org Valois valois: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - valois

Synonyms -

Cytological map position - 38A6--E9

Function - scaffolding protein

Keywords - oogenesis, posterior group, pole plasm assembly

Symbol - vls

FlyBase ID: FBgn0003978

Genetic map position - 2-[54]

Classification - WD domain protein

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations (Schupbach, 1986). Despite its early genetic identification, it has not been characterized at the molecular level until now. vls encodes a divergent WD domain protein and the three available EMS-induced point mutations cause premature stop codons in the vls ORF. A null allele was identified that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules (Cavey, 2005).

The embryonic body axes are specified in Drosophila during oogenesis, when cytoplasmic determinants localize to different regions of the developing oocyte. This initiates the formation of positional information centers, which define polarity and pattern the body plan along the anteroposterior (AP) and dorsoventral (DV) axes during embryogenesis. This developmental control mechanism is based on mRNA localization and anchoring to specific subcellular compartments. In conjunction with tight translational control of localized mRNAs this is an efficient means with which to generate a local source of polarity determinants, one that is widely used throughout phyla for various purposes. In Drosophila oocytes, posteriorly localized oskar (osk) mRNA is locally translated starting in mid-oogenesis (stage 8-9) and nucleates the assembly of the pole plasm (or germ plasm). The pole plasm specifies the germline at the posterior end of the embryo, and it patterns the abdomen along the AP axis (Cavey, 2005).

The osk ribonucleoprotein (RNP) complex has been characterized, and many conserved factors are known to function in mRNA localization and/or translational control in different systems across phyla. Because restriction of osk activity to the posterior is crucial for normal development, both pre- and post-translational control mechanisms regulate Osk protein accumulation. Osk protein is actively degraded by the ubiquitin-proteasome pathway, but protected from it by Par-1 phosphorylation, specifically at the posterior. Translational control of osk involves the coordinate action of repressors and derepressors interacting with discrete elements of osk transcripts during transport and at the posterior pole. Additional factors that do not function as derepressors are also required for stimulating osk translation. In addition, Oo18 RNA-binding protein (Orb) polyadenylates osk transcripts at the posterior pole once derepression has been achieved (Cavey, 2005 and references therein).

Two isoforms of Osk (Long and Short Osk) are produced by initiation at two different in-frame start codons. Short Osk has long been known as the active isoform for pole plasm assembly which recruits downstream components of the pathway such as Vasa (Vas), and recently, Long Osk has been shown to be responsible for anchoring osk mRNA and Short Osk at the posterior. Short Osk is likely to anchor Vas directly at the posterior. Vas is an ATP-dependent RNA-helicase from the DEAD-box family and has been implicated in translational activation of several maternal transcripts, including osk. tudor (tud) acts downstream of vas and is followed in the cascade by additional genes whose products localize to the pole plasm and mark the separation of germline establishment and abdominal patterning activities. Pole cell formation depends on the localization of germ cell less (gcl) mRNA and mitochondrial large ribosomal RNA. Abdominal patterning relies on the vas- dependent translation of nanos (nos) mRNA at the posterior pole. This results in a concentration gradient of Nos protein along the AP axis, which acts as the primary posterior morphogen (Cavey, 2005 and references therein).

One more posterior group gene, valois (vls), had been identified in the initial screen for maternal-effect steriles (Schupbach, 1986), but has neither been cloned nor studied genetically in detail yet. Based on three EMS-induced alleles of vls, it was classified as a member of the 'grandchildless-knirps-like' group that also includes vas, stau and tud. Their phenotype is characterized by a lack of pole cells at the posterior and various degrees of abdominal segment deletions. Pole cell transplantation experiments have demonstrated that vls functions in the germline (Schupbach, 1986) and vls mutants were shown to have a non-functional pole plasm (Lehmann, 1991). Until now, the position of vls in the posterior pathway has remained controversial. vls was tentatively placed downstream of osk and vas, but upstream of tud. This was based on the observation that osk mRNA and Vas protein are initially correctly localized to the posterior of the oocyte in vlsEMS mutants. Vas then detaches from the posterior of the embryo soon after fertilization (Ephrussi, 1991; Hay, 1990; Lasko, 1990) and Tud localization is disrupted in embryos from vls mothers (Bardsley, 1993). However, conflicting data were reported subsequently. Assembly of an ectopic pole plasm at the anterior of the oocyte, caused by overexpressing osk (6xosk) or by targeting osk transcripts specifically to the anterior margin, results in progeny embryos with ectopic pole cells and duplication of the abdomen at the anterior. vls function was found to be required for the expression of the 6xosk phenotype, confirming its position downstream of osk, but not for the expression of the osk-bcd3'UTR phenotype (Ephrussi, 1992; Smith, 1992; Cavey, 2005 and references therein).

