The capsuléen gene (csul; CG3730) is required for pole cell formation in the pathway controlled by osk. To identify Csul-interacting proteins active in pole plasm, a yeast two-hybrid screen was performed. The bait construct consisted of the sequence of the Csul protein (610 amino acid residues) fused to the DNA binding domain (BD) of Gal4. By screening a Drosophila cDNA library, six independent interacting clones were obtained of which four of them corresponded to the CG10728 coding sequence predicted by the Drosophila genome project. Conceptual translation of CG10728 cDNA revealed a protein of 367 amino acids. Two cDNAs encoded a nearly full-size protein lacking the first 10 amino acid residues, whereas the other two isolates encoded a protein in which 37 residues were missing from the N terminus. Confirmation of the interaction was obtained by performing the reciprocal two-hybrid assay, showing that BD-CG10728 could strongly bind the activation domain of Gal4 fused to Csul. Sequence analysis indicated that CG10728 contains four WD repeats (Anne, 2005).
The CG10728 gene is located at chromosomal band 38B2. Since vls was genetically assigned to this region (Schüpbach, 1986), whether the CG10728 sequence was altered in vls mutants was examined. To determine the lesions in vls1, vls2 and vls3, genomic DNA fragments encompassing the CG10728-coding sequence were amplified by PCR, then the the amplified fragments from each mutant was cloned and sequenced. For each vls mutant single nucleotide substitutions were found that result in premature termination of the coding sequence. In vls1 and vls2 the nucleotide substitutions changed a TGG Trp codon into TAG and TGA stop codons, producing truncated proteins of 227 and 52 amino acids, respectively. In vls3, the nucleotide substitution transformed a CGA Arg codon into a TGA stop codon producing a truncated protein of 69 amino acids. To confirm that vls was cloned, two transgenes were constructed containing the presumed vls sequence, including its 5' regulatory sequences. The first transgene contained a genomic DNA fragment comprising the CG10728 transcription unit. The second transgene harboured the same sequence to which was fused six copies of the hemaglutinin tag (HA), in frame with the vls-coding sequence. Western blots of proteins extracted from ovaries producing HA-Vls probed with anti-HA antibodies displayed a single polypeptide with the expected mass of 50 kDa. Both transgenes restored full viability and fertility to embryos produced by vls1 and vls3 homozygous females. Taken together, these results demonstrate that CG10728 corresponds to vls (Anne, 2005).
Among the nearest homologues of Vls identified in protein databases was the methylosome protein 50 (MEP50). MEP50, the human homologue of Vls, contains six WD repeats and interacts with PRMT5 (Friesen, 2002), the human homologue of Csul. The finding of an interaction between Csul and Vls in yeast prompted analysis of whether such binding occurs in Drosophila ovaries. To this end, transgenic flies were constructed bearing both the HA-vls transgene and a Tandem Affinity Purification (TAP) tagged csul transgene (TAP-csul) shown to restore csul development. Protein extracts of P{TAP-csul}; vls3; P{HA-vls} ovaries were immunoprecipitated using rabbit IgG-sepharose beads and the bound Csul-complexes were released by treatment with recombinant TEV protease. Blots of released proteins probed with anti-HA antibodies showed an immunoreactive band of 50 kDa exhibiting the predicted mass of HA-Vls fusion protein. Reciprocally, blots of ovarian proteins immunoprecipitated with anti-HA antibodies and probed with IgG antibodies displayed an immunoreactive band of the mass predicted for TAP-Csul (90 kDa). These results indicate that Csul and Vls can associate in a protein complex in Drosophila ovaries (Anne, 2005).
To further characterize the interaction between Csul and Vls, the binding domains in each protein were mapped by using a GST-pull down assay. For this purpose Vls or fragments of Vls were fused to GST, and Csul or fragments of Csul to S•Tag In vitro translated S•Tag-Csul polypeptides were incubated with immobilized GST-Vls proteins and, after washing, the bound S•Tag-Csul proteins were detected by using S-protein coupled to alkaline phosphatase. Csul binds specifically to immobilized full-length Vls, or to the 30 amino acid C terminal region of Vls, but not to the rest of the protein. Conversely, the region of Csul mediating the interaction with Vls mapped to the C terminal region of Csul. Neither the N-terminal region nor the central region of Csul showed strong binding to Vls. Thus, the data indicate that Csul and Vls interact through their C-terminal regions (Anne, 2005). -
The presence in Vls of four WD repeats able to interact with other proteins prompted an examination of whether Vls could physically interact with proteins localized in the nuage. For this purpose, pull-down assays were performed using tagged Tud, Vas and Gustavus (Gus) (Anne, 2005).
