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 vas^O14 and vas^O11 alleles, are severely reduced for binding to an artificial RNA substrate, and a third form, encoded by vas^D5, was defective for RNA unwinding but not for binding. Although vas^D5 leads to defects in oogenesis, vas^O11 phenotypically resembles vas^PD, and vas^O14 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 vas^PD;P{vas^Delta617} 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 vas^PD;P{vas^Delta617} and vas^PD;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 vas^PD;P{vas^Delta617} 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 vas^O14, osk³⁰¹ and tud^WC, 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 vas^Delta617 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 vas^PD;P{vas^Delta617} 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-tRNA_i^Met 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).

The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage

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; Tejas), and TDRD7 (Tapas) 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. It was shown that, 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 (Jeske, 2017).

This study provides molecular insight into the function of animal LOTUS domain proteins, factors involved in diverse germline functions. The DEAD-box helicase Vasa interacts with the LOTUS domains of Oskar, TDRD5/Tejas, and TDRD7/Tapas but not with MARF1. In Drosophila, interaction with LOTUS domain proteins is required for Vasa localization to the nuage and germ plasm. Structural and functional analyses of the LOTUS-Vasa interaction uncovered a key role of a C-terminal extension present in only a subset of LOTUS domains, pointing to two LOTUS domain subclasses with distinct functions in animals. The eLOTUS domain of Oskar, TDRD5, and TDRD7 not only interacts with Vasa but also stimulates its helicase activity. The mLOTUS domains present in MARF1 lack this extension and very likely have a distinct role within the germline that will need to be addressed in the future. While Drosophila TDRD5 (Tejas) and TDRD7 (Tapas) contain a single eLOTUS domain, some TDRD5 and TDRD7 proteins from other animals harbor mLOTUS domains in addition to their N-terminal eLOTUS domain. Whether the mLOTUS domains from MARF1, TDRD5, and TDRD7 have related activities or are functionally distinct remains to be determined (Jeske, 2017).

The Drosophila eLOTUS domain proteins Oskar, Tejas, and Tapas have been considered to be scaffolding proteins whose function is to recruit Vasa and other germline factors to germ plasm or the nuage. While LOTUS domains were originally predicted to be RNA-binding domains, attempts to detect any RNA-binding activity of the eLOTUS domain of Oskar have failed. The present study uncovered a conserved function of eLOTUS domains in binding and stimulating a DEAD-box RNA helicase, thus attributing an active regulatory role to Oskar, Tejas, and Tapas in the germline. The stimulation of the ATPase activity of Vasa by the eLOTUS domain seems universal, but its consequence and function within the germline are unknown. In Drosophila, Vasa stimulation by Tejas and/or Tapas in the nuage might be involved in the piRNA pathway, whereas Vasa stimulation by Oskar in the pole plasm likely has a distinct role. Vasa was suggested to activate translation of mRNAs in the egg chamber through recruitment of eIF5B, which catalyzes ribosomal subunit joining to form elongation-competent ribosomes. Vasa has been shown to physically interact with eIF5B in yeast two-hybrid assays and pull-down experiments from lysates. A Vasa region that extends C-terminally from the helicase core was shown to be required for the eIF5B interaction, which raised the question of whether eLOTUS and eIF5B jointly or mutually exclusively bind to Vasa. Attempts were made to test this in GST pull-down assays with recombinant proteins. However, surprisingly, no interaction of Vasa with GST-eIF5B or any change in Vasa's ATPase activity in the presence of eIF5B was detected. It is concluded that Vasa and eIF5B do not physically interact and that the recruitment of eIF5B by Vasa might be mediated through RNA or other proteins. It is equally plausible that Vasa's role in translation might be that of a DEAD-box RNA helicase involved in remodeling RNA-protein complexes. Given its importance in germline biology, the mechanism by which Vasa promotes translation of mRNAs merits thorough re-examination (Jeske, 2017).

In the nuage, Vasa is essential for the secondary piRNA biogenesis pathway, also known as the Ping-Pong cycle. Bombyx Vasa associates with the Piwi proteins Siwi and Ago3, two major players in the Ping-Pong cycle in the germ plasm. Within the Ping-Pong cycle, Siwi is loaded with piRNAs, and the complex binds and cleaves transposon mRNAs in an orientation antisense to piRNAs. The cleavage products are then loaded into Ago3, and the complex recognizes and cleaves piRNA cluster transcripts, leading to specific amplification of piRNAs that target transposon mRNAs present in the cell. Vasa is required for the safe handover of transposon mRNA fragments from Siwi to Ago3. Furthermore, the ATPase activity of Vasa is necessary for the release of transposon RNAs from Siwi-piRNA complexes after cleavage. It is therefore possible that stimulation of Vasa by the Tejas and/or Tapas eLOTUS domains is required for high efficiency of the Ping-Pong cycle. The higher activity of Tejas compared with Tapas that was detected might be reflected in vivo by its dominant role in transposon silencing within the nuage (Jeske, 2017).

