Pull down experiments from head extracts of the Elav-Gal4>UASAgo1::GFP flies demonstrated a highly reproducible enrichment of a subset of miRNAs in neurons. We are currently using this technique to examine miRNA populations in specific neuronal subtypes with the goal of understanding which miRNAs are important in specific neurons. This newly adapted technology can be used in combination with any GAL4 driver line to examine cell-type specific miRNA expression under different mutant, physiological, and pharmacological conditions.