vls has now been cloned and characterized. A null mutant for vls has been created that shows stronger phenotypes than the presently available vlsEMS alleles. In contrast to previous models, this tool allows the demonstration that vls acts upstream of vas. Furthermore, vls dramatically affects the levels of Osk protein, even though localization of osk mRNA and initial accumulation of Osk do not require vls function. vls encodes a novel protein with significant similarity to WD domain proteins. The presented data suggest that Vls may act as a co-factor in assembling protein-protein and/or protein-RNA complexes (Cavey, 2005).

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 (Friesen, 2002). 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 (Callebaut, 1997), 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).

Thus the posterior gene vls encodes a maternal protein and is essential for the late localization of Vasa to the posterior of the oocyte as well as for the accumulation of Osk, which orchestrates pole plasm assembly. Unlike many other members of the posterior pathway, vls transcripts and Vls protein are not localized to the posterior but accumulate uniformly in the nurse cells and oocyte throughout oogenesis. Similar to vas, vls transcripts and proteins are also detected in adult males even though they have no essential function in males or fly spermatogenesis (Lasko, 1990; Snee, 2004). By contrast, in mice and probably other mammals, vas is important for male gametogenesis and has no essential function in female fertility. It would thus be interesting to know whether the same is true for vertebrate vls (Cavey, 2005).

Specification of the germline in Drosophila is more sensitive to pole plasm activity than is abdominal patterning. This is illustrated by the fact that weak alleles of posterior group mutants display a grandchildless phenotype caused by the lack of pole cells, while stronger alleles cause additional abdominal patterning defects that result in embryonic lethality (Lehmann, 1991). In this study, the hemizygous EMS alleles vlsPG65, vlsRB71 and vlsHC33 are only partially maternal-effect lethal and 100% grandchildless. vlsnull, however, is 100% maternal-effect lethal. The stronger phenotype of the null mutant suggests that the EMS alleles may be hypomorphs. However, the initial work on vls produced strong genetic evidence that the EMS alleles are actually nulls (Schupbach, 1986). It is therefore also possible that the EMS allele stocks accumulated maternal-effect modifiers that allow them to survive to adulthood (Cavey, 2005).

Although vlsEMS alleles contain premature stop codons in the vls ORF, the corresponding mutant mRNAs seem to escape nonsense-mediated mRNA decay mechanisms (NMD). Even though premature stop codons are recognized differently in Drosophila and vertebrates, the NMD components are conserved. Given that vls+ is translated during oogenesis, it seems unlikely that the mutants are protected because of lack of translation. It would thus be interesting to find out why vlsEMS transcripts accumulate to normal levels (Cavey, 2005).

Because all aspects of the vls mutant phenotype observed in embryos, including abdominal segment deletions, lack of pole cells, gastrulation defects and weak ventralization are rescued completely by a vls transgene and not even partially by a chk2 transgene, it is concluded that vls alone has a developmental requirement. Furthermore, chk2 function is only clearly required upon activation of cell cycle checkpoints. The vls phenotypes are reminiscent of a collapse of pole plasm assembly that seems to occur around stage 10 of oogenesis in vlsnull mutants. vas is crucial for the pole plasm to assemble properly and recruit the mRNAs and proteins required for pole cell specification and abdominal patterning. Genetic evidence implicates vas in the translational activation of several targets during oogenesis, including osk, grk and, in particular, nos at the posterior pole of the embryo. Vasa levels directly correlate with pole plasm activity, pole cell formation being more vulnerable to decreased Vasa levels than is abdominal patterning. Immunostaining for Vasa has been reported to show indistinguishable Vasa accumulation at the posterior pole of vls mutant and wild-type oocytes, and young embryos. These studies, performed with the homo- and hemi-zygous EMS mutants, showed a loss of posterior localization in the embryos from vls mothers sometime between fertilization and pole cell formation (Hay, 1990; Lasko, 1990). The current study used vas-eGFP transgenes to assess the posterior localization of Vasa in vlsnull and hemizygous EMS alleles in detail. Maximal localization was still very weak and was found in oocytes and embryos from vlsEMS mothers. In vlsnull mutants a nearly complete failure to localize Vas-eGFP at the posterior pole was observed. This failure coincides with the collapse of the pole plasm and is probably the cause for the various embryonic phenotypes mentioned above. Consistent with this, the observed Vasa localization defects parallel the severity of the phenotypes of these vls alleles. The weak accumulation of Vasa at the posterior of vlsPG65 hemizygous oocytes gives rise to a grandchildless phenotype, whereas the almost complete absence of Vasa from the posterior of vlsnull oocytes results in a fully penetrant maternal-effect lethal phenotype (Cavey, 2005).

