vasa: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - vasa

Synonyms -

Cytological map position - 35C1-2

Function - assembly of pole plasm

Keyword(s) - posterior group - oocyte protein

Symbol - vas

FlyBase ID:FBgn0283442

Genetic map position - 2-[51]

Classification - RNA helicase - DEAD box

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Wang, S. C., Hsu, H. J., Lin, G. W., Wang, T. F., Chang, C. C. and Lin, M. D. (2015). Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation. Sci Rep 5: 14703. PubMed ID: 26419889
Summary:
Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. Glutamine (Gln) 527 within HELICc of DmVas was identified as critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.
Jeske, M., Muller, C. W. and Ephrussi, A. (2017). The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage. Genes Dev [Epub ahead of print]. PubMed ID: 28536148
Summary:
DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. This study shows that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. In vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved.
BIOLOGICAL OVERVIEW

Vasa protein is essential for the assembly of the pole plasm, a special cytoplasm found in the posterior portion of the egg and early embryo. The pole plasm is required for localization of the mRNA of the posterior determinant Nanos, and serves as the cytoplasm of pole cells, the zygotic cells reserved in development for the establishment of gonads. Nearly a dozen maternal genes, belonging to the posterior group, are involved directly or indirectly in assembly of the pole plasm.

Vasa is an RNA binding protein with an RNA dependent helicase. The helicase activity unwinds RNA, a crucial process since a three dimensional tangled or self annealed structure of RNA would otherwise prevent transcription. Modification of RNA tertiary structure is therefore an important aspect of post-transcriptional regulation.

Vasa has been associated with two other developmental processes. The first involves assembling the perinuclear region of the oocyte. Perinuclear cytoplasm is the precursor of the pole plasm. In the middle phase of oogenesis, it moves from the perinuclear region to the pole, along with Vasa protein. The integrity of the cell's cytoskeleton is crucial for the first occasion of Vasa localization in the periplasmic cytoplasm, as well as the second occasion in the pole plasm.

The second process with which Vasa is associated is the helicase function. The helicase function of Vasa is not required for Vasa localization, but it is required for the assembly of pole plasm. Posterior localization of Vasa depends most likely on an interaction with Oskar. Oskar can successfully localize to the posterior pole without Vasa, but Oskar by itself cannot assemble the pole plasm. Both Oskar and Vasa activity are necessary for Nanos mRNA localization at the posterior pole (Liang, 1994). The function of VASA is to overcome the repressive effect of Nanos translational control element, an evolutionarily conserved dual stem-loop structure in the 3' untranslated region which acts independently of the localization signal to repress translation of Nanos mRNA (Gavis, 1996).

While the activities of pole plasm components such as Vasa have been most thoroughly studied with respect to their function in pole cell formation and specification of the posterior soma, it is clear that some genes involved in pole plasm assembly also function in other stages of germline development. For instance, females homozygous for either of two strong nanos alleles exhibit defects in germ cell proliferation. Furthermore, pole cells lacking maternal nos function fail to complete migration and do not associate with the embryonic gonadal mesoderm, indicating a role for nos in the transition from pole cell to functional germ cell. Similarly, various vas alleles have defects in oogenesis and lay few or no eggs. Females trans-heterozygous for vas deficiency alleles, are blocked in early vitellogenic stages of oogenesis. Analysis of whether this phenotype is caused solely by loss of vas function has been confounded by the fact that these trans-heterozygous deficiency lines are haploid for a large number of genes; however, aside from large deficiencies, a clearly null allele of vas had not been identified. A new vas null allele, vas PH165, was generated by imprecise P-element excision, to investigate in detail the role of vas in oogenesis events prior to pole plasm assembly. Abrogation of vas function results in defects in many aspects of oogenesis, including control of cystocyte divisions, oocyte differentiation, and specification of posterior and dorsal follicle cell-derived structures. In vasa-null ovaries, germaria are atrophied, and contain far fewer developing cysts than do wild-type germaria. This is a phenotype similar to (but less severe than) that of a null nanos allele. vas PH165 oocytes only weakly concentrate many oocyte-localized RNAs, including Bicaudal-D, Orb, Oskar, and Nanos mRNA. Some oocyte-specific molecules, including Gurken RNA, remain concentrated in the oocyte in vas mutant ovaries. However, in the case of Grk, translation is severely reduced in the absence of vas function. This provides evidence that Vasa is involved in translational control mechanisms operating in the early stages of oogenesis (Styhler, 1998).

