arrest
See the embryonic expression pattern of aret at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
Bruno possesses sex specific isoforms. There are three female-specific transcripts of 2.7, 3.3 and 3.7 kb, as well as a single male-specific transcript of 4.0 kb. These transcripts are present in ovaries and testes, respectively, but are not detectable in the remaining somatic tissue. The three ovarian mRNAs are abundant in ovaries and in 0-2 hour embryos, but are extremely reduced or absent during the rest of embryogenesis and the larval stages of the cell cycle. Two pupal transcripts are evident. One migrates slightly slower than the male-specific message and one slightly faster than the 3.3-kb female specific message. The structural bases of these differences in mRNA sizes is not yet known (Webster, 1997).
During oogenesis, BRU mRNA is first expressed in all of the germ cells in region 2A of the germarium and continues to be found throughout the cytoplasm of both the nurse cells and oocyte as oogenesis progresses. Bru protein is also expressed throughout the nurse cells. In contrast, the distribution of Bru protein in the oocyte is highly restricted, showing striking colocalization with OSK mRNA: at stages when OSK transcripts accumulate in discrete regions of the oocyte, Bru protein is highly concentrated in the same regions. Bru protein first appears in all germ cells in region 2A of the germarium and rapidly becomes concentrated in the presumptive oocyte. Bru quickly resolves as a crescent at the oocyte posterior, following a dynamic pattern similar to that of OSK mRNA, including a transient accumulation at the anterior of the oocyte infrequently detected during stages 7 and 8 of oogenesis. In early embryos, however, although OSK mRNA continues to be localized to the posterior pole, Bru protein is no longer detectable in whole-mount tissue. Bru protein is also localized to a distinct anterodorsal zone in stage 10 oocytes, a region where OSK mRNA does not appear. This localization is intriguing, as it coincides with the position of Gurken mRNA. Although Bru protein binds in vitro to GRK mRNA, the significance of this interaction is unknown (Webster, 1997).
Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. GFP-tagged versions of Vasa and Aubergine were used to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. Perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes (Snee, 2004).
Perinuclear nuage contains, in addition to Vas and Aub, the Maelstrom (Mael), and Gustavus (Gus) proteins. Another component, Bruno (Bru), is a protein that acts in translational repression of osk and gurken (grk) mRNAs. By immunolocalization and expression of a GFP-tagged version of this protein, it was found that Bru is concentrated in perinuclear clusters, similar to the distribution of known nuage components. Double labelling experiments with GFPAub confirmed that Bru colocalizes with nuage. However, Bru is also present at high levels in the cytoplasm, raising the question of whether the colocalization reveals an association with nuage or simply reflects random overlap of an abundant protein with the more narrowly distributed nuage. Evidence that Bru is specifically associated with nuage comes from analysis of Bru distribution in vas mutants: as for other nuage components, the perinuclear clusters of Bru are strongly reduced. Given this identification of Bru as a nuage-associated protein, arrest (aret) mutants (the aret gene encodes Bru) were included in a genetic analysis of nuage. The other genes tested were vas, tud, aub and spindle E (spnE), each of which encodes a nuage component or has been shown to be required for nuage formation, or both (Snee, 2004).
Live imaging was used to better characterize the perinuclear nuage defects seen in static images and to extend the analysis to include cytoplasmic nuage particles. GFPAub was used as the nuage marker to test the role of vas, aret and tud, and VasGFP was used to test the roles of aub and spnE. The live imaging confirmed, for the most part, the basic observations from analysis of fixed samples. In vas mutants perinuclear nuage is almost completely absent, with only a few nuage clusters visible. Loss of spnE activity has a less extreme effect: the perinuclear nuage clusters are largely missing, but a perinuclear zone of VasGFP remains. Consistent with the results by using fixed samples, the persistent perinuclear zone of VasGFP is qualitatively different from wild type, appearing almost completely uniform and lacking any visible discontinuities. Similar results were obtained with the aub mutant, except that the VasGFP perinuclear clusters remain present up to stage 8 of oogenesis, after which they disappear. In aret and tud mutants no significant alteration of perinuclear nuage was detected (Snee, 2004).
In mutants whose perinuclear VasGFP is uniform (spnE- and later stage aub-), the protein undergoes rapid exchange with cytoplasmic pools, just as for VasGFP in perinuclear clusters of wild-type egg chambers. In photobleaching experiments the fluorescence-recovery half-time is 50 seconds in aub- and 48.5 seconds in spnE-, similar to the t1/2=59 seconds for wild type (Snee, 2004).
