Novel mechanisms regulating translation elongation during male germ cell differentiation

PROJECT SUMMARY Male fertility is reliant on the proper regulation of mRNA translation during male germ cell development. This is achieved by the concerted actions of RNA binding proteins (RBPs). In meiotic male germ cells, RBPs are often localized to RNA dense cytoplasmic granules termed germ cell RNA granules although many of these granules are of unknown function. Thus, the long-term goal of this work is to define the role of RNA granules in male germ cell differentiation and in doing so, potentially discover new modalities of post-transcriptional regulation governing germ cell development. ADAD2 is a novel RBP of unknown molecular function that forms a unique RNA granule. Loss of ADAD2, and by association the granule, results in severe post-meiotic germ cell loss and complete male infertility. Using genetic models and ribosome isolation procedures, we have shown that a subset of meiotic transcripts have increased ribosome association concurrent with reduced protein abundance independent of total mRNA levels in Adad2 mutants. These seemingly disparate outcomes are indicative of translation elongation defects. Supporting this notion, ADAD2 physically interacts with multiple proteins that influence translation including a component of the eEF1B elongation complex and a regulator of the translation repressor PIWIL1. Together, these observations lead to the hypothesis that the ADAD2 granule acts as a hub for the proper translation of a subset of meiotic mRNAs. Using genetic models, proteomics, and advanced RNA-sequencing analyses, this proposal will define the drivers of altered translation in Adad2 mutants and uncover the role of the ADAD2 granule in spermatogenesis. In Aim 1, how ADAD2 influences translation elongation and the elongation complex itself will be explored. This aim will leverage multiple ‘omics scale translation analysis methods along with quantitative mass spectrometry to define changes in elongation and the elongation complex in Adad2 mutants. Aim 2 will delineate the interaction between ADAD2 and PIWIL1-mediated translation regulation primarily by the application of various RNA-sequencing paradigms. And lastly, Aim 3 will define the protein and RNA components of the ADAD2 granule by leveraging genetic models that impinge on ADAD2 granule formation. RNA granules are incompletely described in terms of composition and function, in spite of their importance for male fertility. This work will connect the male infertility observed in Adad2 mutants to the molecular function of the ADAD2 granule, defining it as a fundamental regulator of mammalian fertility and ultimately building the framework in which to define how translation elongation is regulated in male germ cell development.