InteractiveFly: GeneBrief

capsuleen: Biological Overview | References

Gene name - capsuléen

Synonyms -

Cytological map position - 53A3-53A3

Function - enzyme

Keywords - assembly of nuage, formation of pole plasm, arginine methyltransferase, methylation of residues in spliceosomal Sm proteins

Symbol - csul

FlyBase ID: FBgn0015925

Genetic map position - 2R: 12,152,180..12,154,464 [+]

Classification - PRMT5 arginine-N-methyltransferase

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

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).

Establishment and maintenance of the germline is an essential process for all sexually reproducing organisms. Germ cells, which carry the genetic information to the next generation, are simultaneously totipotent and highly specialized. All germ cells throughout the animal kingdom contain in their cytoplasm a distinct cloud-like structure termed the nuage. One system with a high potential for understanding the assembly and role of the nuage is Drosophila oogenesis. The Drosophila egg chamber consists of a germ line cyst generated from a single cystoblast by four successive mitotic divisions and surrounded by a monolayer of somatic follicle cells. Due to incomplete cytokinesis, the germ cells remain connected to each other through specialized cytoplasmic bridges. The oocyte derives from one of the germ cells, while the remaining 15 cells differentiate into nurse cells. The nuage is concentrated in the perinuclear cytoplasm of nurse cells and can be associated with nuclear pores. It appears as a dense fibrous organelle unbound by membrane and often associated with mitochondrial clusters. Moreover, the nuage interfaces with sponge bodies, which are abundant RNA-rich particles present in the cytoplasm of nurse cells and, to a lesser degree, in the oocyte (Anne, 2007 and references therein).

The majority of the components identified in the nuage are also present in the pole plasm of the oocyte, and more particularly in the polar granules. The pole plasm constitutes the determinant that is both necessary and sufficient to induce germ cell formation during early embryogenesis. The first step in pole plasm formation is the transport of oskar (osk) transcripts synthesized in nurse cell nuclei to the posterior pole of stage 8 oocytes. At this location Osk is synthesized and serves as an anchor to initiate polar granules assembly. In addition to Osk the polar granule components include Vasa (Vas), which interacts directly with Osk and a number of transiently localized factors that are mainly necessary for osk mRNA transport and translation. During late oogenesis and early embryogenesis, the polar granules are maintained at the posterior pole. At the time of blastoderm formation, they are sequestered in the pole cells, the primordial germ cells of the fly, in which they coalesce into a smaller number of large particles, ultimately disappear and are replaced by the nuage. This structure appears to evolve from components of the pole plasm and persists only in established germ cells. The only identified nuage-specific component absent from the polar granules is Maelstrom (Mael) which shuttles between the nucleus and the cytoplasm (Anne, 2007).

Among these proteins, Vas plays a cardinal role in the formation of the nuage. In vas ovaries the nurse cells are devoid of nuage at the ultrastructural level. The function of Tud in the nuage remains unknown but Tud may play a role in the assembly or modification of specific RNP complexes, as indicated by its requirement for the transfer of mitochondrial ribosomal RNAs from the mitochondria to the polar granules. The presence of shared components reinforces the view that the nuage and the polar granules are closely related structures, in which components, such as Vas and Aubergine (Aub), may dissociate from the nuage to reassemble into the polar granules (Anne, 2007).

The mechanisms by which nuage components become assembled at the perinuclear region of the nurse cells remains, however, to be identified. This study reports that the catalytic activity of the Capsuléen (Csul) protein-arginine methyltransferase is required for the localization of specific components of the nuage and pole plasm, and in particular of Tud (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, homologous to Csul, and the recently identified PMRT7 and PRMT9 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. 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 findings 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).

Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen

Using Capsuléen (Csul) methyltransferase as bait in the yeast two-hybrid system, Valois, a protein that acts in pole plasm function, was identified. Vls is homologous to human MEP50, which forms a complex with the PRMT5 methyltransferase -- the human homologue of Csul. Vls and a fragment of Tud interact directly in binding assay. Since the PMRT5/MEP50 complex is involved in ribonucleoprotein complex assembly, it is hypothesized that the Vls complex may play a similar function in assembling the nuage in nurse cells and the polar granules in the oocyte (Anne, 2005).

Given the homologous interaction between human and Drosophila Valois homologs with a methyltransferase, what might be the function of Valois in pole plasm? MEP50 acts as an adaptor binding to a subset of spliceosomal Sm proteins and contributing to their methylation by the PRMT5 methyltransferase (Friesen, 2002). Biochemical assays indicate that MEP50 is necessary for the methyltransferase activity of the methylosome, as evidenced by the observation that anti-MEP50 antibodies significantly reduce the methylation of Sm proteins (Friesen, 2002). However, the precise role of MEP50 remains elusive. Its possible functions include a regulation of the enzymatic activity of PMRT5 and the control of the positioning of the substrate for methylation (Anne, 2005).

The human methylosome complex is involved in the assembly of spliceosomal U-rich small nuclear ribonucleoproteins (snRNPs) mediated by the survival motoneuron (SMN) protein, a gene product that is affected in spinal muscular atrophy. SMN is produced ubiquitously and contains a single Tudor domain that associates with SmB/B', SmD1-D3 and SmE proteins of snRNPs. The assembly of snRNPs mediated by SMN occurs in the cytoplasm and is stimulated by the PMRT5-methylosome complex that converts specific arginine residues in the Sm proteins into dimethylarginines, facilitating the binding of the Sm proteins to SMN and their association with snRNA molecules. Ultimately, the assembled snRNPs are released and targeted to the nucleus, whereas the SMN-PRMT5 complex may dissociate before its components associate again for a new round of assembly (Anne, 2005 and references therein).