Five segments of Tud fused to the S•Tag, including JOZ (amino acid residues 3-273), 9A1 (residues 198-1199), 3ZS+L-N (residues 1198-1981) and 3ZS+L-C (residues 1941-2515) , together comprising the complete Tud protein, were in vitro translated and the synthesized polypeptides were incubated with immobilized full-length and truncated GST-Vls proteins. Of the five Tud fragments, it was found that only the 9A1 fragment could interact with Vls, whereas JOZ and the two subfragments of 3ZS+L showed only weak binding. The 9A1 fragment was further divided into two fragments. The 9A1-N and 9A1-C fragments (residues 198-770 and 751-1199, respectively) displayed a strong binding to Vls (Anne, 2005).
In the reciprocal experiment, it was found that the Tud 9A1-N fragment can bind to the region encompassing residues 90-190 of Vls. When this segment was further divided into two A and B fragments, containing residues 90-139 and 139-192, respectively, it was discovered that only the fragment A was able to bind to Tud 9A1-N. These results indicate that the first WD repeat of Vls can directly interact with Tud (Anne, 2005).
No specific Vls binding with Vasa and Gus polypeptides was detected in GST pull-down assays using GST, GST-Vls and GST-Vls (residues 90-190). Similarly, no interaction could be uncovered between Vls and Vasa in the yeast two-hybrid assay. These data indicate that the binding between Vls and Tud represents a specific interaction (Anne, 2005).
Thus, by analyzing potential interactors of the putative Csul methyltransferase, the vls gene was isoolated. Attempts to isolate additional partners of Vls by using the yeast-two hybrid system were unsuccessful because of the occurrence of a too large number of clones growing on selective medium, presumably reflecting the occurrence of WD repeats that are known to mediate interactions with numerous proteins in eukaryotic cells. By using direct binding assays with proteins involved in pole plasm function, it was found that Vls interacts with Tud (Anne, 2005).
WD-repeat proteins act as scaffolding/anchoring proteins for a number of binding partners. WD-repeat motifs within one protein can simultaneously bind several proteins and foster transient interactions with other proteins. Moreover, WD-repeat proteins occur in relatively stable protein complexes in which they play a structural role. A similar function can be assigned to Vls, a WD-repeat motif protein, in promoting either permanent or transient interaction with other proteins (Anne, 2005).
vls mutations causing a grandchild-less phenotype are characterized by agametic larvae exhibiting defects in abdominal patterning (Schüpbach, 1986). Eggs laid by homozygous vls females are devoid of polar granules and consequently the embryos produce no pole cells (Schüpbach, 1986). In these embryos, Tud is absent from the posterior pole (Bardsley, 1993), and Vasa rapidly disappears from this location during the period of nuclear cleavage (Hay, 1990; Lasko, 1990. The current analysis reveals that localization of Tud is already absent from the nuage surrounding the vls nurse cell nuclei. However, the occurrence of Vasa and Mael in the nuage of vls nurse cells indicates that aspects of this structure can be made independently of Tud (Anne, 2005).
A pivotal role in the organization of the nuage was assigned to Vas on the basis of its involvement in localizing specific components such as Aub and Mael in this structure. To confirm a Vasa-dependence of Tud localization in the nuage, Tud distribution was examined in vas egg chambers and the localization of Tud around nurse cell nuclei was found to be fully abolished. Similarly the absence of Tud in the nuage of vls nurse cells shows that Tud localization in the nuage is vls dependent. However, significant amounts of Tud is detected in vls oocytes and at their anterior border, indicating that Tud accumulation in the nuage is not required for Tud transport into the oocyte and its anterior margin. Furthermore, since Vls-HA does not accumulate in early oocytes nor localize at their anterior margin, it is concluded that the interaction between Vls and Tud should be spatiotemporally regulated (Anne, 2005).
Vls is shown to be a component of the nuage and pole plasm. Only a limited number of proteins display a similar pattern of distribution, including Vasa, Aub and Tud (Bardsley, 1993). The dual location of these proteins indicates that they either perform distinct functions at each location or exert a function in the nuage required for their accumulation in the pole plasm. The finding that Vasa absence in the pole plasm correlates with its absence in the nuage supports the latter possibility (Anne, 2005).
Inactivation of vls function exerts a further effect on the production of the short form of Osk protein. Since osk mRNA localization and amount seems normal in vls embryos (Ephrussi, 1991, it is assumed that the lower amount of this form detected by immunoblotting in vls ovaries corresponds to either a defect in Osk synthesis or stability. It was noticed, however, that the level of Osk abundance varies considerably between individual vls oocytes with an apparently normal level in a small number of oocytes and a markedly reduced level in the majority of oocytes. The lower amount of Vas detected at the posterior pole of stage 10 vls or vls2/Df(2L)TW2 oocytes (Hay, 1990), can be interpreted as a consequence of the reduced amount of Osk protein, since Vasa is absent from the posterior pole of osk oocytes (Hay, 1990; Lasko, 1990; Anne, 2005 and references therein).