LOTUS domains are not restricted to animals but are also present in bacteria, fungi, and plants-organisms without a Vasa ortholog. From sequence alignments, it appears that bacterial, fungal, and plant LOTUS domains lack the particular C-terminal extension, and it will be interesting to investigate and compare their function with that of mLOTUS domains of animal proteins, such as MARF1 (Jeske, 2017).

Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos

The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA pathway component, but has additional important developmental functions. This study addresses the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. Transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. Elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. Suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. These findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development (Durdevic, 2018).

This study shows that a transient loss of vas expression during early oogenesis leads to up-regulation of transposon levels and compromised viability of progeny embryos. The observed embryonic lethality is because of DNA DSBs and nuclear damage that arise as a consequence of the elevated levels of transposon mRNAs and proteins, which are transmitted from the mother to the progeny. This study thus demonstrates that transposon silencing in the nurse cells is essential to prevent maternal transmission of transposons and DNA damage, protecting the progeny from harmful transposon-mediated mutagenic effects (Durdevic, 2018).

The finding that suppression of Chk2-mediated DNA damage signaling in loss-of-function vas mutant flies restores oogenesis, and egg production demonstrates that Chk2 is epistatic to vas. However, hatching is severely impaired, because of the DNA damage sustained by the embryos. The defects displayed by vas, mnk double mutant embryos resembled those of PIWI (piwi, aub, and ago3) single and mnk; PIWI double mutant embryos. Earlier observation that inactivation of DNA damage signaling does not rescue the development of PIWI mutant embryos led to the assumption that PIWI proteins might have an essential role in early somatic development, independent of cell cycle checkpoint signaling. By tracing transposon protein and RNA levels and localization from the mother to the early embryos, it is suggested that, independent of Chk2 signaling, de-repressed transposons are responsible for nuclear damage and embryonic lethality. This study indicates that transposon insertions occur in the maternal genome where they cause DNA DSBs that together with transposon RNAs and proteins are passed on to the progeny embryos. Transposon activity and consequent DNA damage in the early syncytial embryo cause aberrant chromosome segregation, resulting in unequal distribution of the genetic material, nuclear damage and ultimately embryonic lethality. This study shows that early Drosophila embryos are defenseless against transposons and will succumb to their mobilization if the first line of protection against selfish genetic elements in the nuage of nurse cell fails (Durdevic, 2018).

A recent study showed that in p53 mutants, transposon RNAs are up-regulated and accumulate at the posterior pole of the oocyte, without deleterious effects on oogenesis or embryogenesis. It is possible that the absence of pole plasm in vas mutants results in the release of the transposon products and their ectopic accumulation in the oocyte. Localization of transposons to the germ plasm may restrict their activity to the future germline and protect the embryo soma from transposon activity. Transposon-mediated mutagenesis in the germline would produce genetic variability, a phenomenon thought to play a role in the environmental adaptation and evolution of species. It would therefore be of interest to determine the role of pole plasm in transposon control in the future (Durdevic, 2018).

Transposon up-regulation in the Drosophila female germline triggers a DNA damage-signaling cascade. In aub mutants, before their oogenesis arrest occurs, Chk2-mediated signaling leads to phosphorylation of Vasa, leading to impaired grk mRNA translation and embryonic axis specification. Considering the genetic interaction of vas and mnk (Chk2) and the fact that Vasa is phosphorylated in Chk2-dependent manner, it is tempting to speculate that phosphorylation of Vasa might stimulate piRNA biogenesis, reinforcing transposon silencing and thus minimizing transposon-induced DNA damage. The arrest of embryonic development as a first, and arrest of oogenesis as an ultimate response to DNA damage, thus, prevents the spreading of detrimental transposon-induced mutations to the next generation (Durdevic, 2018).

