BIOLOGICAL OVERVIEW

Tudor domains are found in many organisms and have been implicated in protein-protein interactions in which methylated protein substrates bind to these domains. Evidence is presented for the involvement of specific Tudor domains in germline development. Drosophila Tudor, the founder of the Tudor domain family, contains 11 Tudor domains and is a component of polar granules and nuage, electron-dense organelles characteristic of the germline in many organisms, including mammals. This study investigated whether the 11 Tudor domains fulfil specific functions for polar granule assembly, germ cell formation and abdomen formation. It was found that even a small number of non-overlapping Tudor domains or a substantial reduction in overall Tudor protein is sufficient for abdomen development. In stark contrast, a requirement was found for specific Tudor domains in germ cell formation, Tudor localization and polar granule architecture. Combining genetic analysis with structural modeling of specific Tudor domains, it is proposed that these domains serve as 'docking platforms' for polar granule assembly (Arkov, 2006).

tud was the first member of the posterior group of genes identified in Drosophila. The hallmark of this group of maternal effect genes is their dual role in abdomen development and germ cell formation (Boswell, 1985; Thomson, 2004; Thomson, 2005). Germ cells are formed in a specialized embryonic cytoplasm, called germ plasm, which contains characteristic electron-dense organelles, the polar granules. The Tud protein is a component of polar granules (Amikura, 2001; Bardsley, 1993), and they are severely reduced in number and size in strong tud mutants (Amikura, 2001; Boswell, 1985; Thomson, 2004). Based on genetic interactions and its protein localization pattern in other mutants affecting germ plasm, tud acts downstream of oskar and vasa in germ plasm assembly (Bardsley, 1993; Ephrussi, 1992). Recently, Tud protein was shown in vitro to interact with Valois, which is a component of the methylosome in Drosophila (Anne, 2005), suggesting that Tud, like other proteins in the family, may bind to methylated substrates (Arkov, 2006).

Tudor (Tud) domains were initially identified as common protein motifs found in the Drosophila Tud protein and in other proteins from a wide variety of organisms and different kingdoms, including fungi, plants and animals (Maurer-Stroh, 2003; Ponting, 1997; Talbot, 1998). Tud domains are related to plant Agenet, Chromo PWWP and MBT domains, which together form the Tud domain 'Royal Family' (Maurer-Stroh, 2003). Tud domain proteins have been shown to interact with other proteins and efficient binding requires methylated arginine and lysine residues in the target protein (Brahms, 2001; Côté, 2005; Huyen, 2004; Kim, 2006; Sprangers, 2003). The Tud domain of the Survival Motor Neuron (Smn) protein binds directly to spliceosomal Sm proteins during spliceosome assembly (Brahms, 2001; Bühler, 1999; Selenko, 2001; Sprangers, 2003). Several Tud domain proteins have been shown to interact with modified histones. In particular, 53BP1 has tandem Tud domains that bind histone H3 on methylated Lys79 and this may be a molecular device for the recognition of DNA double-strand breaks during checkpoint responses (Huyen, 2004). Subsequently, Tud domains of several proteins were shown to bind to histones H3 and H4 (Huang, 2006; Kim, 2006). The recently identified structure of the N-terminal domain of the Fragile X Mental Retardation Protein (Fmrp) revealed two repeats of a Tud domain, and one of these domains was shown to interact with methylated lysine and with an Fmrp nuclear-interacting protein, 82-FIP (Ramos, 2006). Structural analysis of Tud domains from different proteins revealed that these domains can either fold into a single barrel-like structure composed of five ß strands (Selenko, 2001) or form an intertwined structure consisting of two Tud domains (Huang, 2006) (Arkov, 2006).

Phenotypical analysis of tud mutants revealed abdomen-patterning defects, suggesting that tud is involved not only in germline specification but also in abdomen formation (Boswell, 1985). However abdomen defects are not seen in all of the RNA null mutant embryos (Thomson, 2004), demonstrating that tud is not absolutely required for formation of the abdomen. A likely reason for abdomen development defects is the reduced localization of nanos (nos) RNA (Thomson, 2004; Wang, 1994) and the decreased amount of Nos protein (Gavis, 1994) in tud mutant embryos (Arkov, 2006).