The mechanism by which Vls regulates Osk synthesis and/or stability remains unknown. However, on the basis of Vls localization during oogenesis, it is envisaged that vls could regulate the production of the short form of Osk by two distinct mechanisms: (1) vls could regulate Osk synthesis by recruiting specific enhancing factors in the pole plasma, and (2) Osk synthesis may be dependent on events mediated by vls occurring in the nuage. Similarly, efficient osk mRNA translation in the pole plasm could also be mediated by Aub in the nuage. Furthermore, recent data point out that the nuage may function in assembling or reorganizing ribonucleoprotein complexes, particularly those involving localized or translationally regulated mRNAs (Anne, 2005).
The formation of polar granules fully depends upon vls activity (Schüpbach, 1986, but only partially upon tud function, since polar granules in reduced number and altered morphology are observed in amorphic tud pre-blastoderm embryos. This raises the question of what the targets of vls function are in addition to tud and osk. Further experiments will reveal the components required for vls-dependent formation of polar granules (Anne, 2005).
Since the human methylosome is formed by MEP50 and PMRT5 homologous to Drosophila Vls and Csul, respectively, the finding that Vls can specifically bind to Csul indicates that it is the orthologue of MEP50 and not a divergent WD protein. Restricted and dynamic Vls distribution during oogenesis is found, first in the nuage and then at the posterior pole of the growing oocyte. Finally, Vls is preferentially incorporated in the forming pole cells. These findings show that vls may crucially act in the nuage, germ plasm and pole cells, and are consistent with the vls mutant phenotype. A lower amount of Osk was detected at the posterior pole of growing vls oocytes, Osk levels were found to be already lower in stage 9 egg chambers (Anne, 2005).
In conclusion, it has been demonstrated that Vls can interact physically with at least two proteins, Csul and Tud, which are specifically involved in germ-line determination. Vls, in association with Csul, constitutes the first example of a partner of a dimethylarginine protein methyltransferase whose function has been characterized in vivo. These findings reinforce their cardinal function in a pathway first elucidated through genetic investigations. This work sets the basis for further investigations on the role of Vls, its dependence upon Csul and its involvement in specific localization of cytoplasmic proteins during the formation of a functional pole plasm (Anne, 2005).
Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine methyltransferases (PRMTs) in specific biological processes remain to be clarified. In this study the Drosophila PRMT Capsuléen, homologous to human PRMT5, has been characterized. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In particular, the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are abolished in csul mutant germ cells. The spliceosomal Sm proteins have been identified as in vivo substrates of Capsuléen and it is demonstrated that Capsuléen, together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues in Sm proteins. Finally, Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating that Tudor localization and Sm methylation are separate processes. These results thus reveal the role of a PRMT in protein localization in germ cells (Anne, 2007).
csul encodes a Type II PRMT, which transfers methyl groups from S-adenosyl-L-methionine to the guanidinium group of arginyl residues. PRMTs can be divided into two major categories, catalyzing the synthesis of aDMA (Type I) or sDMA (Type II) residues, respectively. The mammalian PRMT5 (Pollack, 1999; Lee, 2000; Branscombe, 2001), homologous to Csul, and the recently identified PMRT7 and PRMT9 (Lee, 2005; Cook, 2006) are responsible for Type II methylation (Anne, 2007 and references therein).
By using alpha-SYM10 antibodies that recognize proteins harbouring two spaced sDMA-glycine motifs four major reactive proteins bands were identified as specific targets of Csul. These proteins are distinct from aDMA-containing proteins, whose methylation is independent of Csul. Among the sDMA proteins, it was genetically confirmed that the spliceosomal components SmB and SmD3 are Csul targets. The corresponding mammalian targets have been identified for PMRT5. Since alpha-SYM10 may only recognize a subset of sDMA proteins methylated by Csul, further proteomic analysis of ovarian Csul complexes may identify additional targets of Csul (Anne, 2007).
As indicated by the physical interaction of Csul with Valois (Anne, 2005) and the size of the native Csul complexes, with a molecular mass of ~500 kDa, Csul is part of a large protein complex. In the present work Vls, the Drosophila homolog of human MEP50, itself a partner of PRMT5 (Friesen, 2002), is also shown to be required in sDMA synthesis on identical target proteins. However, in the case of pIcln, a component of the human methylosome of yet unknown function, no interaction was detected between Drosophila pIcln and Csul in pull-down assays. Furthermore, both Csul and Vls were found to interact with the N-terminal moiety of SmB. This is in contrast to PRMT5, which appears to bind to the RG-rich C-terminal domain of Sm proteins. Differences in protein interaction and quaternary structure between the human and Drosophila methylosome may reflect divergences in the activities of the methylosome between the two species (Anne, 2007).
