oskar
Mutations in vasa, pumilio and nanos display no effect on Oskar mRNA localization, while capuccino, spire and staufen all show defects in Oskar mRNA localization (Kim-Ha, 1991).
Localization of Oskar mRNA is an elaborate process: first the transcript must move into the oocyte from adjacent interconnected nurse cells and then across the length of the oocyte to its posterior pole. RNA regulatory elements that direct this localization map within the oskar 3' untranslated region and affect different steps in the process: the early movement into the oocyte, accumulation at the anterior margin of the oocyte and finally localization to the posterior pole. This use of multiple cis-acting elements suggests that localization may be orchestrated in a combinatorial fashion, allowing localized mRNAs, despite their ultimately different destinations, to employ common mechanisms for shared intermediate steps (Kim-Ha, 1993).
OskarmRNA localization and translational repression map to the Oskar 3'UTR. Upon localization of Oskar mRNA and protein at the posterior pole, Oskar protein is required to maintain localization of Oskar mRNA throughout oogenesis. Stable anchoring of a transgenic reporter RNA at the posterior pole is disrupted by oskar nonsense mutations. Initially, localization of Oskar mRNA permits translation into Oskar protein; subsequently Oskar protein regulates its own RNA localization through a positive feedback mechanism (Rongo, 1995).
Localization of Bicoid mRNA to the anterior of the Drosophila oocyte and Oskar mRNA to its posterior is critical for
embryonic patterning. Previous genetic studies have implicated exuperantia in Bcd mRNA localization, but its role in this process is not
understood. Exu has been biochemically isolated and shown to be a part of a large RNase-sensitive complex that contains at least seven
other proteins. One of these proteins has been identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the
ExuñYps complex contains OSK mRNA. This biochemical result led to a reexamination of the role of Exu in the localization of OSK mRNA. exu-null mutants are defective in OSK mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and OSK mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. It is proposed that Exu is a core component of a large protein complex involved in localizing mRNAs, both within nurse cells and the developing oocyte (Wilhelm, 2000).
A role for Exu in OSK mRNA localization is consistent with several other findings: (1)
Exu accumulates at both the anterior and posterior poles of the oocyte; (2) OSK mRNA transiently
accumulates at the anterior pole along with BCD mRNA before its transport to the posterior; (3) One of the effects
of exu mutants is to disrupt the localization of OSK mRNA to apical patches within nurse cells. This defect is identical to the nurse cell localization defect described for BCD mRNA in exu mutants and suggests that this step in the localization pathway may be common to both transcripts (Wilhelm, 2000 and references therein).
One of the reasons that exu mutants have not been examined previously for defects in OSK mRNA localization is that only a small percentage of embryos from exu
mothers display posterior patterning defects. The examination of exu mutants reveals that the amount of OSK mRNA localized
to the posterior pole is decreased in these mutants, suggesting that exu plays a role in localizing OSK mRNA within oocytes. However, since this defect is only partially
penetrant, Exu-dependent posterior localization within the oocyte may be redundant with other localization mechanisms. In addition, the posterior patterning defects
associated with the decrease in OSK mRNA localization in exu mutants during stages 9 and 10 of oogenesis may be rescued by localization of OSK mRNA during
cytoplasmic streaming later in oogenesis. In support of this idea it has been shown that injected, fluorescently labeled OSK mRNA can be localized to
the posterior at the time when cytoplasmic streaming occurs. Such localization most likely occurs by random motion during cytoplasmic streaming and specific
anchoring of OSK mRNAs that come in contact with the posterior pole. These multiple mechanisms of localizing OSK mRNA account for the fact that exu mutants do
not display pronounced defects in abdominal patterning (Wilhelm, 2000 and references therein).
The biochemical studies linking Yps to Exu and OSK mRNA suggest that this RNA-binding protein plays a role in posterior mRNA localization. This assertion is
further supported by immunofluorescence studies showing that Yps and OSK mRNA have strikingly similar localization patterns throughout oogenesis: both
accumulate in the early oocyte, transiently localize to the oocyte anterior during stages 8 and 9, and then assume their final positions at the posterior pole during stages
9 and 10. What role might Yps play in the localization complex? Yps belongs to the cold shock domain family
of RNA-binding proteins that have been implicated in regulating translation and mRNA secondary structure. A
notable example is FRGY2, which is complexed with mRNAs in the Xenopus oocyte and is thought to be important for translational silencing. Yps may serve a similar role, since OSK mRNA is translationally repressed until it reaches the posterior
pole. Interestingly, Yps must also serve a function without Exu, since yps is
expressed broadly, whereas exu expression is limited to the germ line. It is possible that
Yps is a component of the mRNA localization machinery outside the germ line, since other components of the oocyte mRNA localization machinery, such as Staufen,
are also used for mRNA localization in somatic tissues. Determining the precise involvement in transport and/or
translational regulation of Yps in the oocyte and other tissues will be resolved in the future by mutational studies (Wilhelm, 2000 and references therein).
Several lines of evidence indicate that OSK mRNA, which encodes a primary organizer of the germ plasm, is a target of Ypsilon schachtel (Yps) activity. (1) OSK mRNA coimmunoprecipitates with both Yps and Exu proteins from ovary extracts (Wilhelm, 2000). (2) OSK mRNA colocalizes with Yps and Exu throughout oogenesis. (3) There is a robust genetic interaction between yps and orb: keeping in mind orb's known regulation of OSK mRNA translation and localization, the yps null allele rescues orb-associated defects in OSK mRNA localization and translation (Mansfield, 2002).
In intermediate allelic combinations of orb, OSK mRNA fails to localize to the posterior pole of the oocyte, and Osk protein is not translated. The localization and translation of OSK mRNA is subject to a complex autoregulatory loop, whereby OSK mRNA must first be localized to the posterior pole of the oocyte to be translated, and subsequently Osk protein is required to maintain the localization of its own mRNA. Because the localization and translation processes are so entwined, it can be difficult to establish which process a regulatory factor affects. In the case of Orb, however, evidence suggests that its primary function may be translational regulation of OSK. In Xenopus, CPEB, which is virtually identical to Orb in its RNA-binding domain, regulates translation of stored maternal mRNAs by binding a U-rich region of 3'UTRs (the cytoplasmic polyadenylation element) and promoting cytoplasmic polyadenylation. The role of OSK's poly(A) tail in translation is controversial. Results from in vitro systems developed to study translation in Drosophila ovaries suggest that the length of OSK's poly(A) tail is not critical for regulating its translation. However, in vivo studies of OSK mRNA suggest that poly(A) tail length does affect its translation. These latter results indicate that polyadenylation of the OSK transcript is dependent on the function of orb, as is accumulation of Osk protein, suggesting that Orb serves a similar function to that of CPEB. In addition, Orb binds specifically to the OSK 3'UTR. Given this evidence, it appears that Orb may function as a translational enhancer of Osk, although a direct role in OSK mRNA localization cannot be ruled out (Mansfield, 2002 and references therein).
The orb genotypes that are rescued in double mutant combinations with ypsJM2 all include the orbmel mutation, a hypomorphic allele that produces some functional Orb protein. In contrast, females homozygous for a null allele (orbF343) or a strong allele (orbF303) show no rescue by the ypsJM2 mutation. These results indicate that rescue by ypsJM2 requires the presence of some functional Orb protein, and that Yps may normally act antagonistically to Orb. In the presence of Yps, the low amount of functional Orb protein present in orbmel mutants is not capable of promoting normal OSK mRNA localization and translation, whereas in the absence of Yps, the reduced Orb protein is sufficient (Mansfield, 2002).