The vas PH165 mutant is null for vasa. At a low frequency (approx. 1% for each), defects are observed in germline differentiation and oocyte determination in vas PH165 mutant ovaries, including tumorous egg chambers, egg chambers with 16 nurse cells and no oocyte, others with two oocytes, and again others with a mislocalized oocyte. Far more frequently the normal 15 nurse cells and one oocyte are present; however, by at least two criteria, the oocytes produced in vas PH165 egg chambers are not fully differentiated. In wild-type development, the nurse cell nuclei endoreplicate during pre-vitellogenic oogenesis and become highly polyploid, whereas the oocyte nucleus remains diploid and condenses into a tight karyosome. However, in vas PH165 the oocyte nucleus appears more diffuse than does the wild-type oocyte nucleus. This would be consistent either with a failure to form the karyosome structure, perhaps involving a premature meiotic arrest in diplotene rather than in metaphase, or an increase in ploidy in vas PH165 oocytes. A very similar nuclear morphology has been observed in spindle (spn) mutant oocytes, which has been interpreted as resulting from a delay in oocyte determination (see Homeless). In addition, vas PH165 oocytes do not efficiently accumulate at least four oocyte-localized RNAs. In wild-type ovaries, the Bicaudal-D, ORB, OSK and NOS mRNAs all accumulate efficiently in the oocyte within the germarium and remain concentrated therein throughout the early stages of oogenesis. Oocyte accumulation of these RNAs is much less pronounced in vas PH165 egg chambers in which Bic-D RNA localization is essentially undetectable, and the concentration of ORB RNA in the oocyte is only slightly above the levels in the nurse cells. In vas PH165 egg chambers, OSK RNA is also not observed to accumulate in the oocyte in the germarium or first vitellarial stages of development. Rather, OSK RNA tends to concentrate in a somewhat diffuse manner in the center of the egg chamber. Finally, loss of vas function has the least pronounced effect on the accumulation of NOS RNA into the oocyte, but even in this case oocyte localization is incomplete and poorly maintained (Styhler, 1998).

Gurken translation is severely reduced in the absence of vas function. The phenotypes of late-stage vasa PH165 null mutant oocytes suggest that the Gurken signaling pathway may be inactive in these ovaries. This observation led to an investigation of the expression and distribution of GRK mRNA and protein in vas PH165. Unlike Bicaudal-D, ORB, OSK and NOS mRNA, localization of GRK mRNA to the oocyte remains highly efficient in vas PH165 egg chambers. However, while the distribution of GRK mRNA forms an obvious posterior crescent in wild-type oocytes from about stage 2-3, GRK mRNA remains tightly concentrated in an extremely small area in vas PH165 oocytes. Later in oogenesis, GRK RNA becomes anteriorly localized in both wild-type and vas PH165 oocytes, although in the mutant its distribution may extend further ventrally. Despite the relatively normal accumulation of GRK RNA in vas PH165 oocytes, the effects of a loss of vas activity are striking with regard to Grk protein accumulation: essentially no localized Grk is observed in vas PH165 oocytes. Furthermore, as measured on western blots, the level of Grk protein is greatly reduced in vas PH165 ovaries as compared with wild-type. Since grk is required for specification of dorsal and posterior follicle structures, the duplicated micropyles and dorsal appendage defects found in vas PH165 eggs (and in eggs produced by other vas alleles) are likely to be caused by the reduced level of Grk protein in vas mutants. These results also suggest that Vas may activate GRK translation in wild-type oocytes (Styhler, 1998).

Interaction with eIF5B is essential for Vasa function during development

The DEAD-box RNA helicase Vasa (Vas) is required for germ cell development and function, as well as for embryonic somatic posterior patterning. Vas interacts with the general translation initiation factor eIF5B (cIF2, also known as dIF2), and thus may regulate translation of specific mRNAs. In order to investigate which functions of Vas are related to translational control, the effects of site-directed vas mutations that reduce or eliminate interaction with eIF5B were examined. Reduction in Vas-eIF5B interaction during oogenesis leads to female sterility, with phenotypes similar to a vas null mutation. Accumulation of Gurken (Grk) protein is greatly reduced when Vas-eIF5B interaction is reduced, suggesting that this interaction is crucial for translational regulation of grk. In addition, reduction in Vas-eIF5B interaction virtually abolishes germ cell formation in embryos, while producing a less severe effect on somatic posterior patterning. It is concluded that interaction with the general translation factor eIF5B is essential for Vas function during development (Johnstone, 2004).

