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 Vase 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).

GENE STRUCTURE

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).

date revised: 20 April 98

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