Developing Tools to Identify Novel Protein Components of Drosophila Germ Granules Using BioID

Abstract

During embryonic development, germ cells remain in a naïve state to maintain their ability to give rise to all cell types. RNAs necessary for preserving germ cell totipotency and for germ cell development and viability are incorporated into ribonucleoprotein (RNP) granules known as germ granules early in development. Germ granules consist of a protein scaffold to which RNAs bind. Some germ granule proteins in Drosophila melanogaster have been identified, including Oskar (Osk), the main organizer protein. We sought to identify novel germ granule proteins using BioID, a proximity-dependent labeling method. We expressed a fusion of EGFP-Osk to the promiscuous biotin ligase BirA* in Drosophila germ cells with the GAL4/UAS system in order to tag germ granule proteins with biotin. Two strategies were used to generate excess germ granules and maximize the yield of biotinylated proteins: expression of EGFP-Osk-BirA* ubiquitously and at the anterior. Although we identified a combination of maternal GAL4 drivers that produced comparable expression of EGFP-Osk-BirA* and the control protein EGFP-BirA* ubiquitously in ovaries, EGFP-Osk-BirA* was poorly expressed in embryos. Alternative constructs that produced anteriorly localized EGFP-Osk-BirA* or Osk-BirA* without the EGFP tag also did not exhibit comparable embryonic expression to EGFP-BirA*. Three constructs remain to be tested to optimize the BioID labeling method for use in Drosophila germ granules. As several known germ granule components are conserved from Drosophila to humans, identifying novel proteins and elucidating their function should further our understanding of the role that RNP granules play in germ cells across many eukaryotic species.