Cytoplasmic nuage particles are affected differently in the vas, aub and spnE mutants. The vas and spnE mutants have few or no cytoplasmic nuage particles. By contrast, aub mutants have no dramatic reduction in the abundance of cytoplasmic nuage particles, even at times well after the disappearance of perinuclear nuage clusters at stage 8, and the particles have a fairly typical size distribution. These particles do not simply represent the default appearance of VasGFP; they are absent in the spnE mutant. Thus, it seems unlikely that perinuclear nuage clusters are required for the formation of cytoplasmic nuage particles, a conclusion consistent with the observation that cytoplasmic particles are produced only infrequently by detachment of perinuclear nuage clusters (Snee, 2004).
The consequences of loss of vas activity were examined in the male germ line. Just as in nurse cells, Vas appears to be concentrated in nuage in spermatocytes. Given the crucial role for Vas in the nuage of other cell types, either male nuage must differ in this requirement or nuage is not essential in the male germ line for fertility. To distinguish between these possibilities vasAS spermatocytes were tested for the presence of nuage, using GFPAub as a marker. Although GFPAub was present in the cytoplasm, there were no visible perinuclear nuage clusters, indicating that nuage does not form in the vas mutant and is therefore not required for spermatocyte function. An alternate and less probable interpretation is that a rudimentary form of nuage, lacking Aub, is present and is sufficient to provide a minimal requirement for nuage in males (Snee, 2004).
In Drosophila, two types of function, not mutually exclusive, have been proposed for nuage. In one model nuage has been suggested to serve as a precursor to polar granules, a view initially based on ultrastructural similarities of the two organelles and supported by the identification of shared components. Another possible role for nuage is based on its position at the periphery of the nucleus, at or near nuclear pores. Specifically, nuage might act in some aspect of remodelling RNPs when RNAs are exported from the nucleus. Analysis of the movements and genesis of nuage particles provides two arguments against the first model: (1) the rate of release of perinuclear nuage clusters in the nurse cells is very low, much lower than expected if the clusters form polar granules; (2) no nuage particles arriving at the posterior pole of the oocyte and becoming incorporated into polar granules were detected. An additional observation that argues against a model where nuage is a precursor for polar granules, is the presence of cytoplasmic nuage particles in aub mutants, despite the fact that these mutants do not assemble polar granules. However, this evidence does not exclude the first model, because the nuage particles in the mutant might not be fully functional. A third argument is provided by the evidence that Osk cannot interact with nuage, leaving de novo assembly of polar granules as the only reasonable option. Overall, the results strongly suggest that nuage is not the precursor to polar granules, and it is believed that the shared features are simply indicative of similar biochemical activities, rather than a precursor-product relationship (Snee, 2004).
The data do not directly test the model that nuage might function as a transition zone in the movements of mRNAs from the nucleus to the cytoplasm, where RNP components might be exchanged or otherwise modified. However, new properties of nuage, and these relate to possible functions, have been identified. It was found that Bruno, an RNA binding protein that acts as a translational repressor of osk and grk mRNAs, is associated with nuage. This extends the correlation of nuage components with factors that act in some aspect on mRNA localization or translational control. Of the previously identified nuage components, Vas and Gus are involved in the regulation of grk mRNA localization and translation, Aub is required for efficient translation of osk mRNA and has also been implicated in RNAi, and mael mutants display defects in the early stages of mRNA localization. Moreover, spnE, which is necessary for normal nuage formation, is required for the localization of multiple mRNAs and acts in RNAi. Thus, every known nuage component has a role in one or more types of post-transcriptional control of gene expression (Snee, 2004).
Genetic analysis of the role of bruno in oogenesis is made difficult by the lack of ovaries in bruno mutants. Flies sensitized to changes in the level of Osk protein were examined for the affects of reducing wild-type Bru protein levels. If Bru acts to repress OSK translation, a reduction in Bru protein might lead to a partial derepression of OSK translation and, subsequently, to elevated Osk activity. A transgene was used which encodes a form of OSK mRNA that retains Bruno response sequences but is mislocalized to the anterior of the oocyte. Flies bearing this genotype produce embryos with modest head defects caused by the misexpressed OSK mRNA. Reduction in Bru level enhances this phenotype, resulting in progeny with extensive anterior deletions, often accompanied by duplication of posterior pattern elements (Webster, 1997).
In females hemizygous for either aretPA62 or aretPD41(alleles encoding missense mutations that alter the first of the three RNA-binding domains) oogenesis appears to proceed normally until approximately stage 9, at which time the egg chambers degenerate. arrestPA62/arrestPD41 transheterozygotes do complete oogenesis and lay eggs, some of which hatch into viable larvae. However the majority of the embryos from these mothers display variable and complex cuticle defects involving partial or complete fusion of adjacent segments (Webster, 1997).
arrest mutants have pleiotropic phenotypes, ranging from an
early arrest of oogenesis to irregular embryonic segmentation defects.