Previous work has shown that, in the minority of orbmel egg chambers in which Osk protein is detectable, Orb protein can be detected at the posterior pole as well. This correlation has been interpreted as evidence of a requirement for Orb for the on-site expression of Osk. When ovaries are doubly mutant for yps and orb, this correlation disappears. While Orb can rarely be detected at the posterior pole of the oocyte in yps;orb mutants, Osk protein is frequently present even in the absence of detectable Orb. However, loss of Yps cannot eliminate the requirement for Orb in Osk expression. It is possible that, in the absence of Yps, a very low concentration of Orb, which is undetectable by immunocytochemistry, is sufficient to localize or enhance the translation of OSK mRNA at the posterior pole. Alternatively, in the absence of Yps, the function of Orb might be accomplished at regions other than the posterior, since in yps;orb double mutants Orb protein is present throughout the oocyte (Mansfield, 2002).
Although OSK translation is significantly rescued in yps;orb ovaries, the amount of Osk present at the posterior appears reduced compared to wild type. In addition, Osk is not reliably detected in yps;orb egg chambers until stage 10. In wild-type ovaries, however, Osk can be detected in stage-9 oocytes, and sometimes as early as stage 8. It is thought that the temporal delay in detecting Osk is due simply to a reduction in Osk expression in yps;orb egg chambers during all stages of oogenesis, such that accumulation of the protein to detectable levels does not occur until stage 10. It is also hypothesized that, due to this reduction in the accumulation of Osk protein in yps;orb ovaries, OSK mRNA localization is not efficiently maintained. In late stage 9, 66% of yps;orb oocytes displayed localized OSK mRNA, while in stage 10 the percentage falls to 45%. This number closely parallels the percentage of yps;orb stage 10 oocytes with detectable Osk protein (43%) and the number of eggs (40%) that hatched from mutant mothers (Mansfield, 2002).
Biochemically an association between Yps and Orb has been detected. Orb protein coimmunoprecipitates with Yps. This association is mediated by RNA, since their coimmunoprecipitation is RNAse-sensitive. Similarly, Orb coimmunoprecipitates with Exu, in an RNA-dependent manner. Exu and Yps also coimmunoprecipitate, but independently of RNA, and bind each other in vitro, indicating that their interaction is probably direct (Wilhelm, 2000). Despite their direct association, Yps is localized normally in exu null ovaries, and Exu protein is localized normally in yps null ovaries. Thus Yps and Exu appear to be recruited independently to this ovarian complex. Do the associations detected by immunoprecipitation reflect biologically significant interactions that occur in vivo? Several other lines of evidence suggest that these proteins interact in vivo, and that OSK mRNA is part of this complex: (1) all three proteins, and OSK mRNA, colocalize throughout oogenesis; (2) OSK mRNA associates with both Exu and Yps (Wilhelm, 2000), and Orb binds directly to OSK mRNA; (3) the genetic results presented in this work are strong evidence for a biologically significant interaction of Yps and Orb in Drosophila ovaries (Mansfield, 2002).
One model supported by the data is that Yps and Orb both bind to OSK mRNA, and have opposite effects on translation: Yps represses, and Orb activates translation. Immunoprecipitation experiments show that both proteins are present in an RNP complex and that their association is mediated by RNA, suggesting that both proteins simultaneously bind a common RNA target. This target is likely to be OSK mRNA. OSK mRNA is a member of this RNP complex (Wilhelm, 2000). Orb is known to bind OSK mRNA, and the genetic results show that a yps loss-of-function mutation suppresses the defects in OSK mRNA localization and translation associated with reduced function orb alleles. Yps could prevent translation by preventing Orb from promoting cytoplasmic polyadenylation. At the posterior of the oocyte, where Orb and Yps both concentrate during mid-oogenesis, and where OSK mRNA is localized and translated, concentration differences between the two proteins could push the complex from being a negative to a positive regulator of translation. Additional factors at the posterior could also interact with either Orb or Yps to modify their functions, as might association with the actin cytoskeleton. This model accounts for why the yps mutation cannot eliminate the requirement for Orb, but can reduce the amount of Orb required for sufficient OSK translation. In the rescued genotypes, there may be enough Orb at the oocyte posterior to allow for on-site cytoplasmic polyadenylation of OSK mRNA, in the absence of negative regulation by Yps. It is also possible that, in the absence of Yps, Orb can stimulate polyadenylation of OSK mRNA before it becomes localized, although it remains subject to translational repression by other factors, such as Apontic and Bruno, until it reaches the posterior pole. Future studies will test this model by determining if Yps and Orb bind competitively to OSK mRNA, and if so, how their combined binding affects the translation of OSK mRNA, and its polyadenylation state. These studies should contribute not only to an understanding of localization-dependent mRNA translation in Drosophila, but also to a better understanding of the biological roles of the widespread family of Y-box proteins (Mansfield, 2002).
The localization of Oskar at the posterior pole of
the Drosophila oocyte induces the assembly of the
pole plasm and thereforedefines where the abdomen and
germ cells form in the embryo. This localization is
achieved by the targeting of Oskar mRNA to the
posterior and the localized activation of its translation. Oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus
end-directed motor kinesin I, but nothing is known
about how the RNA is coupled to the motor. The gene barentsz (CG12878) is required for the localization of Oskar mRNA. In contrast to all
other mutations that disrupt this process,
barentsz-null mutants completely block the posterior localization of Oskar mRNA without affecting Bicoid and Gurken mRNA localization, the organization of the microtubules, or subsequent steps in
pole plasm assembly. Surprisingly, most mutant embryos
still form an abdomen, indicating that Oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with Oskar mRNA, and this localization is
Oskar mRNA dependent. Thus, Barentsz is essential
for the posterior localization of Oskar mRNA and behaves as a specific component of the Oskar RNA transport complex (van Eeden, 2001).
Several lines of evidence indicate that Barentsz associates with
Oskar mRNA and Staufen protein during their movement from the anterior to the posterior of the oocyte. (1)
Barentsz localizes to the posterior pole at the same time
as Oskar mRNA and Staufen and colocalizes with
Staufen in a posterior crescent at stage 9. However,
unlike Staufen, Barentsz does not remain at the posterior
later in oogenesis and only colocalizes with Oskar RNA during the stages when it is being transported to the posterior. (2) Staufen and Barentsz show an identical mislocalization to the center of the oocyte in gurken
mutant egg chambers. Since Oskar mRNA is not
translated in these oocytes, this result argues against a
role for Oskar protein in recruiting Barentsz to the complex. (3) Like Oskar mRNA and Staufen, Barentsz accumulates at the anterior of the oocyte in TmII and in kinesin heavy chain mutants. Thus, Barentsz colocalizes with Oskar mRNA both before and after its transport to the posterior of the oocyte. (4) The posterior localization of Barentsz seems to depend on Oskar RNA. Although it is not possible to examine the localization of Barentsz in oskar RNA-null mutants, overexpression of
oskar induces a corresponding increase in the
amount of Barentsz that localizes to the posterior pole. In conjunction with the lack of Oskar mRNA localization to the posterior in barentsz mutants, these results strongly suggest that Barentsz is an essential component
of the Oskar RNA transport complex (van Eeden, 2001).
Although it has been thought that Staufen is essential for Oskar mRNA localization, these results show that a very small amount of the RNA can still reach the posterior pole at stage 9 in the complete absence of
Staufen protein. Thus, Staufen cannot be the only
RNA-binding protein that recognizes Oskar mRNA and
couples it to the transport machinery. In staufen
mutants, Barentsz shows little if any accumulation at the
anterior of the oocyte where the majority of Oskar
mRNA remains but colocalizes with the tiny fraction of RNA
that reaches the posterior pole. Thus, Staufen seems to be
required to promote or stabilize the efficient association
of Barentsz with Oskar mRNA. However, the Barentsz-Oskar RNA complexes that do form in the absence of Staufen still localize to the posterior (van Eeden, 2001).