vls is thus required during oogenesis for the localization (transport or anchoring) of Vasa to the posterior cortex of the oocyte. The fact that Vls is not specifically enriched at the posterior may suggest that it acts to modify or transport pole plasm components before they reach the posterior pole. Preliminary experiments also failed to produce evidence that Vls and Vasa are part of the same protein complex. This suggests that the mode of action of vls on Vasa localization is transient or indirect. The fact that osk mRNA and protein are initially correctly localized implies that oocyte polarity is normal in vls mutants and that vls is not required for osk mRNA localization. Levels of Osk protein isoforms are then reduced in later stages and Western analysis reveals a much more drastic decrease of overall Osk levels than immunostaining does for both types of vls alleles. This suggests that most of the drop in Osk levels occurs during the late stages of oogenesis, when the vitelline membrane prevents antibody staining for oocyte Osk. Therefore, it seems that shortly after initiating pole plasm assembly, Osk fails to be maintained at the posterior of vls mutants and progressively disappears, concurrent with a complete collapse of the pole plasm (Cavey, 2005).

Several lines of evidence implicate the Short Osk isoform in directly anchoring Vas. Short Osk interacts strongly with Vasa in the two-hybrid system and recruits Vasa when ectopically localized in the oocyte. Because Vas-eGFP mis-localization patterns in stage 10 oocytes are indistinguishable in vls and osk54 mutants, vls could act directly at the level of Osk accumulation (e.g. in stimulating translation of osk), which is necessary for anchoring Vasa at the posterior pole. In contrast, it is also possible that vls acts primarily on Vasa protein localization. Because Vasa also seems to act in a positive feedback loop back on Osk protein accumulation, the lack of Vasa localization in vls mutants would then also preclude maintenance of posterior accumulation of Osk protein. In vls mutants, Osk levels appear to decrease just slightly after Vasa should have localized to the posterior pole, thus it appears that the failure to localize Vasa could be the cause of the pole plasm collapse in vls mutants. To investigate these issues further, Osk levels in vas and tud mutants were compared with those in vls mutants by Western analysis where a more significant drop was detected than by immunostaining. This analysis revealed generally stronger phenotypes for vls than for vas and tud mutants. A comparable decrease of Short Osk levels was observed on Western blots of vls, vas and tud mutant extracts, but with slight differences in the extent of reduction of the hyper- and hypo-phosphorylated forms, both of which are more severely affected in vls mutants. In addition, a clear reduction of Long Osk levels was observed in vls, a minor reduction in tud, but none in vas mutant extracts. However, this analysis is complicated by the fact that the vas and tud alleles that are useful and available, respectively, for these experiments, are not nulls. Their residual activity may therefore maintain Osk at the posterior for a longer period of time. These data are thus consistent with the idea that vls acts on either pathway target, Vasa or Osk, in a process which could involve additional intermediates that remain to be identified (Cavey, 2005).

Why is vls not required for expression of the osk-bcd 3'UTR phenotype? vls was tentatively placed downstream of vas in the posterior pathway based on studies reporting that Vasa localization is correct initially in vlsEMS mutants (Hay, 1990; Lasko, 1990), and because vls was found to be required for the expression of the 6xosk phenotype (Smith, 1992). Surprisingly, however, vls is not required for the expression of the osk-bcd 3'UTR phenotype (Ephrussi, 1992). Since the 3'UTR is present in the 6xosk transgenes but not in the osk-bcd 3'UTR transgene, one explanation for this discrepancy could be that vls is required to relieve translational repression mediated by the osk 3'UTR (Cavey, 2005).