In Drosophila, the Tud protein differs from SMN by containing eight Tudor domains and two Tudor-like domains, whereas SMN contains a single Tudor domain (Ponting, 1997). It is thus possible to envisage that Drosophila Tud may bind different categories of cytoplasmic RNPs through its multiple Tudor domains. However, in contrast to the PMRT5/MEP50 complex that apparently binds SMN through other protein(s) present in the complex, Vls can directly bind to Tud through its first WD repeat. Since the C-terminal tail of Vls binds to Csul, it is possible that the Drosophila Csul/Vls methylosome associates with Tud through Vls. Thus, it is proposed that the association between the methylosome and Tud promotes binding and assembly of specific RNPs on Tud. Further experiments are needed to unravel the relationship between these proteins, the targets of Csul methyltransferase activity and the nature of the RNPs associated to Tud (Anne, 2005).

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. 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, 1992), 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).

Arginine methylation of SmB is required for Drosophila germ cell development

Sm proteins constitute the common core of spliceosomal small nuclear ribonucleoproteins. Although Sm proteins are known to be methylated at specific arginine residues within the C-terminal arginine-glycine dipeptide (RG) repeats, the biological relevance of these modifications remains unknown. In this study, a tissue-specific function of arginine methylation of the SmB protein was identified in Drosophila. Analysis of the distribution of SmB during oogenesis revealed that this protein accumulates at the posterior pole of the oocyte, a cytoplasmic region containing the polar granules, which are necessary for the formation of primordial germ cells. The pole plasm localisation of SmB requires the methylation of arginine residues in its RG repeats by the Capsuléen-Valois methylosome complex. Functional studies showed that the methylation of these arginine residues is essential for distinct processes of the germline life cycle, including germ cell formation, migration and differentiation. In particular, the methylation of a subset of these arginine residues appears essential for the anchoring of the polar granules at the posterior cortex of the oocyte, whereas the methylation of another subset controls germ cell migration during embryogenesis. These results demonstrate a crucial role of arginine methylation in directing the subcellular localisation of SmB and that this modification contributes specifically to the establishment and development of germ cells (Anne, 2010).

Histochemical analysis indicates that the Sm proteins are predominantly present in the nucleus, but studies using cell fractionation techniques reveal that Sm proteins can be isolated as Sm rings in the cytoplasm. Here, it was determined that two Drosophila Sm proteins can specifically accumulate in the pole plasm during the final stage of oocyte differentiation until the formation of pole cells in blastoderm stage embryos. Inclusion of Sm proteins in cytoplasmic structures is a rather novel finding, although they have been detected outside of the nucleus in the germ cells of various species, including C. elegans, Xenopus laevis, rat and mouse (Anne, 2010).

The present data show that access of SmB and SmD3 to the pole plasm depends on (symmetrical dimethylarginine) sDMA methylation. Although SmD1 contains arginine residues that are the targets of sDMA methylation, an accumulation of SmD1 in the pole plasm was not observed. This indicates that sDMA-modified Sm proteins, detected as GFP fusion proteins, are differentially targeted to the pole plasm. As sDMA-modified Sm proteins can interact with the Tud domains of SMN (Côé, 2005) and Tud has been previously shown to bind methylated SmB protein (Anne, 2007), it is possible to envisage that Tud confers specificity to the transport of SmB to the pole plasm. However, this hypothesis is nullified by the finding that SmB and Tud are conjointly detected only when both proteins have reached their destination at the posterior pole of the oocyte. Recent data (Gonzalvez, 2010) indicate that the transport machinery responsible for the pole plasm localisation of SmB and SmD3 might correspond to a cytoplasmic osk mRNA RNP (Anne, 2010).

SmB and SmD3 can form a stable association but the unmethylated form of GFP-SmB was not detected in the pole plasm even in the presence of both endogenous SmB and SmD3. This suggests that the SmB and SmD3 proteins are independently transported to the pole plasm or that they are transported as heteromeric complexes in which the methylation of both Sm proteins is tightly coupled. In favour of a coupling methylation of an SmB-SmD3 complex is the finding (Gonzalvez, 2010) that hypomethylation of SmD3 is accompanied by a decrease in SmB methylation (Anne, 2010).

The present study identifies two functional groups of arginine residues in the tail of SmB: the first group includes the three proximal arginine residues and is required for gonad formation during embryogenesis, whereas the second group comprises the last four arginine residues and is involved in at least two processes: development of the egg chambers within the ovaries and polar granule anchoring in the oocyte. As the phenotype associated with the loss of these arginine residues in SmB is distinct from that produced by capsuléen or valois mutations, it is likely that other arginine methyltransferases (such as Dart7) actively participate in SmB methylation during oogenesis. The existence of a methylated factor that is able to bind to Tud and is necessary for its localisation in nuage and pole plasm is inferred from the presence of Tud in the nuage of SmB ovaries expressing a hypomethylated form of SmB and from previous work (Anne, 2007; Anne, 2010 and references therein).

Tud is also required for SmB localisation in the pole plasm. In a previous report, it was determined that the methyltransferase activity of Csul is necessary for the localisation of Tud at the posterior pole of the oocyte (Anne, 2007). If one considers a possible association between Tud and methylated SmB in the polar granules, this might explain why unmethylated SmB proteins do not strongly accumulate in pole plasm (Anne, 2010).