The sequence of Barentsz gives few clues as to its
biochemical function, although it appears to have
homologs in other species. Some insight into its role
may be provided by the comparison of the Oskar mRNA
localization phenotype in btz mutants with that of
mutants in the heavy chain of kinesin I. In both cases, Oskar mRNA does not localize to the posterior and accumulates instead at the
anterior of the oocyte. Furthermore, khc mutants
block the posterior localization of Barentsz protein,
which remains with Oskar mRNA at the anterior pole.
Since kinesin I is a plus end-directed microtubule motor, a simple explanation for its role in Oskar mRNA localization is that it actually transports Oskar mRNA to the plus ends of the microtubules at the posterior pole. If this model is correct, the mutant phenotype and
localization of Barentsz protein suggest that it acts
somewhere between Oskar mRNA and the kinesin. For example, Barentsz could play a role in coupling the RNA to
the kinesin or in the activation of the motor once the
complex has formed. However, no interaction between Barentsz and the
kinesin heavy chain could be detected in Drosophila ovary extracts,
although this may be due to the fact that only a fraction
of the total Barentsz protein localizes with Oskar
mRNA, and this only occurs in stage 9 and 10 egg chambers, which represent a small proportion of the egg chambers in the ovary (van Eeden, 2001).
Most of the conclusions about Barentsz are
also likely to apply to Mago nashi, which seems to serve a
closely related function in Oskar mRNA
localization. mago nashi mutants cause a very similar failure in the translocation of Oskar mRNA from
the anterior to the posterior of the oocyte. Furthermore, the results
confirm that Mago protein also localizes transiently to the posterior pole, although the amounts are too low to detect by antibody staining. Finally, Mago and Barentsz depend on each other for their localization to the posterior, since the localization of Mago is abolished in barentsz mutants and vice versa. Some clue to the relationship between
the two may be provided by the fact that mago
mutants disrupt the perinuclear localization of Barentsz
in the nurse cells. This suggests that Mago may be
required for the formation of functional Barentsz and that
the two proteins are part of the same complex before they
enter the oocyte. Consistent with this, Barentsz and Mago appear to colocalize at the periphery of the nurse cell
nuclei and at the ring canals between the nurse cells and
the oocyte, although no direct interaction between them has yet been detected (van Eeden, 2001).
Recent results have implicated hnRNP proteins that are predominantly nuclear in the cytoplasmic localization of several RNAs,
suggesting that the nuclear history of a transcript may
determine its fate in the cytoplasm. In this context, it is interesting to note that most Barentsz is associated with
the nuclear membranes of the nurse cells, whereas almost
all Mago nashi is found in the nuclei. Since Oskar
mRNA is transcribed in the nurse cell nuclei, this raises
the possibility that Mago associates with the RNA in the
nucleus and that Barentsz is then recruited to the complex
as it exported into the cytoplasm. Consistent with this,
the human homolog of Mago interacts with RBM8/Y14, a
nucleocytoplasmic shuttling protein that binds to spliced
mRNAs and remains associated with newly exported transcripts in the cytoplasm. Thus, Oskar mRNA may provide another example where factors loaded onto a transcript as it exits the nucleus determine its subsequent cytoplasmic localization. Neither Mago or Barentsz is required for Oskar mRNA
transport from the nurse cells into the oocyte, and they
would therefore have to remain associated with the RNA
during this phase of its localization before directing its
subsequent transport to the posterior of the oocyte (van Eeden, 2001).
The posterior group gene staufen is required both for the localization of maternal determinants to the posterior pole of the Drosophila egg and for Bicoid mRNA to localize correctly to the anterior pole. Staufen protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with OskarmRNA. Staufen appears to tether Oskar mRNA to the posterior pole. Once localized, staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, Staufen protein is also concentrated at the anterior pole, in the same region as Bicoid mRNA (St Johnston, 1991).
Staufen protein also anchors Bicoid mRNA at the anterior pole of the Drosophila egg. Staufen protein colocalizes with BCD mRNA at the anterior, and this localization depends upon its association with the mRNA. Upon injection into the embryo, BCD transcripts specifically interact with Staufen. Mapping the required sequences indicates three regions of the 3'UTR, each of which is predicted to form a long stem-loop. The resulting Staufen-BCD 3'UTR complexes form particles that show a microtubule-dependent localization. Since Staufen is also transported with Oskar mRNA during oogenesis, Staufen associates specifically with both OSK and BCD mRNAs to mediate their localizations, but at two distinct stages of development (Ferrandon, 1994). The orb gene is an ovarian-specific member of a large family of RNA-binding proteins. orb is required for the asymmetric distribution of Oskar and Gurken mRNAs within the oocyte during the later stages of oogenesis. ORB protein localized within the oocyte in wild-type females, is distributed ubiquitously in stage 8-10 orb mutant oocytes. Presumably, ORB is a component of the cellular machinery that delivers mRNA molecules to specific locations within the oocyte and this function contributes to both D/V and A/P axis specification during oogenesis (Christerson, 1994).
Drosophila Staufen protein is required for the localization of Oskar mRNA to the posterior of the oocyte, the anterior
anchoring of Bicoid mRNA and the basal localization of Prospero mRNA in dividing neuroblasts. Analysis of an alignment of the Stau homologs reveals that the only regions of the protein to have been conserved during evolution are the five
dsRBDs and a short region within an insertion that splits dsRBD2 into two halves.
The M. domestica and D. melanogaster proteins show an average of 67% amino acid identity within the
dsRBDs, but less than 15% in the rest of the protein. dsRBD2 and dsRBD5 were originally described as 'half domains' showing similarity to the dsRBD
consensus only over the C-terminal portion of the domain. However, the conservation extends over a region
corresponding to the length of a whole domain, and these domains should therefore be considered as complete, albeit divergent. The only other obvious homology between these proteins is a short region, one which is rich in proline and aromatic amino acids,
within the insertion that splits dsRBD2. Since the regions of the protein
essential for its activity are expected to be conserved during evolution, the dsRBDs and this proline-rich region are likely to mediate all of the
functions of Stau, including its ability to bind both mRNA and the factors that localize Stau-mRNA complexes. dsRBDs 1, 3 and 4 bind dsRNA
in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length
Staufen protein lacking this insertion is able to associate with Oskar mRNA and activate its translation, but fails to localize the RNA to the posterior.
In contrast, Staufen lacking dsRBD5 localizes Oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the
microtubule-dependent localization of OSK mRNA, and dsRBD5 is required for the derepression of Oskar mRNA translation, once localized. Since dsRBD5
has been shown to direct the actin-dependent localization of Prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based
mRNA transport (Micklem, 2000).
It has previously been difficult to investigate the role of Stau in OSKmRNA translation for two reasons: (1) stau null mutations disrupt the localization of OSK mRNA, and it is not translated unless it is localized to the posterior pole; (2) it is difficult to distinguish between the effects of weak stau alleles on translation and anchoring, because Osk protein is required to anchor its own RNA, but the mRNA needs to be anchored at the posterior to be translated. However, StauDeltadsRBD5 seems to have a specific defect in OSK mRNA translation, since OSK mRNA is localized normally to the posterior at stage 10 in these ovaries, but no detectable Osk protein was produced. Furthermore, oskBRE- RNA, lacking the Bruno response element, produces significant amounts of Osk activity in these ovaries, indicating that StauDeltadsRBD5 can function in the translation of derepressed OSK mRNA. Taken together, these results strongly suggest that dsRBD5 is required to relieve Bruno repression once the mRNA has reached the posterior. This requirement cannot be absolute, however, since some Osk protein must be present early in oogenesis to anchor Stau-osk mRNA complexes (Micklem 2000).
Since dsRBD5 does not bind RNA, it presumably mediates its function in Osk translation through protein-protein interactions. Although Miranda binds to this domain, this interaction is unlikely to play any role during oogenesis, since miranda null germline clones have no phenotype. Thus, dsRBD5 presumably interacts with other proteins to regulate OSK translation. A very similar translation defect is observed in osk transgenes that lack binding sites for 68 and 50 kDa proteins in the 5'UTR, whereas Stau is thought to associate with the localization signal in the 3'UTR. Thus, derepression is likely to involve cooperation between proteins bound to both ends of the RNA (Micklem 2000).
