Gene name - tudor
Cytological map position-57C8-57C9
Function - scaffolding
Keywords - oogenesis, polar granule assembly, germ cell formation
Symbol - tud
FlyBase ID: FBgn0003891
Genetic map position - 2R
Classification - tudor domain protein
Cellular location - cytoplasmic
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 tudA36 and tudB42, 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 tudB42 and tudB45 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 tudB42 and tudB45 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).
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
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.