SmB is first detected in the pole plasm during the early stages of vitellogenesis and persists at this location until pole cell formation. The effect of an absence of SmB methylation could only be uncovered when the maternal contribution of methylated SmB is depleted. The first zygotic abnormality resulting from the arginine substitution in the tail of SmB, and therefore from a defect in SmB methylation, is exposed when the germ cells initiate their migration towards the gonadal mesoderm during late embryogenesis. The function of SmB during these processes remains elusive, but a parallel can be drawn with the occurrence of Sm proteins in the spreading initiation centres (SICs) during the initiation of cell spreading. SICs contain numerous ribonucleoprotein complexes and exist temporarily during the very early stages of cell spreading as precursors of focal adhesions. Cell-matrix attachments play an essential role in many vital cellular processes, including motility, differentiation and survival. Drosophila germ cells leave the midgut and migrate towards the lateral mesoderm, displaying a highly polarised morphology, with a broad lagging edge and an unusually long and relatively stable rear protrusion. Whether Sm proteins are present in this cell protrusion remains to be determined (Anne, 2010).

After the transport of the osk mRNA to the posterior pole of the oocyte, this transcript is translated from in-phase alternative initiation codons into two isoforms: a long form and a short form. Owing to its unique ability to promote pole cell formation, only the short Osk isoform can direct the assembly of polar granules. Once polar granule components are recruited by the short form, they must be retained in place to ensure proper assembly of the pole plasm and their inheritance by the pole cells. Polar granule maintenance requires the long Osk form, which is necessary for the efficient anchoring of the short Osk form at the oocyte cortex. Importantly, each Osk isoform exhibits a distinct localisation at the posterior pole: the short Osk is mainly present in the polar granules, whereas the long Osk form is attached to endocytic membranes. The anchoring of the other pole plasm components to the cortex requires an Osk-dependent induction of endocytic activity. Western blot analysis revealed that both isoforms of Osk are synthesised in SmB; p-{UAS-SmBR4-7} ovaries, suggesting that the defective anchoring phenotype cannot be attributed to a failure to produce the long Osk form (Anne, 2010).

The current results suggest that the absence of SmB in pole plasm modifies the organisation of the F-actin cytoskeleton and affects its ability to anchor polar granules at the posterior cortex. Although disruption of the microfilaments by treatment with the actin-depolymerising drugs cytochalasin D or latrunculin A exerts only a mild effect on the posterior anchoring of Osk, several lines of evidence suggest that the F-actin cytoskeleton anchors polar granules to the subcortical posterior region of the oocyte. In particular, the actin-binding proteins Moesin and Bifocal are required for this process. The cortical localisation of Bifocal in the oocyte depends on an intact F-actin cytoskeleton and Bifocal binds directly to F-actin filaments in vitro, suggesting that Bifocal acts to stabilise F-actin filaments. The endocytic pathway may also function downstream of long Osk to anchor the pole plasm components at the cortex by regulating the dynamics of F-actin. Projections of F-actin into the ooplasm appear to emanate from the F-actin bundles overlying the posterior cortex of the oocyte. These projections are osk-dependent and become detectable from stage 10 onward, when anchoring is required, and are of sufficient length to span the distance between the plasma membrane and the underlying polar granules. In the SmB; p-{UAS-SmBR4-7 or R4-7K} egg chambers, there is a correlation between the loose anchoring of the polar granules and the disorganisation of the F-actin cytoskeleton. Although the possibility that SmB is also present at the cortex cannot be excluded at this stage, a role of SmB in the polar granules in establishing a link between these granules and the cortical cytoskeleton network is plausible. The existence of a positive-feedback loop maintenance mechanism, in which polar granules, possibly in concert with long Osk, enhance their own anchoring at the posterior pole, has indeed been proposed (Anne, 2010).

Loss of dart5 disrupts localization of Tudor

The C-terminal tails of spliceosomal Sm proteins contain symmetrical dimethylarginine (sDMA) residues in vivo. The precise function of this posttranslational modification in the biogenesis of small nuclear ribonucleoproteins (snRNPs) and pre-mRNA splicing remains largely uncharacterized. This study examined the organismal and cellular consequences of loss of symmetric dimethylation of Sm proteins in Drosophila. Genetic disruption of dart5, also termed Capsuleen, the fly ortholog of human PRMT5, results in the complete loss of sDMA residues on spliceosomal Sm proteins. Similarly, valois, a previously characterized grandchildless gene, is also required for sDMA modification of Sm proteins. In the absence of dart5, snRNP biogenesis is surprisingly unaffected, and homozygous mutant animals are completely viable. Instead, Dart5 protein is required for maturation of spermatocytes in males and for germ-cell specification in females. Embryos laid by dart5 mutants fail to form pole cells, and Tudor localization is disrupted in stage 10 oocytes. Transgenic expression of Dart5 exclusively within the female germline rescues pole-cell formation, whereas ubiquitous expression rescues sDMA modification of Sm proteins and male sterility. This study has shown that Dart5-mediated methylation of Sm proteins is not essential for snRNP biogenesis. The results uncover a novel role for dart5 in specification of the germline and in spermatocyte maturation. Because disruption of both dart5 and valois causes the specific loss of sDMA-modified Sm proteins and studies in C. elegans show that Sm proteins are required for germ-granule localization, it is proposed that Sm protein methylation is a pivotal event in germ-cell development (Gonsalvez, 2006; full text of article).