A mutant form of Vas, VasDelta617, was analyzed that has greatly reduced ability to interact with eIF5B. Since residue 617 is not involved in binding to any known Vas-interacting protein other than eIF5B, and it is outside the region of Vas that contains the well-characterized catalytic domains that are present in all DEAD-box proteins, it is clear that VasDelta617 specifically disrupts the Vas-eIF5B interaction, and that this mutation can be used to identify developmental processes that are sensitive to an association between Vas and the general translational machinery. The VaseIF5B interaction is crucial for the progression of oogenesis, for correct dorsoventral patterning of the egg, and for expression of high levels of Grk in the developing oocyte. These results are most easily explained if grk is a target for Vas-mediated translational activation acting through its association with eIF5B (Johnstone, 2004).

A role for Vas in positively regulating grk translation is consistent with previous work. Vas-mediated regulation of grk is in turn regulated in response to a meiotic checkpoint, activated when DNA double-strand break (DSB) repair is prevented during meiotic recombination. In response to this checkpoint, Vas is post-translationally modified, and Grk accumulation is reduced. It will be important to understand the nature of the DSB-dependent modification of Vas, and to determine whether it affects the Vas-eIF5B interaction, in order to gain insight into the mechanism connecting cell cycle regulation with oocyte patterning (Johnstone, 2004).

The RNA-binding and unwinding activities of wild-type Vas and several mutant forms of Vas have been assessed through previous in vitro assays. Two mutant forms of Vas, encoded by the vasO14 and vasO11 alleles, are severely reduced for binding to an artificial RNA substrate, and a third form, encoded by vasD5, was defective for RNA unwinding but not for binding. Although vasD5 leads to defects in oogenesis, vasO11 phenotypically resembles vasPD, and vasO14 is a weak temperature-sensitive allele. In the light of the present results, it is surprising that a mutant form of Vas that cannot interact with RNA would nevertheless support oogenesis. Perhaps in vivo, the RNA-binding and helicase activities of Vas are stimulated or enhanced through a co-factor or through posttranslational modifications, and the in vitro assay used in an earlier study may not accurately reflect Vas activity in vivo (Johnstone, 2004).

Are there target RNAs for Vas-eIF5B regulation in the pole plasm? Reduction of the Vas-eIF5B interaction by expressing VasDelta617 severely reduces pole cell formation. This happens despite the ability of vasPD;P{vasDelta617} oocytes to accumulate Osk, Vas and Tud at the posterior pole, demonstrating an essential role for Vas in pole cell specification that is dependent upon its association with eIF5B, and that cannot be substituted by Osk and Tud. The simplest interpretation of these results is that Vas derepresses translation of a localized RNA required for pole cell specification, in a manner analogous to what appears to be the case for grk (Johnstone, 2004).

The possibility is considered that the Vas-eIF5B interaction could target osk mRNA. It has been shown that whereas Osk protein accumulates normally in vas mutant ovaries, Osk levels are severely reduced at the posterior of vas mutant embryos, suggesting a role for Vas in posterior accumulation of Osk after its initial recruitment, and/or in stabilizing Osk at the posterior. Comparable and substantial levels of Osk are observed at the posterior in vasPD;P{vasDelta617} and vasPD;P{vas+} embryos, arguing against a direct role for the VaseIF5B interaction in activating translation of osk mRNA. A requirement for Vas in Par1-mediated phosphorylation and stabilization of Osk has been suggested. Since VasDelta617 localizes normally and is able to interact with Osk, this mutation would not be expected to have any effect on this Osk modification pathway. Thus, the findings are consistent with a model whereby Vas influences Osk activity through effects on phosphorylation, anchoring and/or stability, perhaps through Par1, rather than directly regulating osk translation (Johnstone, 2004).