One function of arrest is in translational repression of oskar mRNA;
this biochemical activity is presumed to be
involved in other functions of arrest. To identify genes that
could provide insight into how arrest contributes to
translational repression or that may be targets for
arrest-dependent translational control, deficiency
mutants were screened for dominant modification of the arrest phenotype.
Only four of the many deficiencies tested, which cover ~30% of the genome,
modified the starting
phenotype. One enhancer, identified fortuitously, is the Star
gene. Star interaction with arrest results in excess
Gurken protein, supporting the model that gurken is a target
of repression. Two modifiers were mapped to individual genes. One is
Lk6,
which encodes a protein kinase predicted to regulate the
rate-limiting initiation factor eIF4E. The second is Delta.
The interaction between arrest and Delta mimics the
phenotype of homozygous Delta mutants, suggesting that
arrest could positively control Delta activity. Indeed,
arrest mutants have significantly reduced levels of Delta
protein at the interface of germline and follicle cells (Yan, 2004).
A screen of third chromosome deficiencies was screened for dominant modifiers of
aret mutants. About three-quarters of the third chromosome was screened,
corresponding to ~30% of the genome.
Only four deficiencies dominantly modified the aret mutant phenotype,
suggesting that the total number of genes in the genome with this property is
small. For two of the four deficiencies the gene
responsible for the interaction was identified, and a third
interacting gene was fortuitously discovered while preparing for the screen.
It was anticipated that two
different types of modifiers might be detected by the screen: those in genes
that act in the same process as Bru and those in genes that are themselves
regulated by Bru or act in a process in which a limiting component is regulated
by Bru. Characterization of the interacting genes suggests that
examples of each type of modifier were discovered (Yan, 2004).
Bru has been proposed to translationally regulate grk mRNA. The
supporting evidence includes (1) binding of Bru to grk mRNA in
vitro and indirect evidence of binding in vivo; (2) rare dorsoventral
patterning defects as a consequence of
overexpression of Bru, and enhancement of this phenotype by reduction of
grk gene dosage, and (3) evidence that localized Grk is present at
reduced levels when Bru is overexpressed, although unlocalized Grk appears more
abundant. However, there has been no evidence of excess
Grk protein in aret mutants. Star is required for grk
activity, and it acts post-translationally in either trafficking or secretion of
Grk protein. When flies were both homozygous for aret and
heterozygous for S1 they accumulated Grk protein in nurse
cells, while ectopic accumulation could not be detected in either aret
mutants or S1 heterozygotes alone. This synthetic effect on
Grk protein accumulation is simple to rationalize. In aret mutants Grk
protein is excessively translated, but an S-dependent delivery step could
efficiently clear the protein from the nurse cells. When S activity is
reduced, a detectable level of Grk remains in the nurse cells. The distribution
of the ectopic Grk, both in cytoplasm and at the nurse cell boundaries, could
correspond to the sites where the protein might stall during delivery. The
actual site of S action is not known, and two different sites of S
concentration, in endoplasmic reticulum or on the plasma membrane, have been
reported. Although this explanation has some appeal, it is
important to note that none of the evidence firmly establishes a role for Bru in
translational repression of grk mRNA, and it remains possible that Bru
could, for example, influence the site of translation rather than its
efficiency (Yan, 2004).
Although the combination of S1 and aret mutations does
affect Grk expression or distribution, there are no precedents that clearly
demonstrate how excess or ectopic Grk would enhance the oogenesis arrest
phenotype of aret mutants. Thus the explanation for the enhancement
remains unknown and could involve the effects on grk or on other genes
that are subject to regulation by Bru (Yan, 2004).
The eIF4E protein binds to the cap at the 5' end of mRNAs. It is a rate-limiting
component of translational initiation, and its activity is under tight control. One form
of regulation is phosphorylation, which is thought to control the mRNA
cap-binding activity of eIF4E. Several lines of correlative
evidence suggest that this phosphorylation is important for cell proliferation,
and mutation of the Drosophila eIF4E to prevent phosphorylation results in
reduced viability and poor growth (Yan, 2004 and references therein).
A transgene expressing a mutant and constitutively activated version of eIF4E,
in which the regulatory phosphorylation is mimicked by an amino acid change, can
suppress the aret phenotype. This result raises the possibility that Bru
has a positive role in initiation of translation. Specifically, in the
aret mutant one or more target mRNAs that require Bru for activation of
translation may be underexpressed, and increasing translation suppresses this
defect (Yan, 2004).
