PROJECT SUMMARY / ABSTRACT Translation produces the proteins necessary for a cell’s function and relies on the ribosome, a highly conserved protein and RNA complex. Post-meiotic male germ cells are especially reliant on translation regulation, and consequently ribosome function. The long-term goal of this project is to determine the molecular mechanism by which ADAD1, a nuclear post-meiotic male germ cell-specific RNA binding protein, impacts translation. Previous work from our laboratory demonstrated Adad1 is required for male fertility as mutation leads to defects in post-meiotic germ cell differentiation. Although previously proposed as an RNA editing enzyme, our evidence demonstrates ADAD1 loss does not influence mRNA editing, making the molecular function of ADAD1 unknown. In preliminary data from our laboratory, we demonstrate ADAD1 loss leads to abnormal ribosome association of transcripts important for post-meiotic germ cell differentiation, reduced ribosomal RNA modification, and aberrant expression of ribosomal proteins and ribosome biogenesis factors. Together, these observations lead to the hypothesis that ADAD1 influences translation of post-meiotic transcripts by modulating nuclear ribosome biogenesis. The goal of this proposal is to generate the genetic tools and datasets necessary to robustly test this hypothesis through two aims. Aim 1 will define ADAD1 interacting proteins and RNAs in post-meiotic germ cells by generating an epitope tagged Adad1 allele and combining it with immunoprecipitation mass spectrometry and RNA sequencing. These efforts will shed light on ADAD1’s molecular mechanism of action. Aim 2 will quantify ribosome protein composition in Adad1 mutants and during germ cell development using quantitative mass spectrometry coupled to immunoprecipitation of a cell-specific epitope-tagged ribosome protein. Together, these analyses will quantify ADAD1’s impact on ribosome protein composition as well as define, for the first time, the meiotic and post-meiotic germ cell ribosome proteome. Historically, the ribosome was considered invariant across tissues and cells, however recent experimental evidence indicates ribosomal protein or RNA composition can vary, resulting in a specialized ribosome with unique translation dynamics. Whether germ cells leverage ribosome variation to control translation is an unexplored question that could have a dramatic impact on our understanding of germ cell biology. The studies proposed here will lay the foundations for defining the role of ADAD1 as well as identifying the male germ cell as a model for ribosome-level translation control.