Deciphering the role of Tudor in the Drosophila Brain

Deciphering the Role of Tudor in the Drosophila BrainTudor (Tud) is an evolutionarily conserved molecular scaffold protein that contains 11 Tud domains that bind to symmetrically dimethylated arginine residues in protein targets, including Piwi protein Aubergine. Aubergine uses piRNAs, a distinct class of small ncRNAs, to target RNA retrotransposon intermediates for cleavage and degradation. Together the Tud-Aub-piRNA pathway is a pivotal part of the developmental program charged with protecting germ cell genomic integrity against mobile genetic elements. Although the role of Tud in the germline is well characterized, its function in the brain is completely unknown. Therefore, the goal of our research is to decipher the function of Tud in the fly brain. To determine if Tud contributes to the piRNA pathway, NGS (Next Generation Sequencing) was performed using wild-type control and tud mutant brains (no Tud is expressed). Preliminary data indicate that over 1400 piRNA species are differentially expressed in tud mutant flies. Additionally, over 300 of these piRNAs map to known genes. A further NGS project is ongoing to determine if mRNAs are being regulated by the differentially expressed piRNAs. In parallel to the sequencing projects, we are using the genetic engineering methodology, CRISPR/Cas9, to tag tud with GFP and HA peptide tags to determine the distribution of endogenous Tud in the brain. Furthermore, HA-Tud complexes and GFP-Tud complexes will be purified and subjected to mass spectrometry analysis to determine Tud interacting partners in the brain. Since we know the Tud interactome in the germline, this will shed light on similarities between the germline and brain.