Drosophila Tud protein contains 11 Tud domains (Talbot, 1998) and, until now, their function in germ cell specification or abdomen formation remained unknown. Slow progress on understanding Tud was in part due to the large size of the protein, which consists of 2515 amino acids (Golumbeski, 1991). As a result of an extensive screen designed to find mutants with germ cell formation defects, 15 new tud alleles were obtained. Characterization of these alleles, as well as the analysis of transgenic lines expressing Tud versions lacking different Tud domains, provided the first evidence for the involvement of specific Tud domains in germline development and in the maintenance of polar granule architecture. On the basis of the structural analysis of Tud domains, it is proposed that the germline specification and architecture of polar granules are dependent on specific protein-protein interactions between these domains and other polar granule components (Arkov, 2006).

Sequence analysis of tud mutants demonstrates that mutations within a single Tud domain can cause a mutant phenotype. This suggests either that single Tud domains act in concert to provide full function, or, alternatively, that specific Tud domains may have specific functions. Tud is required for both abdomen and germ cell formation (Boswell, 1985). Tud function in abdominal development is mediated via its role in nos RNA localization and translation (Gavis, 1994; Wang, 1994). Because tud is a strict maternal effect gene, embryos derived from females mutant for a particular allelic combination will be referred to as 'mutant embryos'. In strong tud mutant embryos, nos RNA localization to the posterior pole is reduced when compared with the wild type, and Nos protein synthesis is decreased (Gavis, 1994; Thomson, 2004; Wang, 1994). However, in contrast to other genes that affect germ plasm assembly, such as oskar or vasa, Tud protein is not absolutely necessary for nos RNA localization and translation, since females carrying a tud null mutation produce embryos with some nos RNA localization, and 15% of these embryos develop into normally segmented larvae (Thomson, 2004). By contrast, Tud function is absolutely required for germ cell formation, since embryos from females mutant for any of the strong alleles lack germ cells (Boswell, 1985; Thomson, 2004; Arkov, 2006 and references therein).

To determine the role of individual Tud domains in abdomen and germ cell formation, the mutant phenotype of new tudor alleles were characterized in detail. In addition, several mini-tud transgenes expressing Tud fragments that lack different parts of Tud were analyzed. In particular, mini-tud Delta1 produces Tud domains 1-6, minitud Delta2 produces domains 10 and 11, and mini-tud Delta3 produces domain 1 and domains 7-11. As a control, a full-length Tud transgene showed complete rescue of abdomen and germline defects in a tud¹ mutant background and co-localized with the polar granule marker Vasa in the germ plasm. All tud alleles that lack protein expression by Western blot show a phenotype very similar to that described for the tud loss-of-function mutation: larvae have segmentation defects and mutant embryos completely lack germ cells. By contrast, females mutant for any one of the alleles that produce Tud protein generate embryos that are normally patterned. Because these mutations affect different parts of the Tud protein, this suggests that any part of Tud may be sufficient to provide nos localization and translation function (Arkov, 2006).

Analysis of Tud domains 1 and 10, both of which carry a point mutation in the same arginine residue in tud^A36 and tud^B42, respectively, predicts that this arginine faces the solvent and that mutations in this residue do not affect the overall structure of the domains. Furthermore, the arginine is in close proximity to the cluster of hydrophobic amino acids that in Smn form a binding pocket for interaction with other proteins (Selenko, 2001; Sprangers, 2003). In Smn, target recognition is dependent not only on the hydrophobic cluster but also on E134, a glutamate located nearby. Tud-domain proteins can interact with flexible peptides carrying methylated amino acids and it is possible that charged amino acids close to the hydrophobic pocket, like the arginines in Tud domains 1 and 10, and glutamate 134, act as a gateway, contributing to the recognition of specific targets. Recently, a new structure of Tud domains was identified in the protein JMJD2A, which revealed an intertwined folding of two Tud domains (Huang, 2006). Other tandem Tud domain structures have been reported (Charier, 2004; Huyen, 2004; Ramos, 2006), and the analysis of sequences from these domains show that the two domains are separated by no more than 20-30 amino acids. Since individual Tud domains in Tud are separated by no less than ~100 amino acids, and because it is possible to create functional proteins after the deletion of large parts of Tud protein, there is presently no evidence predicting such dual domains in Tudor (Arkov, 2006).