A truncated form of a transcription factor Mamo activates vasa in Drosophila embryos

Expression of the vasa gene is associated with germline establishment. Therefore, identification of vasa activator(s) should provide insights into germline development. However, the genes sufficient for vasa activation remain unknown. Previous work showed that the BTB/POZ-Zn-finger protein Mamo is necessary for vasa expression in Drosophila. This study showed that the truncated Mamo lacking the BTB/POZ domain (MamoAF) is a potent vasa activator. Overexpression of MamoAF was sufficient to induce vasa expression in both primordial germ cells and brain. Indeed, Mamo mRNA encoding a truncated Mamo isoform, which is similar to MamoAF, was predominantly expressed in primordial germ cells. The results of these genetic and biochemical studies showed that MamoAF, together with CBP, epigenetically activates vasa expression. Furthermore, MamoAF and the germline transcriptional activator OvoB exhibited synergy in activating vasa transcription. It is proposed that a Mamo-mediated network of epigenetic and transcriptional regulators activates vasa expression (Nakamura, 2019).

Although it is well recognised that maternal translational and transcriptional repressors play essential roles in establishing PGCs in Drosophila, the genes sufficient for vas activation in PGCs remain unknown. This study identified two types of vas activators: full-length Mamo, a weak but specific inducer of vas, and Mamo short isoform, a potent inducer of vas activation. To clarify the molecular mechanisms of vas activation, biochemical and genetic analyses were conducted using MamoAF-induced vas expression; two cofactors of MamoAF, CBP and OvoB, are both involved in activation of vas in PGCs. Thus, MamoAF-induced vas expression is useful for identifying cofactors of vas activation in PGCs. MamoAF can induce vas expression in both PGCs and brain. In both cellular contexts, the transcriptional activator OvoB is necessary for MamoAF-induced vas expression. Moreover, overexpression of both MamoAF and OvoB is sufficient to induce vas expression in VNC. Thus, the Mamo-OvoB axis is essential for directing vas activation. These data revealed that MamoAF functions as a molecular hub: it collaborates with CBP to epigenetically activate the vas locus, and physically interacts with OvoB to stimulate vas transcription. Consistent with this notion, a reporter assay demonstrated that these factors worked together to stimulate transcription. It is concluded that the Mamo-mediated network of epigenetic and transcriptional regulators directs vas activation in Drosophila embryos (Nakamura, 2019).

MamoAF directly activates vas expression through the vas-A element in the first intron, which is essential for endogenous vas expression in PGCs. However, MamoAF could also activate vas transcription through other cis-elements that remain to be identified. It has been previously reported that Mamo has a role in the regulation of chromatin structure. Therefore, Mamo may regulate chromatin structure, in addition to transcriptional activation, to promote vas expression (Nakamura, 2019).

A previous study using reporter assays showed that a 40-bp element in the 5' flanking region of vas, the up-40 element, is sufficient to recapitulate germline-specific expression during oogenesis and embryogenesis. The up-40 element does not contain consensus sequences for either Mamo or Ovo. This implies that different transcriptional factors must control vas expression through the up-40 element. Because removal of the vas-A element decreases endogenous vas expression in PGCs, vas-A and up-40 elements may act in a partially redundant manner to upregulate vas expression in PGCs. Therefore, multiple enhancers may act in parallel to activate vas expression in germ cells. However, it remains unclear whether the up-40 element is necessary for endogenous vas expression (Nakamura, 2019).

This study focused on MamoAF to investigate the mechanisms of vas activation due to its potent activity for vas activation. Thus, it remains unclear how full-length Mamo activates vas expression. Understanding the mechanism requires the identification of factors regulating the nuclear localisation of full-length Mamo. Maternal full-length Mamo in the nuclei of early PGCs may interact with OvoB, which is maternally provided and enriched in PGC nuclei1. However, it will be necessary to investigate the interaction between full-length Mamo and OvoB under conditions in which full-length Mamo is not converted into short derivatives (Nakamura, 2019).

Both full-length and short form Mamo mRNAs are expressed in germ cells in ovaries. However, full-length Mamo rescues the differentiation of mamo mutant germline clones more efficiently than MamoAF. It was found that full-length Mamo can be converted to truncated derivatives. Thus, full-length Mamo has the potential to complement the short isoform. By contrast, the short form of Mamo does not appear to complement the function of the full-length protein owing to the lack of the BTB/POZ domain. During oogenesis, the transcriptional activity of full-length Mamo may be regulated through interactions with epigenetic regulators via the BTB/POZ domain, as reported for other BTB/Zn-finger transcription factors. Full-length Mamo is enriched in the nuclei of the nurse cells in egg chambers after oogenic stage 6, but MamoAF is only weakly enriched. Thus, full-length Mamo may play a role in regulating gene expression in nurse cells and promoting the differentiation of oocytes into mature eggs (Nakamura, 2019).