In addition to its role in derepressing OSK translation at the posterior, Stau is required for the efficient expression of derepressed oskBRE- RNA. Since neither the insert in dsRBD2 nor dsRBD5 is necessary for this activity, it presumably depends on the dsRBDs that bind RNA. It is possible that these dsRBDs also interact with other proteins, since only one face of the domain contacts RNA, and several amino acids on the other faces of these domains have been conserved during evolution. Alternatively, the binding of Stau may enhance OSK mRNA translation indirectly, for example, by altering the folding of the RNA so that other factors can bind more efficiently (Micklem, 2000).
Since Stau has been conserved throughout animal evolution, it seems likely that the homologs will fulfil similar functions in mRNA localization and translational control in other organisms. In support of this view, recent evidence indicates that mammalian Stau mediates mRNA transport along microtubules in neurons. The mouse and human Stau genes share an extra region of homology (not found in the insect homologs) that resembles the microtubule-binding domain of MAP1B, and this region of HsStau binds to microtubules in vitro. It will therefore be interesting to see whether this domain or the insertion in dsRBD2 is required for the microtubule-dependent movement of Stau in neurons (Micklem, 2000).
The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved
in RNA maturation and localization. To determine how this domain recognizes RNA, the third dsRBD
from Drosophila Staufen has been studied. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and
the amino acids required for this interaction have been identified. By mutating these residues in a staufen transgene, it has been
shown that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of Bicoid and
Oskar mRNAs. Using high-resolution NMR, the structure of the complex between dsRBD3 and an RNA stem-loop was determined. The
dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between
loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop
that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of
dsRBD proteins (Ramos, 2000).
The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. This study shows that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. These results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization (Irion, 2006).
Asymmetric localization of mRNAs is a common mechanism for targeting proteins to the regions of the cell where they are required. This process is particularly important in the developing oocytes of many organisms, where localized mRNAs function as cytoplasmic determinants. This has been best characterized in Drosophila, where the localization of bicoid (bcd) and oskar (osk) mRNAs to the anterior and posterior poles of the oocyte defines the primary axis of the embryo. bcd mRNA is translated after fertilization to produce a morphogen that patterns the head and thorax of the embryo, whereas osk mRNA is translated when it reaches the posterior of the oocyte, where Oskar protein nucleates the assembly of the pole plasm, which contains the abdominal determinant nanos mRNA, as well as the germ line determinants. Localized mRNAs can also function as determinants during asymmetric cell divisions. For example, the asymmetric inheritance of mating type switching in budding yeast is controlled by the localization of Ash1 mRNA to the bud tip, which segregates the repressor ASH1p into only the daughter cell at mitosis. Similarly, prospero (pros) mRNA localizes to the basal side of Drosophila embryonic neuroblasts and is inherited by only the smaller daughter cell of this asymmetric cell division, where Prospero protein acts as a determinant of ganglion mother cell fate (Irion, 2006).
To be localized, an mRNA must contain cis-acting localization elements that are recognized by RNA-binding proteins, which couple the mRNA to the localization machinery. This process is only well understood for ASH1 mRNA, which contains four localization elements that are recognized by She3p, which then links the mRNA to the myosin motor complex Myo4p/She2p so that it can be transported along actin cables to the bud tip. Biochemical and genetic approaches have led to the identification of a number of RNA-binding proteins that associate with localized mRNAs in higher eukaryotes, but it is not known how these interactions target the mRNA to the correct region of the cell (Irion, 2006).
One of the best candidates for an RNA-binding protein that plays a direct role in mRNA localization is the dsRNA-binding protein Staufen (Stau). Staufen was first identified because it is required for the localization of osk mRNA to the posterior of the oocyte and co-localizes with it at the posterior pole. This localization depends on the polarized microtubule cytoskeleton and the plus end-directed microtubule motor kinesin, suggesting that Staufen may play a role in coupling osk mRNA to kinesin, which then transports the osk mRNA complex along microtubules. The posterior localization of osk mRNA also requires the exon junction complex components Mago nashi (Mago), Y14, eIF4AIII and Barentsz (Btz), as well as HRP48, which is needed for the formation of Staufen/osk mRNA particles (Irion, 2006).
Staufen homologues seem to play a similar role in the microtubule-dependent localization in vertebrates. GFP-Stau particles have been observed to move along microtubules in cultured neurons, and the protein is a component of large ribonucleo-protein complexes that contain kinesin and dendritically localized mRNAs. In addition, a Xenopus Staufen homologue associates with Vg1 mRNA and is required for its microtubule-dependent localization to the vegetal pole of the oocyte, which is also thought to be mediated by a kinesin (Irion, 2006).
As well as this possible conserved role in kinesin-dependent transport, Drosophila Staufen is also required for the last phase of bcd mRNA localization and co-localizes with the mRNA at the anterior of the oocyte from stage 10B onwards. Furthermore, when the bcd 3′ UTR is injected into the early embryo, it recruits Staufen into particles that move in a microtubule-dependent manner to the poles of the mitotic spindles, consistent with minus end-directed microtubule transport (Irion, 2006).
Staufen also binds to prospero mRNA and is required for its localization to the basal side of the embryonic neuroblasts. In contrast to the other examples of Staufen-dependent mRNA localization, this process depends on the actin cytoskeleton and the adapter protein Miranda (Mira) (Irion, 2006).
The varied functions of Staufen raise the question of how the same protein can function in both actin- and microtubule-dependent mRNA localization, as well as in the targeting of osk and bcd mRNAs to opposite ends of the same cell. Some insight into this comes from the analysis of Staufen protein, which contains five conserved dsRNA-binding domains (dsRBDs). In all Staufen homologues, dsRBD2 is split by a proline-rich insertion in one of the RNA-binding loops, and deletion of this insertion disrupts the localization of osk mRNA, but not that of prospero mRNA, leading to the proposal that this domain couples Staufen/mRNA complexes to a kinesin-dependent posterior localization pathway. In contrast, removal of dsRBD5 prevents the localization of prospero mRNA, whereas osk mRNA localizes normally but is not translated at the posterior of the oocyte. Indeed, dsRBD5 binds directly to Miranda to couple Staufen/prospero mRNA complexes to the actin-based localization pathway. The localization of bcd mRNA also requires dsRBD5, although the loss of the insert in dsRBD2 also affects its localization slightly (Irion, 2006).
The results above suggest that different domains of Staufen couple mRNAs to distinct localization pathways, raising the possibility that the fate of Staufen mRNA complexes may depend on which Staufen-interacting proteins are present in the cell. To test this hypothesis, the effects of expressing Miranda during oogenesis were examined to determine whether it can influence the localization of bcd or osk mRNAs (Irion, 2006).
Although Miranda is not required during oogenesis, its ectopic expression causes a striking defect in anterior–posterior axis formation that reveals several important features of the mechanisms that control the targeting and translation of localized mRNAs. Firstly, these results provide strong support for the idea that the destination of Staufen/mRNA complexes is determined by the Stau-interacting factors that are present in the cell. During wild type oogenesis, Staufen associates with osk mRNA to mediate its kinesin-dependent localization to the posterior of the oocyte at stage 9, and this requires the insertion in Staufen dsRBD2, suggesting that this domain couples Staufen/osk mRNA complexes to the posterior localization pathway. However, the expression of Miranda is sufficient to target a proportion of these complexes to the anterior. This localization is mediated through the binding of Miranda to dsRBD5 of Staufen because deletion of this domain abolishes anterior localization without affecting the transport to the posterior pole. By contrast, deletion of the insert in dsRBD2 in the presence of Miranda results in the localization of all Staufen/osk mRNA complexes to the anterior pole. Thus, these two pathways act through different domains of Staufen to direct localization to opposite ends of the same cell. These pathways compete with each other, resulting in the partitioning of the Miranda/Staufen/osk mRNA complexes to the anterior and posterior poles, but each is capable of localizing all of the complexes when the other pathway is compromised. exu and swa mutants abolish the Miranda-dependent anterior localization, and osk mRNA now localizes exclusively to the posterior, whereas btz, mago and TmII mutants block the posterior localization pathway, resulting in the localization of all osk mRNA at the anterior cortex and the formation of reverse polarity embryos (Irion, 2006).
