Abstract
The adult Drosophila intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. Here, we describe a new proximity-based profiling method that relies upon a GAL4 driver, termed intestinal-kickout-GAL4 (I-KCKT-GAL4), that is exclusively expressed in intestinal progenitor cells. This method uses UV crosslinked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by I-KCKT-GAL4 When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identifies 98.8% of transcripts found after progenitor cell sorting, and has low background noise despite being derived from whole animal lysate. We also mapped the targets of the more selective RNA binder, Fragile X mental retardation protein (FMRP), using enhanced crosslinking and immunoprecipitation (eCLIP), and report for the first time its binding motif in Drosophila cells. This method will therefore enable the RNA profiling of wild-type and mutant intestinal progenitor cells from intact flies exposed to normal and altered environments, as well as the identification of RNA-protein interactions crucial for stem cell function.
Keywords:
CLIP; FMRP; Intestinal stem cell; PABP; eCLIP.