Abstract
Scaffold proteins play crucial roles in subcellular organization and function. In many organisms, proteins with multiple Tudor domains are required for the assembly of membraneless RNA-protein organelles (germ granules) in germ cells. Tudor domains are protein-protein interaction modules which bind to methylated polypeptides. Drosophila Tudor protein contains eleven Tudor domains, which is the highest number known in a single protein. The role of each of these domains in germ cell formation has not been systematically tested and it is not clear if some domains are functionally redundant. Using CRISPR methodology, we generated mutations in several uncharacterized Tudor domains and showed that they all caused defects in germ cell formation. Mutations in individual domains affected Tudor protein differently causing reduction in protein levels, defects in subcellular localization and in the assembly of germ granules. Our data suggest that multiple domains of Tudor protein are all needed for efficient germ cell formation highlighting the rational for keeping many Tudor domains in protein scaffolds of biomolecular condensates in Drosophila and other organisms.