Since dsRBD5, which is not an RNA-binding domain, is necessary and sufficient for the interaction of Staufen with Miranda, the anterior localization of osk mRNA by Miranda provides a simple in vivo assay for the binding of Staufen to osk mRNA. This reveals that neither the insert of dsRBD2 nor the RNA-binding residues of dsRBD3 are required for the stable association of Staufen with the RNA. The lack of a requirement for the insert in dsRBD2 is consistent with the observation that dsRBD2Δloop binds dsRNA in vitro when expressed on its own, whereas the full-length dsRBD2 does not. It is more surprising, however, that the mutations in dsRBD3 have no effect on Staufen binding to osk mRNA since this domain binds to dsRNA with the highest affinity in vitro, and these mutations in the five key amino acids that contact the RNA abolish the domain's RNA-binding activity in vitro. The two other functional dsRNA-binding domains in Staufen (dsRBD1 and 4) must therefore be sufficient to form a stable complex with osk mRNA (Irion, 2006).
The specific effect of a quintuple mutant in dsRBD3 on posterior localization, but not on RNA binding of full-length Staufen, further suggests that these five amino acids play a role in coupling Staufen/osk mRNA complexes to the posterior localization pathway. Although it is possible that these residues are required for an interaction with a trans-acting factor, it seems more likely that it is the association of dsRBD3 with the RNA that is important because this affects either the folding of the RNA or the conformation of Staufen protein. For example, it has been suggested that the binding of Staufen dsRBDs1, 3 and 4 to osk mRNA presents a double-stranded region of the RNA to dsRBD2, which induces a conformational change in dsRBD2 that brings together the two RNA-binding regions of the domain and loops out the large insertion, which is then exposed to interact with the transport machinery. The effect of the point mutations in dRBD3 is consistent with this model and the idea that dsRBD2 functions as an RNA-binding sensor that couples Staufen/osk mRNA complexes to factors that target it to the posterior (Irion, 2006).
Although all mRNAs that accumulate in the oocyte localize at least transiently to the anterior, several lines of evidence indicate that Miranda links Staufen and osk mRNA specifically to the bcd localization pathway. (1) All other anterior mRNAs, except bcd and hu li tai shao (hts), localize to the anterior only during stages 9–10A and become delocalized at stage 10B when rapid cytoplasmic streaming begins. In contrast, Miranda maintains osk mRNA at the anterior throughout oogenesis, so that it is still localized in a tight anterior cap in the freshly laid egg. (2) Miranda, Staufen and oskar undergo the same change in their anterior localization at stage 10B as bcd mRNA: they initially localize as a ring around the anterior cortex and then move towards the middle of the anterior when the centripetal follicle cells start to migrate inwards. (3) Like bcd, the anterior localization of osk mRNA by Miranda requires Exu, Swallow and Staufen, whereas hts mRNA localization is independent of Exu and Staufen. Since the anterior localization does not require bcd mRNA itself, Miranda cannot simply hitchhike on the bcd mRNA localization complex, and it therefore presumably links osk mRNA to the same microtubule-dependent anterior transport pathway used by bcd mRNA (Irion, 2006).
In addition to its role in osk mRNA localization, Staufen associates with bcd mRNA during the late stages of oogenesis to mediate the final steps in its localization to the anterior cortex of the oocyte. Since this localization requires the Miranda-binding domain of Staufen and Miranda couples Staufen/mRNA complexes to the bcd localization pathway, it is attractive to propose that Miranda normally mediates the late anterior localization of bcd mRNA. mira mutants have no phenotype during oogenesis, however, although the protein is expressed in late oocytes. Thus, if Miranda does play a role in bcd mRNA localization, it must function redundantly with another unidentified factor. This is perhaps to be expected given the previous evidence for redundancy in the localization of bcd mRNA. For example, none of the small deletions within the bicoid localization signal abolishes its anterior localization, indicating that it contains redundant localization elements, and two distinct bcd mRNA recognition complexes have been purified biochemically from ovarian extracts (Irion, 2006).
The elucidation of the role of Miranda in bicoid mRNA localization will require the identification of other factors that couple Staufen/bicoid mRNA complexes to the anterior localization pathway, which may function redundantly with Miranda. There are no obvious candidates for these factors, however, since Staufen is the only known protein that is specifically required for the final step of bicoid mRNA localization. Indeed, one reason why such factors may have been missed in genetic screens for mutants that disrupt bicoid mRNA localization is because they are redundant with Miranda and have no phenotype on their own. For these reasons, it is hard to address the question of redundancy using a genetic approach, but further analysis of how Miranda targets Staufen/mRNA complexes to the anterior may help resolve this issue. For example, mapping the Miranda domains that direct anterior localization may provide a clue as to the molecular nature of the unidentified factors that also fulfil this function, while screens for proteins that interact with this domain could identify other components of the anterior localization pathway (Irion, 2006).
These results reveal that Miranda, like Staufen, has the capacity to mediate both microtubule- and actin-dependent localization, raising the question whether the former plays any role in its well-characterized function during the asymmetric divisions of the embryonic neuroblasts. The localization of Miranda to the basal side of the neuroblast is actin-dependent. However, the protein also accumulates at the apical centrosome during both embryonic and larval neuroblast divisions, and this localization is even more prominent in l(2)gl or dlg mutants. Furthermore, Miranda was independently identified as a component of the pericentriolar matrix and co-localizes with γ-tubulin on all of the centrosomes at syncytial blastoderm stage. Although the centrosomes disappear in the female germ line, the anterior cortex is the major site for microtubule nucleation and γ-tubulin localization in the oocyte. Thus, Miranda may localize to the anterior of the oocyte by the same mechanism as it localizes to centrosomes (Irion, 2006).
The phenotype of mira-GFP also provides insights into the translational control of osk mRNA. In wild type ovaries, osk mRNA is translationally repressed before it is localized, and this repression is then specifically relieved once the mRNA reaches the posterior pole. In principle, translational activation of osk mRNA could occur by a specific signal at the posterior, but it could also be due to some other consequence of localization, such as the concentration of the RNA in a small region or its association with the oocyte cortex. Evidence in favor of a specific posterior signal comes from an experiment in which a LacZ reporter gene under the control of the oskar 5′ region and the first 370 nt of the 3′ UTR was targeted to the anterior by the bcd localization element. Since this anterior RNA was not translated, concentration at the cortex appeared to be insufficient to relieve BRE mediated repression. However, it has recently emerged that this reporter RNA lacks the two clusters of insulin growth factor II mRNA-binding protein (IMP) binding elements in the distal oskar 3′ UTR that are essential for oskar translational activation at the posterior, making it hard to draw any conclusions from the lack of translation of this reporter RNA at the anterior. Mira-GFP provides an alternative way to test this hypothesis because it directs the anterior localization of wild type osk mRNA, with all of its translational control elements intact. This anterior mRNA is not translated during stages 9–13, despite being efficiently localized to the cortex, whereas the osk mRNA at the posterior of the same oocytes is translated normally. Thus, concentration at the cortex is not sufficient to de-repress translation, strongly supporting the idea that activation depends on a specific posterior signal (Irion, 2006).