Despite the virtual lack of polar granules in tud^B42 and tud^B45 mutants, substantial (albeit reduced) germ plasm-specific accumulation of polar granule component pgc RNA was observed, although previous results that failed to find pgc RNA localized to the germ plasm of strong tud mutants (Nakamura, 1996; Thomson, 2004). Because pgc RNA can accumulate in germ plasm that lacks clearly discernable polar granules, it is concluded that some localization and anchoring of RNA to the germ plasm can occur independently of complete polar granule assembly and that smaller particles containing germ plasm components may be sufficient to tether RNA. The role of Tud in germ plasm formation may be to assemble these pre-particles into a larger order granule. Because abdomen formation and nos RNA localization were normal in tud^B42 and tud^B45 mutants, it is proposed that these 'pre-particles' may be sufficient to promote nos localization and translational derepression at the posterior pole (Arkov, 2006).

For germ cell formation, specific Tud domains are essential and it is likely that these individual domains interact with specific partner proteins. Similar to Smn protein and other Tud domain proteins, these partners are likely to be methylated. Indeed, two germline proteins, Valois and Capsuléen, are components of the Drosophila methylosome and required for germ cell formation (Anne, 2005; Cavey, 2005; Schüpbach, 1986). In particular, Capsuléen is a homolog of the mammalian PRMT5 methyltransferase that has been recently implicated in germline specification in the mouse (Ancelin, 2006). Anne (2005) identified a particular region in Valois that interacts with Tud in vitro and that analysis suggests that the interaction of Tud with the methylosome may tether Tud to the posterior pole, possibly via specific methylated binding partners (Anne, 2005). Analysis of transgenes lacking different Tud domains showed that mini-tud Delta3 is sufficient for germ cell formation and abdomen segmentation. This transgene construct lacks the Tud segment that is responsible for the strong interaction with Valois protein in vitro (Anne, 2005). The ability of mini-tud Delta3 to induce germ cell formation indicates that the Tud-Valois interaction may not be absolutely necessary for germ cell formation. However, this interaction may be required for efficient germline development, since mini-tud Delta3 could not generate a normal number of germ cells. Alternatively, the weak binding detected between Valois and other Tud fragments that overlap with regions present in mini-tud Delta 3 (Anne, 2005) may be sufficient for the formation of some germ cells (Arkov, 2006).

Tud protein localizes to both the nuage, an electron-dense material associated with nurse cell nuclei, and the germ plasm (Bardsley, 1993). Besides Tud, three other proteins, Vasa, Aubergine and Valois are found in both the nuage and the germ plasm, and it has been suggested that the nuage forms a precursor stage of germ plasm assembly during oogenesis. This notion is supported by the finding that Vasa localization to both the nuage and the germ plasm is equally affected in vasa mutants. However, analysis of mini-tud transgenes shows that nuage localization is not necessary for Tud localization to the germ plasm or for germ cell formation. Thus, Tud localization to the germ plasm and its function in germ cell formation can be uncoupled from its association with the nuage during oogenesis. These results are consistent with the finding that posteriorly localized Aubergine is not transported to the germ plasm as a protein associated with nuage particles. Thus, the role of the perinuclear nuage and the function of Tud in this organelle remain to be elucidated (Arkov, 2006).

Protein components of ribonucleoprotein granules from Drosophila germ cells oligomerize and show distinct spatial organization during germline development

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 super-resolution microscopy, this study showed 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, this study showed that Aubergine forms homo-oligomers and using all purified Tudor domains, it was demonstrated that multiple domains, distributed along the entire Tudor structure, interact with Aubergine. These 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 (Vo, 2019).