It is proposed that Mamo short isoform is a potent vas activator. The C2H2 Zn-finger domains of Mamo short isoform are homologous to those of human Sp1;20 Sp1-related transcription factors regulate gene expression in germ cells in vertebrates. For example, chicken Sp1 promotes vas expression in PGCs. Moreover, Ovo has conserved roles in germline development in mouse and Drosophila. Accordingly, Sp1-related Zn-finger proteins and Ovo may also be key transcriptional activators of vas in other animals, including mouse and human. It is anticipated that these results will facilitate understanding of the molecular mechanisms that regulate germline development in animals (Nakamura, 2019).

Comparative Proteomics Reveal Me31B's Interactome Dynamics, Expression Regulation, and Assembly Mechanism into Germ Granules during Drosophila Germline Development

Me31B is a protein component of Drosophila germ granules and plays an important role in germline development by interacting with other proteins and RNAs. To understand the dynamic changes that the Me31B interactome undergoes from oogenesis to early embryogenesis, this study characterized the early embryo Me31B interactome and compared it to the known ovary interactome. The two interactomes shared RNA regulation proteins, glycolytic enzymes, and cytoskeleton/motor proteins, but the core germ plasm proteins Vas, Tud, and Aub were significantly decreased in the embryo interactome. Follow-up on two RNA regulations proteins present in both interactomes, Tral and Cup, revealed that they colocalize with Me31B in nuage granules, P-bodies/sponge bodies, and possibly in germ plasm granules. It was further shown that Tral and Cup are both needed for maintaining Me31B protein level and mRNA stability, with Tral's effect being more specific. In addition, evidence is provided that Me31B likely colocalizes and interacts with germ plasm marker Vas in the ovaries and early embryo germ granules. Finally, it was shown that Me31B's localization in germ plasm is likely independent of the Osk-Vas-Tud-Aub germ plasm assembly pathway although its proper enrichment in the germ plasm may still rely on certain conserved germ plasm proteins (McCambridge, 2020).

To summarize, although Me31B's localization to the posterior of an oocyte is likely independent of Osk, Aub, and Dart5, its proper enrichment at the site may still rely on Aub. Together with a previous report that Me31B's localization pattern is not affected in vas and tud mutants, it is speculated that Me31B's localization in a developing oocyte may be independent of the Osk-Vas-Tud-Aub assembly pathway, but its proper enrichment at the posterior germ plasm may still depend on certain conserved germ plasm proteins like Aub. (McCambridge, 2020).

This speculation, together with earlier conclusions in this study, led to the proposal of a hypothetical model for Me31B localization and enrichment process in the germline cells (see Hypothetical model of Me31B localization and enrichment into germ plasm). In this model, Me31B and conserved germ plasm proteins, Osk-Vas-Tud-Aub, exist in distinct granules in the germ plasm, Osk-Vas-Tud-Aub in germ plasm granules and Me31B (possibly associated with Tral and Cup) in separate granules but in close proximity. Me31B granules use an Osk-Vas-Tud-Aub-independent mechanism to localize to the cortex and the posterior of a developing oocyte, then the posteriorly localized Me31B granules interact with the germ plasm granules, which is necessary for proper Me31B granule enrichment in the germ plasm. In the early embryos, Me31B proteins begin to degrade rapidly and become dispersed in the cytoplasm (McCambridge, 2020).

Tejas functions as a core component in nuage assembly and precursor processing in Drosophila piRNA biogenesis

PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown. This study conducted a detailed analysis of the subcellular localization of fluorescently tagged nuage proteins and behavior of piRNA precursors. The results demonstrate that Tej functions as a core component that recruits Vasa (Vas) and Spindle-E (Spn-E) into nuage granules through distinct motifs, thereby assembling nuage and engaging precursors for further processing. This study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, it is proposed that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating the mobility of nuage components (Lin, 2023).

Transposons (transposable elements, TEs) are mobile genetic elements that exist in the genomes of all eukaryotic organisms and they occupy a substantial portion of genomes. They directly impair genomes by causing double-strand breaks, promoting ectopic recombination, and abolishing gene expression. PIWI-interacting RNAs (piRNAs), a class of 23-29-nt gonad-specific small RNAs, protect genome integrity by mitigating any catastrophes in germline cells that will be transmitted to the next generations. piRNAs are quite conserved and widely found among animals, and the model animal system, Drosophila, has been used to investigate and dissect the molecular mechanisms of piRNAs (Lin, 2023).