Although the anterior osk mRNA is not translated at the normal time, the repression system breaks down at the very end of oogenesis, and the mRNA is very efficiently translated in mature oocytes. This suggests that some key component of the repression system disappears at this stage, and a good candidate is the BRE-binding protein Bruno. Bruno is highly expressed during oogenesis but is not detectable in embryos. Furthermore, the addition of Bruno is sufficient to cause the repression of exogenous osk mRNA in an embryonic translation system. These results indicate that Bruno is degraded at the end of oogenesis, whereas all other components necessary for translational repression of osk mRNA are still present in the embryo. Thus, the translation of anterior osk mRNA in mira-GFP oocytes is most probably triggered by the disappearance of Bruno (Irion, 2006).
Once it is translated at the posterior of the oocyte, Oskar protein nucleates the formation of the pole plasm with its characteristic electron-dense polar granules, which gradually assemble during stages 9–14 of oogenesis. This appears to be a stepwise process, in which Oskar protein recruits some polar granule components as soon as it is translated at stage 9, such as Vasa and Fat facets, while other components are added in sequence during the rest of oogenesis. For example, Tudor, Capsuleen and Valois are recruited during stage 10A, whereas nanos, Pgc and gcl mRNAs only become enriched at the posterior at stages 10B–11. It is therefore surprising that the anterior Oskar protein, which is only synthesized in stage 14 oocytes, can still nucleate fully functional pole plasm that induces the formation of anterior pole cells. Thus, although the pole plasm normally assembles in an ordered fashion over the last 5 stages of oogenesis, this whole process can still occur once oogenesis is complete. This indicates that the assembly of the pole plasm does not depend on the order of addition of its components, all of which must still be present and freely diffusible in mature oocytes (Irion, 2006).
mRNA localization is a powerful and widely employed mechanism for generating cell asymmetry. In Drosophila, localization of mRNAs in the oocyte determines the axes of the future embryo. Oskar mRNA localization at the posterior pole is essential and sufficient for the specification of the germline and the abdomen. Its posterior transport along the microtubules is mediated by Kinesin I and several proteins, such as Mago-nashi, which, together with Oskar mRNA, form a posterior localization complex. It was recently shown that human Y14, a nuclear protein that associates with mRNAs upon splicing and shuttles to the cytoplasm, interacts with MAGOH, the human homolog of Mago-nashi. Drosophila Y14 interacts with Mago-nashi in vivo. Immunohistochemistry reveals that Y14 is predominantly nuclear and colocalizes with Oskar mRNA at the posterior pole. In y14 mutant oocytes, Oskar mRNA localization to the posterior pole is specifically affected, while the cytoskeleton appears to be intact. These findings indicate that Y14 is part of the Oskar mRNA localization complex and that the nuclear shuttling protein Y14 has a specific and direct role in Oskar mRNA cytoplasmic localization (Hachet, 2001).
Y14 and Mago-nashi are highly conserved proteins, from unicellular eukaryotes to vertebrates. MAGOH and Mago-nashi share 89% identity, and human and Drosophila Y14 share 59% identity. However, the Y14 homologs differ noticeably in one respect: Drosophila Y14 lacks most of the SR domain present in the human form. To test whether the Y14:MAGOH interaction is conserved in Drosophila, the Drosophila y14 gene was cloned and an interaction test was designed, making use of the LexA two-hybrid system. An interaction between Mago-nashi and Drosophila Y14 was detected in both assay orientations, using two independent Mago-nashi baits that differ with regard to inclusion of a nuclear localization signal (NLS) (Hachet, 2001).
To address the physiological relevance of the Y14:Mago-nashi interaction, an antibody was raised against Y14 and coimmunoprecipitations were performed from ovarian extracts of myc-mago transgenic flies, which produce a Myc-mago protein in a wild-type background. Y14 can be coimmunoprecipitated with the Myc-mago protein when using the anti-Myc antibody. Mago-nashi coimmunoprecipitates with Y14 when wild-type ovarian extracts are treated with anti-Y14 antibody, showing that the in vivo interaction is detected in both orientations. More than 50% of Mago-nashi coimmunoprecipitates with Y14. This indicates that the Y14:Mago-nashi interaction is remarkably robust and that a large proportion of Y14 is associated with Mago-nashi in the ovary (Hachet, 2001).
To investigate the subcellular localization of Y14 in Drosophila egg chambers, in situ immunostaining was performed using affinity-purified anti-Y14 antibody. The in situ localization study highlights the predominantly nuclear localization of Drosophila Y14, as is the case for the human protein. This distribution correlates with that of Mago-nashi and is consistent with the detected protein interaction. Interestingly, Y14 also shows a cytoplasmic distribution. During the early stages of oogenesis, Y14 is enriched in the posterior half of the oocyte, like Mago-nashi. After stage 7, Y14 localizes to the posterior of the oocyte. Thus, Y14 is asymmetrically distributed in the oocyte, where it colocalizes with Mago-nashi at the posterior pole. This suggests that Y14 might be part of the oskar mRNA localization complex (Hachet, 2001).
To test whether Y14 shuttles with the Oskar mRNA posterior transport machinery, the localization of Y14 was examined in a genetic context in which Oskar mRNA and its partner, the double-stranded RNA binding protein, Staufen, were mislocalized. To do so, a par-1 mutant combination (9A/w3) was employed in which Oskar mRNA is targeted to an ectopic site, due to misorientation of the microtubule network. In this genetic background, as in the wild-type, Y14 colocalizes with Staufen, indicating that Y14 travels with the Oskar mRNA localization complex. This supports the idea that Y14 may be part of this complex (Hachet, 2001).
To analyze the function of Y14 in vivo, a genetic analysis of the y14 gene was performed. Through database searches, a transposable element (EP element) inserted 70 base pairs upstream of the y14 start codon was identified in the EP(2)0567 line. This line is homozygous lethal, and lethality is observed when the EP element is placed over three independent chromosomal deletions covering the y14 locus. Hence, the lethality is closely associated with the EP element insertion. In addition, the lethality can be rescued by mobilizing the transposable element. This indicates that the EP(2)0567 insertion affects the expression of an essential gene. Transposable element insertions are known to interfere with the expression of downstream coding sequences. Since this mutation is lethal, in order to test whether y14 expression is affected by the EP insertion, homozygous clones were generated in mosaic tissue, making use of the FLP/FRT system. Homozygous mutant clones were marked by the absence of a GFP signal, whereas heterozygous and wild-type cells were marked by a GFP signal of proportional intensity. This clonal analysis revealed that the level of Y14 expression is reduced in heterozygous compared to wild-type cells. Importantly, no Y14 protein was detected in EP(2)0567 homozygous cells, either in somatic or germline clones. This shows that EP(2)0567 strongly reduces or even completely abolishes the expression of y14 and thus constitutes a strong y14 allele. This is confirmed by the fact that the excision of the EP element restores Y14 expression (Hachet, 2001).
Because Y14 is likely to belong to the Oskar mRNA localization complex, whether Y14 has a role in Oskar mRNA localization was investigated. The effect of the y14EP(2)0567 allele on the distribution of three localized mRNAs, Oskar, Gurken, and Bicoid, was investigated by in situ hybridization. For this purpose, homozygous y14EP(2)0567 germline clones were generated by using the FLP/FRT ovoD system, which allows only homozygous mutant germline clones to proceed through oogenesis. Interestingly, the analysis of Oskar mRNA distribution revealed that the early transport of Oskar mRNA is not affected by the y14EP(2)0567 mutation. From stages 3 to 5, Oskar mRNA accumulates in the oocyte, showing that its transport from the nurse cells to the oocyte does not depend on Y14. At stage 7, when the microtubule network is reoriented, while Oskar mRNA is transported toward the posterior in the wild-type, in y14EP(2)0567 egg chambers, Oskar mRNA fails to localize at the posterior. This defect in Oskar mRNA posterior localization is persistent, since Oskar mRNA never accumulates at the posterior pole and eventually diffuses throughout the oocyte during the ooplasmic streaming that occurs at stage 10b. In contrast, both Gurken and Bicoid mRNAs reach their final destination in y14 mutant egg chambers, even if the amount of localized mRNA seems reduced. This shows that their localization competence per se is not altered in the absence of Y14. As Staufen colocalizes with Oskar mRNA at the oocyte posterior, the effect of y14EP(2)0567 on Staufen protein distribution was examined. As expected, Staufen accumulates properly in the oocyte during the early stages, but is not observed at the posterior pole after stage 9, in contrast to the wild-type. Instead, Staufen is detected throughout the oocyte and at the anterior pole, confirming the Oskar mRNA mislocalization phenotype. Staufen localization is rescued by excision of the P element. In addition, no Oskar protein was detected at the posterior of y14EP(2)0567 egg chambers, consistent with the defect in Oskar mRNA localization, because Oskar mRNA translation is only activated at the posterior pole. The in situ localization study reveals that Y14 is specifically required for Oskar mRNA posterior localization, suggesting that Y14, like Mago-nashi, may have a direct function in the posterior transport of Oskar mRNA (Hachet, 2001).