Pre-messenger-RNA splicing, a hallmark feature of eukaryotic cells, is carried out by a large ribonucleoprotein (RNP) complex called the spliceosome. Numerous gene products are therefore dedicated to the task of building functional spliceosomes, the cellular machines that mediate the removal of intronic sequences. Small nuclear RNPs (snRNPs), central components of the spliceosome, are assembled in a highly orchestrated and sequential manner, involving maturation steps in both the nucleus and cytoplasm of the cell. The U1, U2, U4, and U5 spliceosomal snRNPs each contain a common set of seven core Sm proteins: SmB/B′, SmD1, SmD2, SmD3, SmE, SmF, and SmG. These proteins bind to a common sequence motif within the U snRNAs and form a heteroheptameric ring structure (Gonsalvez, 2006).

Assembly of the Sm ring takes place in the cytoplasm and, in vivo, requires the activity of the survival of motor neurons (SMN) protein complex. Mutations that reduce the level of SMN, the central member of this complex, result in a human neurogenetic disorder called spinal muscular atrophy (SMA). Importantly, cells from SMA patients display a reduced capacity for Sm core assembly. Collectively, the available data are consistent with the idea that SMA results from a general reduction in snRNP biogenesis, with motor neurons being particularly susceptible to reduced snRNP levels. However, the possibility that SMN functions in a novel cell-specific pathway has not been conclusively ruled out (Gonsalvez, 2006).

Three of the seven core Sm proteins, SmB/B′, SmD1, and SmD3, contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. The enzymes that catalyze this posttranslational modification are called protein arginine methyltransferases (PRMTs) and have been placed into two categories -- type I and type II. Type I enzymes mediate the more common modification, asymmetric dimethylarginine (aDMA). Type II enzymes are responsible for the less frequent sDMA modification. To date, the only known type II enzymes are PRMT5 and PRMT7, each of which is capable of methylating Sm proteins in vitro. Reduction of PRMT5 levels by RNA interference (RNAi) correlates with a decrease in the level of Sm-protein methylation in vivo. Furthermore, PRMT5 associates, along with MEP50/WD45 and pICln, in a complex that contains Sm proteins in vivo. Both MEP50 and pICln can directly bind to Sm proteins, thus making a strong case for involvement of the PRMT5 complex in Sm-protein methylation. It is not currently known whether PRMT7 plays any role in Sm-protein methylation, and binding partners for PRMT7 have not been described (Gonsalvez, 2006).

The precise role of Sm-protein methylation in snRNP biogenesis remains a poorly understood topic. In vitro, SMN protein preferentially binds to C-terminal peptides, derived from SmD1 and SmD3, that contain sDMA but not aDMA residues. The prevailing view holds that sDMA modification of Sm proteins serves to recruit SMN, thus facilitating efficient transfer of Sm proteins from the PRMT5 complex to the SMN complex for assembly of the Sm core. A prediction that follows from this interpretation is that symmetric dimethylation of Sm proteins is a requirement for efficient snRNP biogenesis. This hypothesis was explored in vivo, with Drosophila melanogaster. For these experiments, a fly strain containing an insertion in the dart5 gene, the fly ortholog of human PRMT5, was used. Lysates prepared from homozygous mutant flies display a complete and specific loss of sDMA modification of Sm proteins. Surprisingly, homozygous disruption of dart5 is not lethal, and the expected number of progeny is recovered. Using additional molecular assays, it was found that spliceosomal snRNP biogenesis was similarly unaffected. Instead, it was found that dart5 males were completely sterile, with defects in spermatogenesis. In contrast to the males, the homozygous mutant females were fertile. However, the progeny obtained from homozygous dart5 mothers were sterile and agametic. Consistent with this finding, embryos from dart5 females were devoid of pole cells, the germline precursors. This is reminiscent of the classic 'grandchildless' phenotype described for a number of genes such as tudor, vasa, and valois. Interestingly, it was recently shown that valois is the Drosophila ortholog of human MEP50/WD45. Like their human counterparts PRMT5 and MEP50, the Valois and Dart5 (also known as Capsuléen) proteins were recently shown to associate in the fly. Plausibly, these two gene products may function in a related and perhaps overlapping pathway that contributes to germ-cell specification. Notably, it was found that, similar to the valois mutant phenotype, Tudor protein was mislocalized in dart5 mutant ovaries. On the basis of these and other findings, it is proposed that sDMA modification of Sm proteins represents a critical step in the specification and maintenance of the germ-cell lineage (Gonsalvez, 2006).

Although Dart5 activity is required for Tudor function, dart5 does not fit the mold of a classical posterior-group gene. In order to be placed directly in the germ-cell specification pathway, upstream of tudor, the dart5 phenotype should be at least as strong as that of tudor. It is not. Similarly, mutations in vasa do not have an appreciable effect on Dart5 activity, as measured by Sm-protein methylation. Thus a revised model of the germ-cell specification pathway is proposed, wherein dart5 (and valois) primarily affect Tudor localization, resulting in a loss of pole-cell formation. However, unlike oskar and vasa mutations, somatic patterning appears to be relatively unaffected. Because 15% of tudor null embryos develop normally, it has been suggested that Tudor is not directly required for posterior patterning. However, tudor null embryos contain fewer polar granules than wild-type embryos and never form pole cells. Thus a fully functional pole plasm may be required for stabilizing the level or maintaining the localization of factors involved in establishing the body plan. In such a scenario, it is not surprising that a subset of tudor null embryos display patterning defects. Given that mutation of dart5 appears to compromise Tudor function, a small fraction of dart5-1 embryos also display patterning defects (Gonsalvez, 2006).