Drosophila piRNAs are processed from long piRNA precursor transcripts derived from genomic loci called piRNA clusters, where inactive or fragmented transposons are deposited. Discrete piRNA clusters are active in gonads, where they produce dual-strand piRNA precursors in germline cells or unistrand piRNA precursors in somatic gonadal cells. In germline cells, nascent piRNA precursors are transported to a unique, germline-specific membraneless structure called nuage in the perinuclear region via the Nxf3-Nxt1 pathway. Nuage consists of precursors and transposon RNAs being processed, two PIWI family proteins-Aub and Ago3-and other relevant components, DEAD-box RNA helicase Vasa (Vas), DEAH box helicase RNA helicase Spindle-E (Spn-E), and a group of Tudor domain-containing proteins (Tdrds), Krimper (Krimp), Tejas (Tej), Tudor, Tapas (Tap), Qin/Kumo, and Vreteno. After loading long piRNA precursors and transposon RNAs onto Aub and Ago3, they are cleaved and sliced into mature piRNAs, leading to the formation of antisense and sense piRNAs with a 10-nt complementarity. These processed piRNAs are further amplified in nuage in a feed-forward amplification cycle called the ping-pong cycle. However, the molecular mechanisms of nuage assembly are still unclear (Lin, 2023).

Although Tdrds are multifunctional, their overall activities are not fully understood. They interact with symmetrically demethylated arginine (sDMA), which is usually present at the N-terminus of PIWI family proteins, through the Tudor domain, thereby promoting aggregate formation in mammalian cells. This behavior implies the importance of molecular associations of Tdrds for nuage formation. Membraneless organelles composed of RNA and proteins are responsible for diverse RNA processing, including P-body and Yb body in Drosophila, which modulate the molecular organization in a process called phase separation. Two Tdrds localized in Drosophila nuage-Tej and Tap-contain an extended Tudor domain (eTudor) and an additional Lotus domain that is conserved from bacteria to eukaryotes. The Lotus domain was previously reported to interact with Vas, which is required for the piRNA pathway (Lin, 2023).

Of these two proteins, Tej/Tdrd5 is one of the key factors in the piRNA pathway in both Drosophila and mice (Patil, 2014; Patil, 2010; Yabuta, 2011). piRNAs are massively reduced with the displacement of other components from nuage in the absence of Tej/Tdrd5; however, the molecular functions of Tej remain elusive. This study identified the domains of Tej that interact with Vas and Spn-E, which are required for proper nuage formation and piRNA precursor processing, in addition to the contribution of the intrinsically disordered region (IDR) to the dynamics of other nuage components. It is proposed that Tej plays a pivotal role in piRNA precursor processing by recruiting Vas and Spn-E for nuage and modulating their dynamics for nuage assembly (Lin, 2023).

The piRNAs in Drosophila germline cells are produced and amplified in the membraneless organelle, nuage, which is assembled by orderly recruitment of the corresponding components to ensure its proper function. Although its precise function has not been clarified, the findings of this study demonstrate that Tej plays a crucial role in recruiting RNA helicases Vas and Spn-E to nuage through distinct domains, namely, Lotus and SRS. The results provide new insights into the regulation of stepwise piRNA precursor processing by Tej, Spn-E, and Vas in the initial phase of piRNA biogenesis prior to the ping-pong amplification cycle. Tej recruits these helicases for the engagement of the precursors involved in further processing of nuage, thereby also controlling the dynamics of these nuage components (Lin, 2023).

The results confirmed that the Tej Lotus domain recruited Vas to nuage, which is consistent with the fact that it enables Vas to hydrolyze ATP for RNA release (Jeske, 2017). This study newly identified that the SRS motif in Tej is responsible for Spn-E recruitment to nuage. Full deletion or single amino acid substitution of SRS significantly disrupted Spn-E recruitment to Tej granules in S2 cells, whereas further deletions of eight amino acids other than SRS, eSRS, were critical for recruiting Spn-E to nuage in the ovaries. This result raises a possibility that Tej, as well as other factors, may assist the recruitment of Spn-E to nuage in the ovaries. Another protein known as Tap, which is a fly counterpart of TDRD7 and harbors Lotus and eTudor domains, has previously been reported to participate in the piRNA pathway and interact with Vas (Jeske, 2017; Patil, 2014). However, since Tap lacks the SRS found in Tej, it is unlikely to be involved in the recruitment of Spn-E. The mouse homolog of Spn-E (TDRD9) is localized in both nuage and the nucleus in prespermatogonia, and might perform different functions that remain elusive. This finding suggests a possibility that the intrinsically nuclear protein Spn-E was deliberately recruited to nuage via Tej to exert a unique function, such as piRNA precursor processing. In contrast, the eTudor domain mainly contributes to Tej aggregation, which is consistent with previous studies showing that the eTudor domain is engaged in granulation by binding to its ligand sDMA (Lin, 2023).