Both human and Drosophila methylosomes lead to sDMA synthesis on Sm proteins. Similarly to the requirement of sDMA synthesis for the association of human Sm proteins with the SMN Tudor domain (Côté, 2005), it was found that Drosophila Sm proteins need to be symmetrically methylated to bind Drosophila Tud. The binding of sDMA-Sm to non-overlapping Tud polypeptides indicates that these proteins may bind to several, if not all Tudor domains in Tud (Anne, 2007).
The association of human SMN protein with the PMRT5 complex suggests direct interactions between PMRT5, MEP50 and SMN (Meister, 2002). Similarly, Drosophila Tud can directly bind to Csul and Vls (Anne, 2005). However, in contrast to Sm, which binds to multiple sites on Tud, Csul and Vls more strongly interact with the N-terminal than the C-terminal moiety of Tud, suggesting a distinct mechanism of association with Tud. Although the specific binding sites of Csul and Vls on Tud remain to be determined, preliminary results indicate that each protein binds to a distinct site (Anne, 2007).
As this work was being completed, another group reported the identification of the csul gene, termed dart5 (Gonsalvez, 2006), and showed that disruption of this gene (mutant e00797 from the Exelixis collection) leads to the absence of sDMA synthesis of spliceosomal Sm proteins without impairing spliceosomal function. This work and the current study confirm the previous finding of Khusial et al. (Khusial, 2005), indicating that sDMA synthesis on Sm proteins is not required for sRNP assembly and transport, a critical process for Drosophila development. In addition, Gonsalvez (2006) also characterized the maternal requirement of csul for pole cell formation (Anne, 2007).
In addition to their role in sDMA synthesis, Csul and Vls are required for Tud localization in the nuage. These data indicate that csul activity is also necessary for the proper nuage accumulation of Vas. However, despite the occurrence of Vas in the nuage of early csul egg chambers, Tud is absent from this structure, suggesting that the activity of Csul in Tud localization is independent from that exerted on Vas (Anne, 2007).
How the Csul/Vls methylosome directs Tud localization in the nuage remains an open question. The restoration of fertility by mutated csul transgenes defective in SmB binding, and hence in sDMA synthesis on SmB, points out the occurrence of a yet unknown protein which should act as a substrate of Csul and specifically function in germline formation. A cytoplasmic association of the Csul/Vls methylosome with this substrate and Tud is favored. Upon methylation the substrate is then transferred to Tud, as indicated by the preferential binding of sDMA-SmB to Tud polypeptides. The interaction between Csul/Vls, the substrate, and Tud may be critical to position Tud in the vicinity of the site where sDMA synthesis takes place, thus facilitating the association of Tud with the sDMA-protein. A similar model has been proposed for the targeting of high-affinity Sm protein substrates to the SMN complex. Following the docking of the sDMA protein on Tud, the Csul/Vls methylosome is released, and the Tud/sDMA protein complex becomes positioned in the nuage. The docking of the sDMA protein might induce an allosteric change in Tud, increasing its affinity for a component of the nuage (Anne, 2007).
Finally, although Vas is not properly localized at the perinuclear region of nurse cells in csul and vls mutant egg chambers it was noticed that its distribution pattern differs in each mutant. In particular, the level of Vas in the nuage is comparatively smaller in csul than in vls mutants (Anne, 2005), suggesting that Csul acts independently of Vls in the localization of Vas to the nuage. Moreover, the finding that Vls specifically accumulates in the nuage and pole plasm whereas Csul displays a ubiquitous distribution suggests that both proteins may exert additional independent functions (Anne, 2007).
Although the functional relationship between the nuage and pole plasm remains unresolved, events occurring in the nuage may affect pole plasm formation. In csul mutant egg chambers, Tud is absent from both the nuage and the pole plasm and, similarly, a reduced amount of Vas in the nuage correlates with a decreased level of this protein in the pole plasm. However, it has been reported recently that a Tud protein containing the Tudor domains 1 and 6-10 could localize to the pole plasm, albeit at a moderate level compared to full-length Tud, but fail to properly localize to the nuage (Arkov, 2006). Additional work on the requirement of Csul for Tud localization in the nuage will be critical for understanding the assembly of this structure, its dynamical relationship with the pole plasm, and the role of arginine methylation in protein targeting (Anne, 2007).
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