It is also possible that differences in osk mRNA levels and concentration at the anterior between the two systems might explain the discrepancy. In fact, Vasa protein accumulation at the posterior pole and the number of pole cells that develop afterwards correlate directly with the osk gene copy number (Ephrussi, 1992). Besides, 6xosk produces lower levels of osk mRNA at the anterior than osk-bcd3'UTR (Smith, 1992). Therefore, the ectopic pole plasm induced by osk-bcd3'UTR mRNA is probably more resistant to defects in localization/anchoring of downstream components such as Vasa or to defects in the maintenance of Osk itself. By contrast, the 6xosk system seems to represent a more sensitized background where the collapse of an ectopic pole plasm is more likely to occur in the absence of vls. Supporting this idea, the bicaudal phenotype of the progeny from transgenic mothers is 100% penetrant with osk-bcd 3'UTR (Ephrussi, 1992), but only 73% penetrant with 6xosk (Smith, 1992). vls might thus function as an enhancer of pole plasm assembly, which is dispensable when osk pole plasm-inducing activity is already extensively deployed at the anterior. This is consistent with the observation that vls dose also correlates with pole plasm activity in the same way that osk does. One copy of a wild-type vls+ transgene rescues almost completely the phenotypes described for vlsnull, but minor defects were sometimes noted compared with wild-type flies, as well as reduced hatching rates of embryos (Cavey, 2005).

vls differs in many respects from the other long-known members of the posterior pathway and seems to encode a co-factor acting on Osk protein accumulation, Vasa localization and possibly on another, yet unknown, component of this pathway. Two lines of evidence suggest that vls facilitates the process of pole plasm assembly but is not absolutely essential: some residual Vasa localization is possible even in the null mutant, and an ectopic pole plasm can assemble in the absence of vls function provided that the system is set up excessively or through different 3'UTR control elements (osk-bcd3'UTR vs. 6xosk). How could Vls perform this function at the molecular level? Vls is a divergent WD domain protein. The ß-propeller structure of WD proteins is thought to arise from the folding of at least four WD domains and to promote several simultaneous protein-protein interactions. Because computer predictions found only two or three such domains in Vls, preliminary experiments tested whether Vls forms homodimers. However, no untagged Vls was detected in immunoprecipitations performed with functional Vls-eGFP and Vls-6xHis fusion proteins. Whether Vls forms heterodimers with other WD domain-containing proteins remains to be tested. Sequence alignments point to a more likely interpretation. Vls and a whole family of Drosophila WD domain proteins show similarities to MEP50, which contains six WD domains and facilitates the interactions between a methyltransferase and its substrates, the Sm proteins (Friesen, 2002). Notably, the regions corresponding to the WD domains of MEP50 are better conserved than the others, suggesting that these domains are under greater selection pressure and may therefore fold in similar structures that can fulfill similar functions. This sequence comparison also shows that Vls might not be the ortholog of MEP50 and that different members of this family might fulfill the function of MEP50 in different Drosophila tissues (Cavey, 2005).

It is therefore possible that Vls also acts as a mediator of molecular interactions between proteins and possibly also mRNAs. Future experiments will have to focus on identifying the interactors of Vls to determine how precisely vls facilitates the pole plasm assembly process. The Vls interactions may turn out to represent an activating step in pole plasm assembly that involves a methyltransferase or another protein modification enzyme and their substrates. With this information it should then also be possible to clarify how directly this mechanism acts on the targets Vasa and Osk (Cavey, 2005).