The elevated hatching frequency of dart5-1 as compared to tudor null embryos and the residual accumulation of Oskar in dart5-1 blastoderm embryos suggest that a partially functional pole plasm is formed in the absence of Dart5. However, this level of functionality is insufficient to mediate germ-cell specification, given that 100% of the embryos that develop are agametic. Because Tudor is only modestly reduced in dart5-1 mutant ovaries in comparison to its complete absence from tudor null ovaries, it is logical that the dart5-1 phenotype would be less severe than the tudor null phenotype. Although Tudor was not enriched at the posterior pole in dart5-1 oocytes, neither was it completely absent from this location. As such, the residual level of Tudor, along with properly localized Oskar and Vasa, might be sufficient to assemble a partially functional pole plasm in the oocyte (Gonsalvez, 2006).

Given their in vivo association, it is not surprising that valois and dart5 share many phenotypes: absence of pole cells, male sterility, and loss of Sm-protein sDMA residues. Despite the similarity of the mutant phenotypes, there are a few differences worth noting. For instance, the spermatocyte maturation defect was less severe in the valois mutant as compared to dart5-1. Additionally, unlike the dart5-1 mutant, the vls3 mutant displayed a rather strong maternal-effect lethal phenotype. This result is consistent with a previous finding that valois mutants displayed pleiotropic defects during cellularization. In addition, whereas valois mutants affect the level of Oskar protein in ovaries, there is no apparent Oskar defect in dart5-1 mutants. Thus Valois may have additional functions outside of its complex with Dart5 (Gonsalvez, 2006).

This report has identified Sm proteins as in vivo targets of Dart5. Furthermore, it was shown that Valois is also required for the sDMA modification of Sm proteins and proper expression of Dart5. In the absence of Dart5 and Valois, germ-cell specification, but not general snRNP biogenesis, is disrupted. These observations point to a model whereby Sm proteins, or more precisely symmetrical arginine dimethylation of Sm proteins, play a critical role in germ-cell specification. Consistent with this hypothesis, Sm proteins are thought to play a specific role, unrelated to splicing, in P granule integrity germ-cell specification in C. elegans. P granules are structurally and functionally related to the nuage of Drosophila. Like the Drosophila nuage, P granules are RNA rich and contain a number of proteins that have critical roles in germ-cell development. Importantly, Valois is localized to, and is required for, the proper formation of the nurse-cell nuage in Drosophila. Another prominent component of the Drosophila nuage is Tudor. In mouse spermatocytes, Mouse-Tudor-Repeat gene1 (MTR-1) is localized to the nuage and specifically associates with Sm proteins therein. Furthermore, the nuage of Xenopus oocytes was also shown to specifically contain Sm proteins. It will therefore be of great interest to determine whether Sm proteins are components of the nuage in Drosophila. In the absence of Dart5 activity, prominent Tudor localization to the nuage is disrupted. Sm-protein methylation may therefore be required for maintaining proper integrity of the Drosophila nuage. In order to more fully explore this hypothesis, ultrastructural analyses will be required (Gonsalvez, 2006).

Tudor is the founding member of a family of proteins that contain Tudor domains. Several lines of evidence point to a function for Tudor domains as methyl binding protein modules: (1) the SMN protein contains a single Tudor domain, mutation of which causes a significant decrease in binding affinity for Sm proteins; (2) molecular modeling studies suggest that Tudor domains are structurally related to other domains, such as the Chromo domain, that are known to bind methylated proteins; (3) SMN binding to Sm proteins decreases upon loss of methylation; (4) it has been shown that two other Tudor-domain proteins, splicing factor 30 (SPF30) and Tudor-domain-containing 3 (TDRD3) interact with Sm proteins in a methylation-dependent manner. Drosophila Tudor contains 11 such protein motifs. Thus, it is plausible that Tudor interacts with sDMA residues within the C termini of Sm proteins and that this interaction is somehow required for Tudor function and, consequently, for germ-cell development. Experiments designed to examine this hypothesis are ongoing. In this regard, it is noteworthy that disruption of dart5 affects the levels of Tudor protein and its localization within the egg chamber (Gonsalvez, 2006).

This study has shown that symmetrical dimethylation of arginine residues within the Sm proteins is lost upon disruption of dart5, the Drosophila ortholog of PRMT5. The possibility cannot be ruled out that, in the absence of Dart5 activity, Sm proteins might contain other posttranslational modifications (e.g., monomethylated or asymmetrically dimethylated arginine residues). However, correlated with the loss of symmetric dimethylation of Sm proteins is a complete failure to develop germ cells in subsequent generations. Expression of myc-tagged Dart5 only in the female germline via a nanos driver rescued pole-cell formation in early embryos and Vasa localization to the developing gonad. One interpretation of these observations is that symmetric dimethylation of Sm proteins plays a central role in specifying the germline. The dart5-1 allele will be a valuable tool in exploring this hypothesis. If Sm proteins do play a role in germ-cell specification, simple mutational or knockout experiments will not be useful in uncovering the mechanism, because these alterations cause somatic-cell lethality. RNAi of Sm proteins in C. elegans, while causing a disruption in the localization and integrity of P granules, is also coupled with embryonic lethality. The available mutations in Drosophila Sm proteins are likewise all lethal (Gonsalvez, 2006).