However, the aret mutant phenotype is also suppressed by a mutation of
Lk6 and enhanced by overexpression of Lk6. Lk6 is the Drosophila
protein most closely related to mammalian mitogen-activated protein
kinase-interacting protein kinase 1 (MNK1), which phosphorylates translation
initiation factor eIF4E after activation by either the p44/42 or p38 MAPKs. Thus mutation of Lk6 might be expected to reduce eIF4E
phosphorylation and thereby decrease translational capacity. By this view the
suppression of the aret phenotype would be consistent with an interaction
between eIF4E and Bru that involves the known function of Bru in translational
repression. In favor of this notion Bru has been shown to
physically interact with Cup, an eIF4E-binding protein that is required for
repression of osk mRNA translation. To explore this possibility further
it was asked if suppression of the aret phenotype by EP(3)0886 was
accompanied by a change in the levels of Osk or Grk proteins, or if homozygous
EP(3)0886 females have abnormal amounts of either protein. No change was
seen in either case. Thus it is not known if the Lk6 mutation
impacts the function of aret in repression of osk or grk
mRNAs (Yan, 2004).
Given the similar consequences on the aret phenotype of the
constitutively active eIF4E and the mutant predicted to reduce eIF4E activity,
the simplest explanation is that Lk6 may affect aret function by a
means other than phosphorylation of eIF4E. Suppression of the aret
phenotype by the mutant eIF4E clearly suggests a link between Bru and the
initiation of translation, although this need not be direct (Yan, 2004).
The combination of aretPD/aretQB with
Dl9P/+ produces a variety of ovarian defects, complicating
interpretation of the phenotype. Nevertheless, one striking feature is the
similarity of many of the defects to those seen when Dl activity is
largely or completely eliminated, suggesting that the aret mutations are
enhancing the Dl phenotype. Dl is a component of the
Notch/Dl signaling pathway, which acts in many signaling events in a wide
range of cell types. In the ovary Dl is
required in the germline cells for control of differentiation and proliferation
of the somatic follicle cells and for setting up anteroposterior polarity. The earliest
and, at least initially, most dramatic consequence of loss of Dl activity
is the fusion of cysts—the phenotype most apparent in the
aretPD/aretQB; Dl9P/+ ovaries (Yan, 2004).
Large germline clones of strong Dl mutant alleles cause a complete fusion
of egg chambers into a single egg chamber with multiple cysts, reminiscent of
the complete fusions described here. Smaller clones retain a more regular
ovariole organization. Individual egg chambers with Dl germline clones
often fuse with the adjacent anterior wild-type egg chamber. Fusion can be
incomplete, resulting in a double layer of follicle cells that separate the egg
chambers, much as observed for the A/P partial fusions reported in this study. However, the
similarities are not perfect. For example, Dl mutant clones upregulate
FasIII in the follicular epithelium, but
aretPD/aretQB; Dl9P/+ egg chambers do
not. Other features of the Dl mutant phenotype, such as
the defects in anteroposterior polarity, are difficult to detect in the
aretPD/aretQB; Dl9P/+ ovaries,
because of their arrest of oogenesis. The lack of perfect correspondence between
the Dl germline clones and the aretPD/aretQB;
Dl9P/+ ovaries is not surprising for several reasons: (1)
there is substantial phenotypic variation even among the Dl germline
clones, if both large and small clones are considered; (2) the clones are
homozygous for Dl–, while in the aret mutant
background one wild-type copy of Dl remains; (3) the Dl-like
defects in aretPD/aretQB; Dl9P/+ ovaries
are superimposed on the aret mutant phenotype (Yan, 2004).
The simplest interpretation of these results is that the aret mutations are
reducing the activity of the N/Dl signaling pathway, which in combination
with mutation of one copy of Dl leads to phenotypes similar to those
resulting from loss of Dl. This model is fully supported by the finding
that in aret mutants the amount of Dl protein concentrated at the border
between germline cells and follicle cells is reduced. What remains unclear is
how this reduction occurs. Assuming that Bru is acting as a translational
repressor, in the aret mutant the target protein should be present at
elevated levels. By this model the target should be a gene that normally has a
negative effect on Dl expression or delivery to the membrane.
Alternatively, Bru could also have a role in translational activation, in which
case Dl could be a direct target. This seems quite unlikely, as the
Dl 3'-UTR lacks any recognizable BREs, the sequences to which Bru is
known to bind. Nevertheless, a role for Bru in translational activation is
possible, and the target could normally have a positive effect on provision of
Dl activity (Yan, 2004).
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date revised: 10 July 2005
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