Abstract
The assembly of large RNA-protein granules occurs in germ cells of many animals and these germ granules have provided a paradigm to study structure-functional aspects of similar structures in different cells. Germ granules in Drosophila oocyte’s posterior pole (polar granules) are composed of RNA, in the form of homotypic clusters, and proteins required for germline development. In the granules, Piwi protein Aubergine binds to a scaffold protein Tudor, which contains 11 Tudor domains. Using a super-resolution microscopy, we show that surprisingly, Aubergine and Tudor form distinct clusters within the same polar granules in early Drosophila embryos. These clusters partially overlap and, after germ cells form, they transition into spherical granules with the structural organization unexpected from these interacting proteins: Aubergine shell around the Tudor core. Consistent with the formation of distinct clusters, we show that Aubergine forms homo-oligomers and using all purified Tudor domains, we demonstrate that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. Our data suggest that in polar granules, Aubergine and Tudor are assembled into distinct phases, partially mixed at their “interaction hubs”, and that association of distinct protein clusters may be an evolutionarily conserved mechanism for the assembly of germ granules.
Highlights
Membraneless RNA-protein structures are central to all aspects of cellular life and the assembly of different types of these structures has been the focus of intense investigation[1,2,3,4,5]
Germ granules, which assemble in germ cells in different organisms and contain RNA and proteins required for germline development[6], provide an important model to study the assembly mechanisms of large and dynamic RNPs
Tud- and Aub-specific clusters assemble into the same polar granules, which vary in size and morphology at this early stage of embryo development, and their diverse 2D appearance and size agreed well with previously published electron microscopy (EM) section images at this stage[12,20,24]
Summary
Membraneless RNA-protein (ribonucleoprotein, RNP) structures are central to all aspects of cellular life and the assembly of different types of these structures has been the focus of intense investigation[1,2,3,4,5]. Using super-resolution confocal microscopy, we focused on the detailed distribution of protein polar granule components, Tud and Aub, in early embryos before and after germ cell formation stage. Since these proteins are direct interacting partners, we expected to see their complete overlap in polar granules. Using size-exclusion chromatography and native-gel electrophoresis, we demonstrated that, irrespective of its methylation status, purified Aub forms homo-oligomers This property of the Piwi family protein to oligomerize may contribute to the formation of this protein’s distinct clusters within the granules and to the assembly of the shell around the Tud scaffold in germ cells. This work suggests that some protein components of polar granules are engaged in the granule assembly by forming distinct phases mixed at certain regions (“interaction hubs”) and this may be a common strategy for the assembly of germ granules in different organisms
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