These results suggest that Sm proteins play at least two distinct roles in the organism, one a general function in pre-mRNA splicing and the other in germ-cell specification and maintenance. The dart5-1 allele is very informative in this regard because it uncouples these two functions: snRNP biogenesis and splicing are ongoing in dart5-1 homozygotes, but germ-cell specification is disrupted. Given the similar phenotypes of the dart5 and valois mutants, the function of the Tudor domain, the delocalization of Tudor in dart5-1 egg chambers, and the available data on the localization of Sm proteins to the nuage in several different species, the strongest interpretation favors a critical role for Sm proteins in germ-cell specification. Although this hypothesis is favored, it is not possible to rule out the possibility that, for example, loss of methylation of some other protein causes the observed phenotypes. Future work should provide much-needed mechanistic insight into this question. In this regard, it will be particularly important to determine whether Sm proteins are components of the nuage and pole plasm in Drosophila. If so, it will be most interesting to elucidate whether they are associated with snRNAs or are complexed with a different class of RNA (Gonsalvez, 2006).

Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability

Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). This study reports that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). Piwi proteins are expressed in Xenopus oocytes and numerous Xenopus piRNAs were identified. This paper reports that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, capsuleen/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. These studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. These findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification (Kirino, 2009).

Piwi family proteins are expressed in the germline and bind ~26 to ~30 nucleotide (nt) piRNAs. Drosophila express three Piwi proteins: Aub, Piwi and Ago3. Mice also express three Piwi proteins termed Miwi, Mili/PiwiL2 and Miwi2/PiwiL4. Tens of thousands of distinct piRNAs have been described and most of them are species-specific. In Drosophila , Piwi proteins and piRNAs (also known as rasiRNAs - repeat associated small interfering RNAs) silence transposons in the germline. A similar function has been found for a subset of mouse and zebrafish piRNAs. An amplification loop of piRNAs has been described but how primary piRNAs are generated is unknown. sDMA modifications occur in sequence motifs composed of arginines flanked by glycines (GRG) or alanines (GRA or ARG) that are often found as repeats. PRMT5 and its cofactors MEP50/WD45 and pICln form the methylosome that methylates Sm proteins. A highly specific monoclonal antibody (17.8) was produced that recognizes Mili by Western blot, immunoprecipitation and immunofluorescence microscopy. By serendipity it was discovered that the widely used Y12 monoclonal antibody recognizes mouse Mili and Miwi proteins and their bound piRNAs. The Sm proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs) constitute the main antigen for Y12. piRNAs were not identified in immunoprecipitates of snRNPs, heterogeneous ribonucleoproteins (hnRNPs) or of the Survival of Motor Neurons (SMN) complex using various antibodies. By Northern blot analysis it was found that piR-1, but not miR-16, an abundant miRNA, was found in Y12 immunoprecipitates, suggesting that Y12 recognizes Piwi but not Ago proteins (Kirino, 2009).

The epitope that Y12 recognizes on Sm proteins consists of symmetrically dimethylated arginines, in the glycine-arginine rich regions of the proteins. It was reasoned that Y12 likely reacted with sDMA-containing epitopes in Mili and Miwi, and arginine residues were sought that could be symmetrically methylated. Intriguingly, it was found that most animal Piwi proteins contain sDMA motifs that are typically clustered close to the amino terminus, while no animal Ago proteins contained such motifs. However, it was found that four of ten Arabidopsis Ago proteins contained sDMA motifs (Kirino, 2009).

To test whether Miwi and Mili contain sDMAs, SYM11 and ASYM24 antibodies, which specifically recognize proteins containing sDMA-glycine or aDMA-glycine repeats, respectively, were used. SYM11, as well as Y12, reacted strongly with endogenous Miwi and Mili, while ASYM24 showed only faint reactivity towards endogenous Miwi. In contrast, recombinant Flag-Mili or Flag-Miwi purified from baculovirus-infected Sf9 cells, were not recognized by Y12 or SYM11 (or ASYM24). This is entirely consistent with the finding that recombinant human Sm proteins expressed in Sf9 cells also do not contain sDMAs because Sf9 cells do not express type II PRMTs and thus cannot produce sDMA modifications. These findings indicate that Mili and Miwi proteins contain sDMAs. The putative sDMA motifs of Miwi are concentrated very close to the amino terminus with the exception of one GRG triplet. Flag-tagged full-length Miwi and two truncated forms of Miwi (aa 68-862 or 1-212) were transfected in 293T cells, by Flag immunoprecipitation and subject to western blot with SYM11 antibody, SYM11 antibody recognizes the amino terminus of Miwi protein (Kirino, 2009).

Next it was asked whether the sDMA modification was conserved in Piwi family proteins from other species. A stumbling block in studying the molecular functions of Piwi proteins and piRNAs has been the lack of suitable cell culture systems. It was reasoned that Xenopus laevis oocytes might express Piwi proteins and piRNAs and thus prove very useful not only to confirm that sDMAs of Piwi proteins are conserved but also as a model to study the function of Piwi proteins and piRNAs. By searching the Gurdon EST database at Xenbase three Xenopus Piwi proteins were identified which were named Xili, Xiwi and Xiwi2. All three Xenopus Piwi proteins contain putative sDMA motifs. Immunoprecipitations with Y12 from X. laevis oocytes (defolliculated, mixed Dumont stages I-VI), testis and liver revealed the presence of two proteins at ~95 kDa and ~110 kDa specifically in the Y12 immunoprecipitates from oocytes and testis that were identified by mass spectrometry as Xiwi and Xili respectively. As shown in the western blots, Y12 recognizes both Xiwi and Xili, while anti-Mili (17.8) reacts only with Xili. In addition, both Xiwi and Xili are recognized by SYM11, indicating that Xiwi and Xili contain sDMAs (Kirino, 2009).

