449A Poster - 05. Reproduction and gametogenesis
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Kwan Yin Lee; Elizabeth R. Gavis

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ

Keywords:
j. RNA binding proteins; u. RNA binding proteins

Mechanisms of mRNA regulation commonly involve the engagement of protein factors capable of influencing transcript biology. Proteins can engage with mRNAs in stable ribonucleoprotein complexes or form more fluid and dynamic structures by coalescing into phase-separated condensates. Currently, how the physical features of these complex structures are associated with mRNA regulation remains poorly understood. We are combining proximity-dependent labeling by APEX2 with the MS2/MCP system to spatially define the molecular environment proximal to specific mRNAs in the Drosophila oocyte. In late-stage oocytes, nanos mRNA is incorporated into phase transitioned condensates known as germ granules that are a conserved feature of germline development. The nanos mRNAs occupying germ granules form spatially distinct clusters within the granule compartment, each containing multiple copies of the mRNA. We aim to understand the role of these clusters in mRNA regulation by defining their surrounding molecular environment using the MS2-based APEX2 targeting system described here. To test the method, we have tethered MCP-APEX2 to nanos mRNA. APEX2 dependent biotinylation is detected together with nanos mRNA at multiple sites across Drosophila egg chambers. Biotinylated proteins nearby nanos mRNA will be isolated and identified by mass spectrometry.