Another candidate target for the Vas-eIF5B interaction is germ cell-less (gcl), the activity of which is important for pole cell specification but not for posterior patterning. Unfortunately, with current reagents Gcl protein cannot be detected even in wild-type embryos prior to pole bud formation, thus the effects on gcl translation of any mutation that abrogates pole cell formation cannot presently be addressed. In addition, effects on gcl cannot fully explain the severe consequences of the VasDelta617 mutation on pole cell formation, because the number of pole cells formed in maternal gcl-null embryos is somewhat higher than in vasPD;P{vasDelta617} embryos. This suggests that even if gcl is a target, the Vas-eIF5B interaction may regulate translation of more than one target RNA involved in pole cell formation (Johnstone, 2004).

Is the Vas-eIF5B interaction required for posterior patterning? Although the Vas-eIF5B interaction is vital for pole cell specification, it is perhaps less so for posterior patterning and establishment of the Nos gradient. Previous analysis of hypomorphic mutations in posterior-group genes, including vas has indicated that a higher level of activity is required for pole cell specification than for posterior patterning. For example, all embryos produced by females homozygous for vasO14, osk301 and tudWC, lack pole cells, but some have normal posterior patterning and are able to hatch. The present results suggest two alternative explanations for these observations. One possibility is that the Vas-eIF5B interaction is required for posterior patterning, but that the residual activity present in VasDelta617 is sufficient to achieve the low activity level that is necessary. Alternatively, the Vas-eIF5B interaction may be dispensable for posterior patterning, and the fact that complete rescue of this phenotype is not observed with the vasDelta617 transgene may be due to an indirect effect of this mutation, resulting from a general destabilization of the pole plasm that occurs in embryos that do not form pole cells. In such embryos, pole plasm components localize initially but become fully delocalized by the blastoderm stage. Consistent with this idea, all of the pole plasm components examined that are downstream of Vas, including nos RNA, could be detected at the posterior of vasPD;P{vasDelta617} embryos, although to variable degrees (Johnstone, 2004).

Previous work has suggested that nos may be a target for Vas-mediated translational regulation. Outside of the pole plasm, nos translation is repressed through the binding of Smg, and possibly other repressors, to its 3' UTR. Smg achieves this regulation at least in part through interaction with the eIF4E-binding protein Cup, thus influencing the cap-binding stage of translation. Within the pole plasm, in complexes with Osk and Vas, nos translational repression is overcome, potentially through a direct interaction between Osk and Smg that may displace Smg-Cup interaction. Analysis of VasDelta617 does not support an important role for the VaseIF5B interaction in activating nos translation in the pole plasm, since it is clear that translation of nos is far less sensitive to the level of this interaction than is translation of grk in early oocytes. The primary function of Vas in nos accumulation may therefore be in anchoring nos mRNA in complexes within the pole plasm, consistent with recent observations that nos mRNA is trapped at the posterior by complexes containing Vas. Of course it remains possible that the low level of residual eIF5B binding provided by VasDelta617 is sufficient to fulfill a role of Vas in activating translation of this transcript (Johnstone, 2004).

How might Vas-eIF5B interaction regulate translation of grk and potentially other target mRNAs? Cap-dependent translation initiation in eukaryotes requires many translation initiation factors, and involves several main steps. Most known mechanisms of translational regulation impinge on the recruitment of the cap-binding complex eIF4F to the mRNA: this represents the rate-limiting first step of initiation. mRNA circularization through proteins such as Cup serves an important role in translational control by allowing 3' UTR-bound regulatory factors to influence translation initiation at the 5' end of the transcript (Johnstone, 2004).

60S ribosomal subunit joining represents the interface between translation initiation and elongation, and the VaseIF5B interaction suggests a distinct mechanism of translational control occurring at this last stage of initiation. Although this step has not historically been considered a target for regulation, several examples have emerged to suggest that subunit joining may in fact be subject to regulation. Translational repression of mammalian 15-lipoxygenase (LOX) mRNA is mediated by hnRNP proteins that bind to a specific 3' UTR regulatory element, and which are thought to act by blocking the activity of either eIF5 or eIF5B. An additional link between mRNA 3' regulatory regions, and eIF5B activity, comes from analysis of two DEAD-box proteins in yeast, Ski2p and Slh1p. These proteins are required to achieve the selective translation of poly(A)+ mRNAs, relative to poly(A)- mRNAs, and genetic experiments suggest that they specifically repress the translation of poly(A)- mRNAs by acting through eIF5 and eIF5B (Johnstone, 2004).