X. laevis piRNAs were isolated and analyzed from Y12 immunoprecipitates. ~26-29 nt piRNAs are present in the Y12 immunoprecipitates and their 3'-termini are not eliminated by periodate oxidation and are thus likely 2'-O-methylated, as seen in piRNAs from other species (Kirino, 2009).

Deep sequencing was performed of X. laevis piRNAs from Y12 immunoprecipitates of oocytes and testis. The nucleotide composition of X. laevis piRNAs shows enrichment of Uridine in the first nucleotide position and of Adenine in the tenth nucleotide position. There is also enrichment for piRNAs whose first 10 nucleotides are complementary to the first 10 nucleotide of other piRNAs. These features indicate that a fraction of X. laevis piRNAs target transposon transcripts and that they also participate in a piRNA amplification loop, as has been described for Drosophila and zebrafish piRNAs and prepachytene mouse piRNAs. By Northern blot XL-piR-3, a representative piRNA, is expressed specifically in oocytes and by in situ hybridization XL-piR-3 is localized predominantly in the cytoplasm of X. laevis oocytes and it is expressed in higher levels in immature oocytes (Kirino, 2009).

Genetic disruption of either Drosophila PRMT5 (dPRMT5; also know as capsuleen - csul- , and dart5) or its cofactor valois, (the Drosophila homolog of MEP50/WD45), results in complete loss of sDMA modifications of Sm proteins in ovaries. However, unlike the situation in mammals, the levels or function of Sm proteins is not affected by loss of sDMAs (Kirino, 2009).

Null or hypomorphic alleles of dPRMT5 (csul, dart5) phenocopy aub null alleles and it was reasoned that dPRMT5 might be the methyltransferase that produces sDMAs in Aub, Piwi and Ago3, in vivo. To test this, ovaries were used from csulRM50/Df(2R)Jp7 females, which give rise to embryos that are genetic nulls for dPRMT5 and w- was used as a wild-type control. Western blots of ovary lysates from wt and maternal null csul showed that there was near complete loss of SYM11 reactivity, indicating dramatic reduction of sDMA modified proteins in csul ovaries. There was no change in ASYM24 reactivity between wt and csul, indicating that aDMA modified proteins were not affected. These findings confirm that dPRMT5 (csul, dart5) activity is required specifically for sDMA modification. Piwi and Aub proteins were immunoprecipitated from wt and csul mutant ovaries and the immunoprecipitates were probed with SYM11 and ASYM24. SYM11 reacted very strongly with Aub and also with Piwi immunopurifed from wt but not csul ovaries; ASYM24 reacted only weakly with Aub from wt ovaries. Immunoprecipitates of Ago3 were also with SYM11 and ASYM24 and it was observed that only Ago3 from wt ovaries reacted with SYM11. These results indicate that, like the mouse and X. laevis Piwi family proteins, Drosophila Piwi, Aub and Ago3 contain sDMAs and that dPRMT5 is the methylase that produces sDMAs of these proteins (Kirino, 2009).

In Aub the four arginines that are putative substrates for symmetrical dimethylation are found in tandem very close to the amino terminus. Site-directed mutagenesis was used to change these arginines into lysines that are not subjected to methylation by PRMTs. Flag-tagged wild-type (WT) or mutant (M) Aub were stably transfected into Drosophila S2 cells (which express dPRMT5), the proteins were purified by Flag immunoprecipitation and subjected to western blot with Flag, SYM11 and ASYM24 antibodies. SYM11 antibody reacted only with wild-type Aub. Next the binding of wild-type and mutant Aub to a synthetic piRNA was assayed. Immunopurified, wild-type or mutant Flag-Aub were incubated with a 5'-end radiolabeled synthetic piRNA containing 4-thio-Uridine at the first position, followed by crosslinking with Ultraviolet light and NuPAGE analysis. There was similar binding between wild-type and mutant Aub proteins. These findings indicate that one or more of the four arginines in the amino terminus of Aub are symmetrically dimethylated and arginine methylation does not impact piRNA binding (Kirino, 2009).

Next, RNAs bound to Piwi and Aub were isolated and analyzed from wt or csul ovaries. piRNAs remain bound to Piwi and Aub proteins in the csul ovaries. There is mild reduction of Piwi-piRNAs and marked reduction of Aub-piRNAs in csul ovaries corresponding to concordant reduction of protein levels of Piwi and Aub. The Piwi associated piRNAs were gel purified and subjected to periodate oxidation followed by β-elimination and it was revealed that Piwi-associated piRNAs purified from csul ovaries retain 2'-O-methylation of their 3' termini. These findings indicate that the lack of sDMA modifications of Piwi and Aub in csul ovaries does not impair the methylation of piRNAs or their binding to Piwi and Aub (Kirino, 2009).