GENE STRUCTURE

cDNA clone length - 1312 (see Entrez Nucleotide)

Bases in 5' UTR - 77

Exons - 3

Bases in 3' UTR - 121

PROTEIN STRUCTURE

Amino Acids - 367

Structural Domains

vls encodes a novel protein and PROSITE predicted the existence of two WD domains. Database searches reveal the best sequence similarity with the human methylosome protein 50 (MEP50; 20.4% identity) and alignment of Vls and MEP50 shows that the two predicted WD domains of Vls correspond closely to the predicted WD domains 2 and 3 of MEP50. With the exception of the WD domain 5, the predicted WD domains of MEP50 show elevated similarity with corresponding Vls regions compared to the alignment of the entire proteins. This suggests that Vls may have five to six domains that have a similar structure or function as WD domains, and it may mean that Vls has evolved from a WD domain protein. The six WD domains of MEP50 are thought to fold into a ß-propeller structure, which serves as a platform for recruiting the Arg-methyltransferase JBP1/PRMT5 and its substrates, the Sm proteins (Friesen, 2002). This event is required for assembling the splicing machinery prior to import into the nucleus (Cavey, 2005).

To test whether Vls may be the Drosophila ortholog of MEP50, the translated Drosophila genome was searched by BLAST for sequences similar to MEP50 and the CLUSTALW multiple alignments tool was used to analyze the results. The search revealed an entire family of Drosophila WD domain proteins with comparable sequence similarity to MEP50 and clearly a greater level of conservation over the WD domains than in regions outside. Although different algorithms give slightly different alignments, the protein products of CG6486, Lis-1 and vls have the highest levels of similarity over the WD domains of MEP50. The observed differences are too small to predict which one of the Drosophila proteins is more likely to be the ortholog of MEP50 (Cavey, 2005).

The feature characterizing the superfamily of WD-repeat proteins is the WD motif, a ~40 amino acid stretch typically containing a GH dipeptide 11-24 residues from its N-terminus and a WD dipeptide at its C terminus, albeit exhibiting only limited amino acid sequence conservation. When present in a protein, the WD motif typically occurs in multiple tandem repeated units. Based on structural analysis, the conformation of the WD repeat is defined as a series of four-stranded anti-parallel ß sheets, which fold into a higher-order structure termed a ß-propeller. At least four repeats are required to constitute a ß-propeller. Vls contains four conserved WD repeats and can potentially form a ß-propeller structure. Interestingly, the nearest mammalian homologue of Vls, the human MEP50 protein, displays six WD repeats. Although the sequence of the two additional repeats is conserved overall in Drosophila Vls, no characteristic GH and WD di-peptides could be found in these repeats (Anne, 2005).


EVOLUTIONARY HOMOLOGS

A large (20 S) protein arginine methyltransferase complex, termed the methylosome, has been described that contains the methyltransferase JBP1 (PRMT5) and the pICln protein. The methylosome functions to modify specific arginines to dimethylarginines in the arginine- and glycine-rich domains of several spliceosomal Sm proteins, and this modification targets these proteins to the survival of motor neurons (SMN) complex for assembly into small nuclear ribonucleoprotein (snRNP) core particles. A novel component of the methylosome, a 50-kilodalton WD repeat protein termed methylosome protein 50 (MEP50), is described. MEP50 is important for methylosome activity and binds to JBP1 and to a subset of Sm proteins. Because WD repeat proteins provide a platform for multiple protein interactions, MEP50 may function to mediate the interaction of multiple substrates with the methylosome. Interestingly, all of the known components of the methylosome bind Sm proteins, suggesting that in addition to producing properly methylated substrates for the SMN complex, the methylosome may be involved in Sm protein rearrangements or pre-assembly required for snRNP biogenesis (Friesen, 2002).

RNA polymerase II transcription is associated with cyclic phosphorylation of the C-terminal domain (CTD) of the large subunit of RNA polymerase II. To date, FCP1 is the only specific CTD phosphatase, that is required for general transcription and cell viability. To identify FCP1-associated proteins, a human cell line expressing epitope-tagged FCP1 was constructed. In addition to RAP74, a previously identified FCP1 interacting factor, it was determined that FCP1-affinity purified extracts contain RNAPII that has either a hyper- or a hypo-phosphorylated CTD. In addition, by mass spectrometry of affinity purified FCP1-associated factors, a novel FCP1-interacting protein, named MEP50, a recently described component of the methylosome complex that binds to the snRNP's Sm proteins, was identified. FCP1 specifically interacts with components of the spliceosomal U small nuclear ribonucleoproteins. These results suggest a putative role of FCP1 CTD-phosphatase in linking the transcription elongation with the splicing process (Licciardo, 2003).


valois: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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