Together with these studies, the current work suggests that in the Drosophila germline, specific translational repression events may target eIF5B and the ribosomal subunit joining step of initiation. Vas, which potentially functions at the 3' UTR through interaction with specific repressor proteins, may act to alleviate a translation block occurring at this step. Such a model is consistent with what is known about translational regulation of grk. For example, grk translation is repressed by Bru, which binds to a Bruno-response element within its 3' UTR. Vas interacts with Bru, suggesting that Vas could function as a derepressor by overcoming Bru-mediated repression of grk translation. However, the inability of a vas transgene to ameliorate the phenotype of nosGAL4VP16-driven overexpression of Bru, might argue against this model. The mechanism by which Bru regulates grk remains unclear. Translational repression of osk by Bru relies on direct interaction with Cup, linking Bru with eIF4E. However, mutations in cup that prevent interaction with Bru do not appear to affect Grk expression, suggesting that Bru may operate through a distinct mechanism to regulate grk translation. In addition, in vitro translation assays have suggested that Bru can mediate translational repression through a cap-independent mechanism. Thus, Bru may be capable of regulating translation at more than one stage. Based on the observations for the mammalian hnRNP proteins on the LOX mRNA, and the Ski2p and Slh1p helicases in yeast, specific translational repressors such as Bru could target the subunit joining step of initiation (Johnstone, 2004).

eIF5B is thought to form a molecular bridge between the two ribosomal subunits, and to play a fundamental role in stabilizing the initiator Met-tRNAiMet in the ribosomal P site. Inhibition of eIF5B activity could occur while the factor is bound to the initiation complex, at the start codon, and block its ability to link or stabilize the ribosomal subunits. Through circularization of the mRNA, this block could be achieved by trans-acting factors at the 3' UTR, and the Vas-eIF5B interaction may be involved in alleviating these specific repression events, potentially through displacement of a repressor protein. Alternatively, Vas could play a role in recruitment of eIF5B to specific transcripts. Since eIF5B is required for all cellular translation, a general mechanism must exist to recruit this factor to all transcripts. However, in a scenario where repressor proteins may be blocking the subunit joining step, either through a direct effect on eIF5B, or another mechanism, it is conceivable that eIF5B could become limiting for translation. In this situation, Vas could play a role in recruiting this factor to specific transcripts (Johnstone, 2004).


GENE STRUCTURE

cDNA clone length - 2.0kb

Exons - seven


PROTEIN STRUCTURE

Amino Acids - 660

Structural Domains

Vasa shows significant sequence similarity to eIF-4A, a translation initiation factor that binds to mRNA, and to other helicases (Hay, 1988 and Lasko, 1988). Vasa contains a DEAD box employed to unwind RNA.

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for theDEAD and DEAH box families ATP-dependent helicases signatures.

Within the superfamily, several subfamilies of related proteins can be distinguished by two criteria: particular signature motifs [e.g., the sequence DEAD (Asp, Glu, Ala, Asp) in segment II] and a higher overall sequence identity throughout the entire helicase domain. On the basis of these criteria, Maleless is a member of a subfamily of putative helicase proteins, the DEAH family. The first described members of this family are three proteins involved in pre-mRNA processing in yeast (PRP2, PRP16 and PRP22). The similarity between Mle and PRP2, PRP16 and PRP22 is extensive throughout the putative helicase domain. Similarity between Mle and DEAH family members extends approximately 100 amino acids beyond the last conserved segment (VI) of the large superfamily. Despite the high overall similarity of Mle to DEAH family members, Mle carries a difference in the proposed signature sequence, with the sequence DEIH rather than DEAH in segment II of the putative ATP-binding motif. Outside of the putative helicase domain, Mle does not show significant sequence similarity to proteins in current data bases. The C-terminal region contains nine imperfect repeats of a glycine-rich sequence. The posterior embryonic determinant Vasa, a member of the superfamily of putative helicases, encodes six copies of a glycine-rich heptad repeat (Kuroda, 1991 and references).


vasa: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 April 98

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