During later stages of oogenesis, the nurse cells degenerate, expelling their entire cytoplasm into the oocyte in a process called 'dumping'. The y14 mutation leads to a dumpless phenotype; laid eggs are smaller than wild-type and are unfertilized. This prevented an analysis of the effect of the mutation on embryonic patterning (Hachet, 2001).
Since human Y14 has been described as imprinting mRNAs upon splicing, it is tempting to imagine that recruitment of the Y14:Mago-nashi complex upon splicing of Oskar mRNA constitutes a critical step in the assembly of the posterior transport machinery. If this were the case, this mechanism would constitute a new function for the splicing event, the deposit of a landmark addressing mRNAs to their cytoplasmic destinations (Hachet, 2001).
In Drosophila, formation of the axes and the primordial germ cells is regulated by interactions between the germ line-derived oocyte and the surrounding somatic follicle cells. This reciprocal signaling results in the asymmetric localization of mRNAs and proteins critical for these oogenic processes. Mago Nashi protein interprets the posterior follicle cell-to-oocyte signal to establish the
major axes and to determine the fate of the primordial germ cells. Using the yeast two-hybrid system, an RNA-binding protein, Tsunagi, has been identified that interacts with Mago Nashi protein. The proteins coimmunoprecipitate and colocalize, indicating that they form a complex in vivo. Immunolocalization reveals that Tsunagi protein is localized within the posterior oocyte cytoplasm during stages 1-5 and 8-9, and this localization is dependent on wild-type mago nashi function. When tsunagi function is removed from the germ line, egg chambers develop in which the oocyte nucleus fails to migrate, Oskar mRNA is not localized within the posterior pole, and dorsal-ventral pattern abnormalities are observed. These results show that a Mago Nashi-Tsunagi protein complex is required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells (Mohr, 2001).
The Mago-Tsunagi complex localizes within the posterior pole of the
oocyte during stages 8 and 9 of oogenesis. Posterior pole localization
of OSK mRNA is first detected during stage 9 of oogenesis. Previous studies reveal that OSK mRNA accumulates within the oocyte but fails to
localize within the posterior pole of egg chambers from
mago1 mutant females, owing specifically to the
inability of the Mago1 protein to localize within the
posterior pole plasm. In mago1 mutant egg chambers Tsunagi protein fails to localize within the posterior end of the oocyte. The results establish that the Mago-Tsunagi complex is detected within the posterior pole prior to and during the time when OSK mRNA is initially sequestered within this discrete cytoplasmic region of the oocyte, and that detection of the complex within the posterior pole is dependent on wild-type mago function (Mohr, 2001).
tsunagi is Japanese for 'connection' or 'link.' Hybridization of a probe from tsu cDNA to polytene chromosomes was used to ascertain the location of tsu in the genome. A single focus of hybridization on the right arm of chromosome 2, in polytene chromosome interval 45A4, was detected. Two genomic DNA contigs in the region, Dbp45A and hig, were examined by PCR for the presence of tsu sequence. A 30-40-kb P1 phagemid clone, DS02099 (BDGP), that maps to the distal end of Dbp45A was found to contain tsu (Mohr, 2001).
Mutational analysis of tsu reveals that mothers homozygous or
heteroallelic for tsu alleles can produce egg chambers in
which OSK mRNA fails to localize within the posterior pole.
Consistent with this observation, embryos from tsu mutant
females that survive to the time of cellularization lack primordial
germ cells. Although posterior pole localization of OSK mRNA
is not detected in tsu mutant oocytes, its anterior pole
accumulation at stage 8 of oogenesis appears normal. A similar result
is observed when germ-line clones lacking tsu+
function are examined for the presence and distribution of OSK mRNA. However, when follicle-cell clones are induced, no apparent abnormalities in OSK mRNA localization are detected. The distribution of Mago protein within mutant tsu egg chambers is indistinguishable from wild type, suggesting that posterior pole localization of Mago protein occurs independently of Tsunagi function. These results show that (1) in mutant tsu egg chambers
OSK mRNA is transcribed and deposited into the oocyte, (2)
anterior localization of OSK mRNA does not require
tsu+ function, (3) posterior pole localization of
Mago protein is independent of tsu+ function, and
(4) posterior pole accumulation of OSK mRNA is dependent on
tsu+ germ-line function (Mohr, 2001).
What role might the molecular interaction between Mago and Tsunagi serve in the posterior localization of the Staufen protein/OSK mRNA complex? Several lines of evidence indicate that Mago protein is required to anchor/localize components within the posterior pole that
mediate the localization of the Staufen protein/OSK mRNA
complex within the pole during stage 9: (1) Mago colocalizes with
the Staufen protein/OSK mRNA complex; (2) Mago protein is mislocalized to the same ectopic site as the Staufen protein/OSK mRNA complex in mutants in which oocyte polarity is disrupted; for example, gurken; (3) localization of Mago within the posterior pole (during stage 8)
precedes the posterior pole accumulation of the Staufen/OSK
mRNA complex and is not dependent on localization of the complex. The evidence indicates that posterior pole localization of Mago occurs independently from the
Staufen protein/OSK mRNA complex and Tsunagi protein. Mago's
ability to interact with a particular cytoplasmic location
independently of the components that it localizes suggests that it may
serve as an adaptor that recognizes a specific site within the
posterior pole of the oocyte (Mohr, 2001).
Given its interaction with Mago protein, it is likely that Tsunagi is
required for anchoring the Staufen protein/OSK mRNA complex
within the posterior pole but not for its transport to this cytoplasmic
destination. In agreement with this conclusion, the distribution of
Tsunagi protein during multiple stages of oogenesis is
indistinguishable from that of Mago protein. If Tsunagi protein were
involved in transporting the Staufen protein/OSK mRNA complex
to the posterior pole, its subcellular distribution would more closely
reflect that of Staufen protein and OSK mRNA, which mirror one
another during oogenesis. In addition,
RNA-binding experiments have failed to reveal interaction between Tsunagi
protein and OSK mRNA (Mohr, 2001).
Some of the spatial cues which direct early patterning events in Drosophila embryogenesis are maternal mRNAs localized in the oocyte during oogenesis. Microtubules, but not microfilaments, are required for localization of these mRNAs during oogenesis. However, the RNAs show a differential sensitivity to microtubule inhibitors. Anterior localization of Bicaudal-D, Fs (1) K10, and Orb mRNAs is completely disrupted following even mild drug treatments. Bicoid mRNA localization is intermediate in its response to microtubule drugs, while Oskar mRNA localization is much more resistant. In addition, the localized mRNAs respond differently to taxol, a microtubule stabilizing agent. The differences among these mRNAs suggest that factors other than microtubules are required to maintain the positions of localized mRNAs in the oocyte (Pokrywka, 1995). It appears that there is an interactin between the actin and tubulin based cytoskeletons. Profilin, encoded by chicakadee, a component of the actin based cytoskeleton, physically interacts with Cappuccino, involved in the microtubule based cytoskeleton. Mutants in chickadee resemble cappuccino in that they fail to localize Staufen protein and Oskar mRNA in the posterior pole of the developing oocyte. Also, a strong allele of cappuccino has multinucleate nurse cells, similar to those previously described in chickadee (Manseau, 1996).