This work focuses on the high-resolution imaging and in vitro analysis of the assembly of interacting proteins, Piwi family protein Aub and scaffold protein Tud, in the RNA-protein granules (polar granules) in the germline. Aub and Tud are the principal components of the granules and since they interact, it was expected that they would be homogenously distributed within the granules. Surprisingly, it was found that, while in the same granules and partially overlapping, these proteins form distinct mutually exclusive clusters in the germ plasm before germ cell formation. In addition, after germ cells form, Aub and Tud show even more striking segregation pattern within the large sphere-like granules that form in the cytoplasm of germ cells. In these granules, Aub is at the surface of the sphere, wrapping around a large Tud core (Vo, 2019).

Classic EM images of the germ granules in Drosophila germ cells from Mahowald's lab demonstrated large spherical cytoplasmic organelles, which in EM sections, appeared as donut- or ring-like structures with electron-dense rim around an electron-lucid core. These granules had the diameter of 0.75 μm - 1 μm in EM sections and are in good agreement with the Aub-Tud large granules described in this study (1.13 μm on average), suggesting that one of the components of the electron-dense rim is Aub. Furthermore, consistent with these data, similar donut-like Aub granules were described in germ cells using confocal microscopy. Although these large spherical granules are quite prominent, they are not as abundant as much more numerous germ plasm-like small polar granules in the cytoplasm of germ cells, therefore, the test of their functional significance awaits further investigation (Vo, 2019).

Interestingly, other proteins can assemble at the surface of large granules, that are different from polar granules, with ring-like distribution visualized in optical sections which resemble electron-dense rim of the corresponding granules imaged with EM. In particular, in Drosophila melanogaster, while Tud and Aub are exclusively cytoplasmic, germ granule proteins Oskar (Osk) and Vasa (Vas) can form granules in the nuclei of germ cells, which are referred to as nuclear bodies. These nuclear bodies are similar in EM sections to cytoplasmic large polar granules described in this study and also show an electron-dense rim with Osk and Vas ring-like distribution with the characteristic donut-like morphology in optical sections. Furthermore, in C. elegans germ granules (P granules), using high-resolution lattice light sheet microscopy, MEG-3 protein was shown to be assembled at the surface of the PGL-3 protein core and these proteins only partially overlap within the P granules. MEG-3/PGL-3 distribution in P granules resembles Aub/Tud assembly in the large cytoplasmic granules in Drosophila germ cells and suggests conservation in the molecular mechanisms of protein assembly in the large germ granules (Vo, 2019).

Interestingly, other 'non-germ' RNP granules, including mammalian P-bodies, stress granules and nucleoli show heterogeneity in their protein distribution within the granules. In particular, there is evidence that Xenopus nucleoli consist of different immiscible liquid-like phases that form core-shell arrangement, with Nucleophosmin NPM1 protein phase (shell) enveloping the FIB1 protein clusters (core). What may be responsible for the shell/core arrangement of the germ and nucleolus RNP granules? Analysis of the nucleolus protein phases provided evidence that their different hydrophobicity and surface tensions result in their distinct incorporation into the nucleolus structure which can be mimicked using different types of oil mixed with water. However, the FIB1 clusters can also 'age' and transition to solid-like state over time. Another mechanism to form distinct compartments within the RNP granules is based on the reentrant phase transition of RNPs which can be controlled by RNA. In this case, titration of an RNA-binding protein with increasing RNA concentration can initially result in the formation of a positively charged RNP droplet (due to a positively charged RNA-binding protein amino acid residues) which subsequently, at higher RNA concentration, leads to the change of the RNP charge from positive to negative. This charge inversion of the RNP granule causes the eventual dissolution of the granule. However, during this process, internal compartments form inside the RNP granules and at certain RNA concentrations these granules can exist for more than two hours, which can be sufficiently long for the time scale of many cellular and developmental processes. The physical principles described above, based on different liquid phases' surface tensions in aqueous environment and the reentrant phase transition, were used to describe behavior of spherical granules and may be contributing to distinct cluster and core-shell architecture of Aub-Tud RNP polar granules reported in this study. In fact, Tud scaffold core may stabilize Aub shell to prevent that from dissolution. However, some important aspects of Tud and Aub distribution as distinct clusters in polar granules before germ cell formation, when these proteins are not embedded into spherical granules but rather overlap in 'interaction hubs' forming amorphous diverse granules, await further biochemical and biophysical analysis (Vo, 2019).

