Project Summary/Abstract: TRIM-NHL proteins (named after their TRIpartite Motif and founding members NCL-1, HT2A, LIN-41) are essential for animal development and control of stem cell fate. Their dysfunction causes cancer, infertility, and neurological disorders including congenital hydrocephalus. The recent discovery that multiple TRIM-NHL proteins bind to mRNAs provided a crucial insight into their molecular functions. The long term goal of this research is to discover how TRIM-NHL proteins regulate gene expression to control crucial developmental processes. This proposal focuses on the Drosophila melanogaster TRIM-NHL protein, Brain Tumor (Brat), which binds to specific mRNAs and functions in neurogenesis, oogenesis, and embryogenesis. In stem cells, Brat suppresses stemness and promotes differentiation whereas loss-of-function leads to over-proliferation in the brain and germline. We pursue the central hypothesis that Brat negatively regulates gene expression by causing mRNA degradation and inhibiting translation to control stem cell fate. Our research plan will determine the effect of Brat on translation, mRNA decay, and stem cell fate during neurogenesis and oogenesis. A major strength of our work is that it integrates both molecular regulatory mechanisms of Brat and its biological functions at the organismal level. First, using quantitative assays that specifically measure Brat activity, we will dissect its repressive domains and identify the required pathways and corepressors in cultured cells. Second, we will identify the network of genes that are regulated by Brat. We will map Brat-binding sites across the transcriptome and measure Bratʹs effect on the abundance and translation status of expressed mRNAs. By integrating the resulting data, our data will provide a comprehensive view of the relationship between Brat-RNA occupancy, location of functional binding sites, and impact on mRNA stability and translation efficiency. Third, we use precision genome editing to interrogate the roles of RNA-binding and repression domains of the brat gene during neurogenesis and oogenesis in Drosophila. We also created tissue specific reporter gene assays that specifically measure Bratʹs repressive activity in stem cells. The results of this research will provide a mechanistic understanding of Brat mediated gene regulation and provide a global view of its impact on gene expression. Our results will establish the role of Brat’s mRNA regulatory activities in the control of the stem cell proliferation-differentiation axis. Brat serves as an archetype for the TRIM-NHL family, and our discoveries will also broadly enhance the understanding of TRIM-NHL protein functions in development and disease.