Two RNA binding proteins, ADAD2 and RNF17, interact to form novel meiotic germ cell granules required for male fertility

Abstract

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in RNA binding protein (RBP) rich non-membrane bound organelles termed RNA germ cell granules. Though known to be required for male germ cell differentiation, little is understood of the relationships between and functions of the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic male germ cells. This work aimed to define the role of ADAD2 granules in male germ cell differentiation and their relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutant mice defined a shared and rare post-meiotic chromatin defect, suggesting shared biological roles. We further demonstrated ADAD2 and RNF17 are dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs including DDX4 and PIWIL1, a subset of the ADAD2-RNF17 granules are likely components of the piRNA pathway. In contrast, a second, morphologically distinct population of ADAD2-RNF17 co-localize with the translation regulator NANOS1 and form a unique cup-shaped structure with distinct protein subdomains. This cup shape appears to be driven, in part, by association with the endoplasmic reticulum. Lastly, a double Adad2-Rnf17 mutant model demonstrated loss of ADAD2-RNF17 granules themselves, as opposed to loss of either ADAD2 or RNF17, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. Together, this work identified a set of novel germ cell granules required for male fertility and sheds light on the relationship between germ cell granule pools. The example described here defines a new genetic approach to germ cell granule study. AUTHOR SUMMARYTo differentiate successfully, male germ cells tightly regulate their RNA pools. As such, they rely on RNA binding proteins, which often localize to cytoplasmic granules. The majority of studies have focused on a single granule type which regulates small-RNA biogenesis. Although additional granules have been identified, there is limited knowledge about their relationship to each other and exact functions. Here, we identify an interaction between two RNA binding proteins, ADAD2 and RNF17, and demonstrate mutants share a rare germ cell phenotype. Further, ADAD2 and RNF17 colocalize to the same germ cell granule, which displays two morphologically unique types. The first subset of ADAD2-RNF17 granules have similar morphologies to other characterized granules and likely play a role in the small-RNA pathway. The second granule type forms a unique shape with distinct protein subdomains. This second population appears to be closely associated with the endoplasmic reticulum. Genetic models further demonstrate the granules themselves, as opposed to the resident proteins, likely drive the mutant phenotypes. These findings not only identify a novel population of germ cell granules but reveal a new genetic approach to defining their formation and function during germ cell differentiation.