The data show that Aub forms homo-oligomers under native conditions. It remains to be determined whether this oligomerization property of Aub is required for the assembly of polar granules. Interestingly, similarly to Aub homo-oligomerization, nucleolar shell NPM1 protein forms pentameric homo-oligomers and this oligomerization is required for NPM1 to form the protein liquid droplets in vitro and to efficiently localize to the nucleolus (Vo, 2019).

This study also demonstrates that Aub homo-oligomers are insensitive to Aub methylation status and form when Aub contains sDMAs required for interaction with Tud domains of Tud protein. This may indicate that in polar granules, Tud binds to methylated Aub oligomers rather than to monomeric Aub. Furthermore, six Tud domains (1, 3, 4, 6, 9 and 11) of Tud can bind to Aub. Consistent with previous methylated Aub peptide-Tud domains 7-11 binding studies, no interaction of domain 10 to full-length Aub was detected presumably due to this domain's incomplete sDMA-binding pocket, and Aub interaction was shwon with domains 9 and 11. Although Aub binding to the other five Tud domains of Tud protein was not detected, it was previously shown that methylated Aub peptides can bind to Tud domains 7 and 8. Therefore, based on this and previous data, it is proposed that one molecule of Tud protein contains at least eight Tud domains that may potentially bind to multiple Aub homo-oligomers. However, future research will be required to determine the precise stoichiometry of Tud/Aub complexes in polar granules (Vo, 2019).

The formation of distinct Tud and Aub clusters in the same polar granules is intriguing and was unexpected since these proteins are bona fide interacting granule proteins. Tud and Aub may undergo phase transitions during the assembly of polar granules, forming, together with their interacting ligands, two distinct phases. The data are consistent with the model that while these Tud and Aub phases are immiscible with the surrounding cytoplasm, they may be partially mixing and wetting each other, thereby partially overlapping in the 'interaction hubs'. Non-spherical shape of Aub- and Tud-labeled polar granules in the germ plasm before germ cells formation may indicate that Aub and Tud phases, at least to some degree, have transitioned to gel-like or solid-like state. In support of this model, it was observed that polar granules in the germ plasm of early embryos are more structured than liquid droplets and seem to contain both liquid and hydrogel-like regions (Vo, 2019).

After germ cells form in the embryo's posterior, the formation of large granules with Aub shell and Tud core may be driven by the concentration-dependent assembly since germ cells actively accumulate polar granule components using dynein-dependent transport. During this process Aub homo-oligomers (which themselves form even at low concentration) may be interacting with each other to stabilize the shell of the granule and free Aub oligomers may be recruited to assemble around Tud scaffold (Vo, 2019).

Previous imaging analysis of the polar granules demonstrated that RNA granule components form homotypic clusters in the granules. This study has shown that interacting protein components of polar granules form distinct clusters within the same granules which can be subsequently distributed into the large shell-core architecture during development. Future studies will establish how the RNA and protein clusters together determine the granule morphology and how this clustering contributes to the function of polar granules in germ cell development (Vo, 2019).

GENE STRUCTURE

cDNA clone length - 8205

Bases in 5' UTR - 385

Exons - 6

Bases in 3' UTR - 272

PROTEIN STRUCTURE

Amino Acids - 2515

Structural Domains

The tudor locus of Drosophila is required during oogenesis for the formation of primordial germ cells and for normal abdominal segmentation. The tud locus was cloned, and its product was identified by Northern analysis of wild-type and tud mutant RNAs. The locus encodes a single mRNA of approximately 8.0 kb. The tud protein has a predicted molecular mass of 285,000 daltons and has no distinctive sequence similarity to known proteins or protein structural motifs. Taken together, these results indicate that the tud product is a novel protein required during oogenesis for establishment of a functional center of morphogenetic activity in the posterior tip of the Drosophila embryo (Golumbeski, 1991).

date revised: 1 August 2007

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