Next the protein levels of Piwi family proteins were compared between wt, heterozygous and homozygous csul ovaries. Western blot analysis showed that there was marked reduction of Aub and Ago3 protein levels and lesser reduction of Piwi levels in csul ovaries, whereas the levels of the miRNA binding protein Ago1 were not affected. Since mRNA levels of Aub, Piwi and Ago3 are the same between wt and csul ovaries, dPRMT5 activity might be required to stabilize the Aub, Ago3 and Piwi proteins most likely by symmetrically methylating their arginines. The level of a representative piRNA (roo-rasiRNA), was decreased in csul ovaries in accord with reduction of Piwi family proteins, while the level of a representative miRNA, miR-8, was not affected. The homozygous csul ovaries showed a 30-fold increase in the levels of the HeT-A retrotransposon transcript, whose expression is most sensitive to mutations that disrupt piRNA-directed silencing in the female germline. Collectively these results indicate that loss of dPRMT5 activity impairs the amounts of Piwi proteins and piRNAs, resulting in disruption of their function of transposon silencing (Kirino, 2009).

Next the localization of Ago3, Aub and Piwi was analyzed by confocal microscopy in wt and homozygous csul early stage egg chambers. Previous studies have shown that Piwi is localized predominantly in the nuclei of follicle and germ cells while Ago3 and Aub are localized in the cytoplasm of germ cells. In oocytes, Aub is concentrated in the germ (pole) plasm. Representative images reveal that the level of Ago3 is markedly reduced in csul early stage egg chambers, while there is only a mild reduction of Aub and Piwi protein levels (Kirino, 2009).

Germ cell (PGC) formation in Drosophila requires that cytoplasmic determinants are localized to the posterior pole of the embryo. Genetic screens have identified grandchildless maternal genes that are required for PGC specification and invariably the protein or RNA products of these genes are concentrated in the pole plasm and are incorporated into the PGCs. Among these genes are Aub, dPRMT5 (csul, dart5), and its cofactor valois and tudor, whose protein product contains eleven tudor domains. The localization of Aub was tested in csul oocytes by confocal microscopy. Representative results show that the levels of Aub in the pole plasm of stage 10 egg chambers are markedly reduced. Western blotting reveals marked reduction of Aub protein levels in csul ovaries while confocal microscopy shows that Aub levels are subtly reduced in early stage egg chambers but markedly reduced in later stage egg chambers, suggesting that lack of sDMAs affects Aub levels at later stages in oogenesis (Kirino, 2009).

These studies show that sDMA modification of Piwi family proteins is a conserved post-translational modification, and the methyltransferase PRMT5 (csul/dart5) is identified as the enzyme that catalyzes sDMAs of Piwi, Ago3 and Aub in Drosophila ovaries, in vivo. Both Aub and csul/dart5 (dPRMT5) are grandchildless genes and the finding that Aub is a substrate for dPRMT5, indicates that an important function of dPRMT5 in pole plasm function and PGC specification involves methylation of Aub. Intriguingly, tudor domains bind to sDMAs and Tudor protein is also a grandchildless gene that is required for pole plasm assembly and function. These findings suggest that pole plasm function may involve an interacting network of genes whose protein products contain sDMAs (Aub), the methylase (dPRMT5) and its cofactor (valois/dMEP50) that produce sDMAs and tudor domain (Tudor) proteins that may bind to sDMA-containing proteins. It is noted that both PRMT5 and tudor-domain-containing genes are found in all species that express Piwi family proteins and knockout of tudor domain containing 1/mouse tudor repeat 1 in mice leads to spermatogonial cell death and male sterility. Furthermore, it is noted that other Drosophila proteins whose genes are required for piRNA accumulation or function, such as Spindle-E/homeless, contain tudor domains. It will be interesting to test whether tudor domain containing proteins interact with sDMA-modified Piwi family proteins and to elucidate their function (Kirino, 2009).


Search PubMed for articles about Drosophila Capsuleen

Anne, J. and Mechler, B. M. (2005). Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuleen. Development 132(9): 2167-77. PubMed ID: 15800004

Anne, J. et al. (2007). Arginine methyltransferase Capsuléen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila. Development 134: 137-146. PubMed ID: 17164419

Anne, J. (2010). Arginine methylation of SmB is required for Drosophila germ cell development. Development 137(17): 2819-28. PubMed ID: 20659974

Arkov, A. L., Wang, J.-Y. S., Ramos, A. and Lehmann, R. (2006). The role of Tudor domains in germline development and polar granule architecture. Development 133: 4053-4062. PubMed ID: 16971472

Bardsley, A., McDonald, K. and Boswell, R. E. (1993). Distribution of Tudor protein in the Drosophila embryo suggests separation of functions based on site of localization. Development 119: 207-219. PubMed ID: 8275857

Côté J. and Richard, S. (2005). Tudor domains bind symmetrical dimethylated arginines. J. Biol. Chem. 280: 28476-28483. PubMed ID: 15955813

Ephrussi, A. and Lehmann, R. (1992). Induction of germ cell formation by oskar. Nature 358: 387-392. PubMed ID: 1641021

Friesen, W. J., et al. (2002). A novel WD repeat protein component of the methylosome binds Sm proteins. J. Biol. Chem. 277(10): 8243-7. PubMed ID: 11756452

Gonsalvez, G. B., Rajendra, T. K., Tian, L. and Matera, A. G. (2006). The Sm-protein methyl transferase, dart5, is essential for germ-cell specification and maintenance. Curr. Biol. 16: 1077-1089. PubMed ID: 16753561

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Biological Overview

date revised: 10 February 2011

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