Despite the unusual nuage granules of Tej-ΔeTudor, it mildly suppressed transposon expression. Notably, Tej-ΔeTudor displays interaction with Vas and Spn-E, albeit to a lesser extent, especially with Spn-E. The CL-IP results also supported these interactions as reported in S2 cells (Patil, 2010). Alternatively, Tej-ΔeTudor possibly may facilitate the association of other components with nuage activity for piRNA processing. Unlike the mutation of precursor transporter, nxf3, and the ping-pong cycle assistant, krimp, tej, as well as spn-E and vas mutants, exhibited the accumulation of piRNA precursors in the perinuclear region and a collapse of the ping-pong amplification. These results suggest that they function upstream during ping-pong amplification. Stalling of piRNA precursors was also observed when the recruitment of Vas or Spn-E to nuage was abolished by the loss of the Lotus or eSRS domains, respectively. Precursor accumulation was concentrated in the malfunctioning nuage or perinuclear region, which would result in a failure in precursor processing and cause TE upregulation (Lin, 2023).

Genetic analysis of nuage organization revealed that Spn-E and Tej occupy a higher hierarchical position than Vas at an earlier stage, which is inconsistent with a previous observation (Patil, 2010), possibly due to the fluctuation of nuage assembly and/or structure at a later stage in the mutants. In contrast, Tej and Spn-E are mutually dependent for the proper assembly of nuage granules because Spn-E is required for the proper localization of Tej within nuage. Moreover, Tej may form a relatively stable scaffold with Spn-E for nuage assembly, while a mobile fraction of Tej may contain Vas. These results suggest that Tej may facilitate the compartmentalization of Vas and Spn-E, as shown in CL-IP experiments and also reported in Bombyx germ cells, while the possibility cannot be excluded of simultaneous binding among these proteins. Further results with S2 revealed that the weak hydrophobic interaction between the proteins may contribute to the formation and regulation of membraneless structures on nuage. DEAD-box RNA helicase family members, including Vas homolog, reportedly form non-membranous, phase-separated organelles in both prokaryotes and eukaryotes, and the large IDR at the N-terminal region facilitates their aggregation by LLPS. In addition, the loss of IDR in Tej significantly suppressed the mobility of Tej and Vas; nevertheless, the TE repression was only mildly attenuated. Thus, Tej-ΔIDR may remain colocalized with Vas and Spn-E, facilitating the processing of piRNAs. Alternatively, the reduction of Vas mobility by the loss of Tej IDR could be compensated by other components in nuage. Only the localization of Vas was remarkably changed upon 1,6-hexanediol (1,6-HD) treatment in S2 cells, further supporting the finding that weak hydrophobic interaction controlled the dynamics of Vas, although a possibility of the unexpected effects by the 1,6-HD treatment cannot be excluded. It also cannot be excluded that 1,6-HD treatment might have impaired kinase and/or phosphatase activity. Hence, localization might have been affected by the changes in their phosphorylation status. The behavior of these proteins is seemingly influenced by their respective binding modes and properties with Tej. The interaction of Vas with Tej is affected by 1,6-HD and IDR region of Tej through the hydrophobic association, whereas that of Spn-E with Tej is more rigid, possibly contributing to the formation of the scaffold of nuage. In conclusion, Tej utilizes the eTudor domain for granule formation, whereas the IDR of Tej appears to maintain the assemble of Tej granules, controlling the mobility of Vas in nuage (Lin, 2023).

Membraneless macromolecular nuage contains more than a dozen components, including Vas and Tej that harbor IDRs, which could contribute to the dynamics of nuage and impact the efficient production of piRNAs. Nuage also contains piRNA precursors and TE RNAs that are processed therein; their unique or specific propensities may affect nuage assembly and function. Further investigation of those proteins and RNA components will shed light on the regulatory mechanisms underlying the formation and dynamics of nuage to promote each sequential step of piRNA biogenesis (Lin, 2023).

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: 25 October 2023

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.