Mutations in genes that reduce Oskar mRNA localization or activity can be recovered as viable sterile adults. In a screen for mutants defective in germ cell formation, nine alleles of the tropomyosin II gene were isolated. Mutations in tropomyosin II virtually abolish Oskar RNA localization to the posterior pole, suggesting an involvement of the actin network in Oskar RNA localization (Erdelyi, 1995).
Movement of Oskar mRNA involves two aspects of major importance to developmental biology, protein-RNA interaction and the cytoskeleton. Interactions between proteins and RNA are involved in RNA subcellular localization and in translational regulation. Oskar mRNA interacts with Gurken protein in the process of Oskar mRNA subcellular localization. The cytoskeleton of the oocyte is critical to the subcellular localization of Oskar mRNA. The protein Cappuccino, which interacts with the oocytic microtubular system is involved in the process of Oskar mRNA subcellular localization. Since there are two major kinds of cytoskeleton, tubulin based microtubules and actin based microfilaments, it is of interest to know which type of cytoskeleton is involved in Oskar mRNA subcellular localization. It appears that both systems are involved, as the two systems act in an interdependent manner. In fact, Profilin (encoded by chickadee), a component of the actin based cytoskeleton, physically interacts with Cappuccino. Profilin, like Cappuccino, is required for the subcellular localization of Oskar mRNA (Manseau, 1996 and references)
Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. A lack-of-function mutation in the cytoskeletal isoform (cTmII) produces zygotic mutant embryos with defects in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. Oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. Nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of Oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton (Tetzlaff, 1996).
Localization of mRNAs is one of many aspects of cellular
organization that requires the cytoskeleton. In Drosophila,
microtubules are known to be required for correct localization of
developmentally important mRNAs and proteins during oogenesis;
however, the role of the actin cytoskeleton in localization is less
clear. Furthermore, it is not known whether either of these
cytoskeletal systems are necessary for maintenance of RNA
localization in the early embryo. The contribution of the actin and
microtubule cytoskeletons to maintenance of RNA and protein
localization in the early Drosophila embryo has been examined.
While microtubules are not necessary, the actin cytoskeleton is
needed for stable association of Nanos, Oskar, Germ
cell-less and Cyclin B mRNAs, as well as Oskar and Vasa
proteins at the posterior pole in the early embryo. In contrast,
Bicoid RNA, which is located at the anterior pole, does not
require either cytoskeletal system to remain at the anterior (Lantz,
1999).
While Cytochalasin D (CD) has a strong affect on maintenence
of posteriorly-localized components, for each gene product
examined, a significant number of embryos still have a small but
detectable level of RNA or protein present at the posterior pole. The
RNA or protein remaining for all posterior components examined is
usually tightly apposed to the cortex and often patchy, in contrast to
control embryos where the RNA/protein is evenly concentrated in a
disclike shape at the posterior. Some components may remain
because there are actin filaments resistant to depolymerization by
CD and latrunculin A. In fact, actin filaments can still be present
after CD treatment in some cells. In this case, however, no actin
filaments are observed following these treatments and
actin-dependent processes are significantly affected. If there is a
resistant population, it must be a minor component of the actin
network that is difficult to visualize. Alternatively, posterior
components may be anchored to another cellular component at the
membrane in addition to the actin cytoskeleton, making this subset
more stably associated with the cortex. A complex of cytoskeletal
proteins, the 95F unconventional myosin and D-CLIP-190, is
enriched at the posterior of the early embryo. This complexes'
posterior enrichment is also affected by treatment with CD. In those
experiments, incubation for 30 min with 10 mg/ml CD caused
strong effects on enrichment. In contrast, longer incubations (45
min) are required to observe similar effects on posterior group gene
products. The apparent difference in sensitivity of these different
components to actin disruption may be the result of differences in
global distribution of the components. 95F myosin and CLIP are
present in the entire cortex, but enriched at the posterior. Upon
disruption of actin, their distribution becomes more uniform. A
small amount of residual protein complex retained with polar
granules at the posterior would likely be obscured by the high level
of general cortical staining. In contrast, the posterior group gene
products are essentially only detected in the cortex at the posterior.
Therefore, the small residual amount that remains is much more
visible. The posterior maintenance of Oskar protein is affected by
disruption of the actin cytoskeleton using two drugs that act in
different ways to depolymerize actin filaments. Cytochalasin D
binds to and stabilizes the barbed end of the actin filament, where
rapid polymerization/depolymerization would normally occur.
Consequently, actin filaments bound by CD depolymerize from the
pointed end where slow polymerization/depolymerization normally
occurs. In contrast, latrunculin A depolymerizes actin filaments by
binding in a 1:1 ratio to actin monomers. Therefore, the actin
cytoskeleton is depolymerized due to turnover of existing actin
filaments and the failure to polymerize new ones. In addition,
latrunculin A is reported to cause more complete and rapid
depolymerization of the actin cytoskeleton. The similarities in the effects
of both drugs suggests that loss of localization is due to disruption
of actin filaments and not secondary drug effects (Lantz, 1999).
Cooperation between different cytoskeletal components appear
to mediate RNA localization. A model is presented for RNA
localization of posterior pole plasm components. During oogenesis,
mRNAs are synthesized in the nurse cells and transported along
microtubules that during early stages are oriented with their plus ends at the
posterior of the oocyte. Later, microtubules are
important for cytoplasmic flow/streaming, which may allow some
mRNAs that are localized later in oogenesis to reach the posterior
pole (e.g. Nanos, Cyclin B and Gcl). At
least for posteriorly localized mRNAs/proteins, it appears that
microtubules are required only for these molecules to reach the
posterior pole and not for them to remain there. Once mRNAs are
localized, the actin cytoskeleton is likely to be required to anchor
mRNAs/proteins in late stage oocytes and then maintain them at
the posterior pole during early embryogenesis. It has been suggested that
during oogenesis, actin filaments are not absolutely required for
posterior accumulation since transport of newly synthesized RNA continues
along microtubules from the nurse cells to the posterior.
This continued transport during mid-oogenesis may mask a
requirement for anchoring via the actin cytoskeleton. This model
would reconcile the somewhat contradictory data that CD
depolymerization of actin has no effect on localization during late
oogenesis, but tropomyosin and profilin mutants have reduced
accumulation of posterior pole plasm components. It is not known
how the association of mRNAs with cytoskeletal elements is
mediated. Two possible players are a myosin (95F unconventional
myosin), and a microtubule-binding protein (D-CLIP-190); both are
concentrated at the posterior of the early Drosophila embryo.
Posterior enrichment of both proteins in the early embryo is
dependent on posterior pole plasm assembly. Their maintenence at
the posterior depends on actin but not the microtubule cytoskeleton.
These two cytoskeletal proteins, which are present in the same
complex, may coordinate interaction between the actin and
microtubule cytoskeletons and hence, may play a role in anchoring of
mRNAs and proteins targeted to the posterior pole (Lantz, 1999).
Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the
same machinery and RNA signals drive specific accumulation of maternal RNAs in
the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos;
interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor
complex that drives transcript localization in a variety of tissues (Bullock, 2001).
During Drosophila oogenesis, specification of the oocyte is associated with selective accumulation of RNA determinants supplied by the neighboring, interconnecting ovarian nurse cells. Subsequently, deposition of mRNA transcripts at selected sites within the
oocyte leads to localized translation of the proteins that establish the
prospective embryonic body axes. gurken (grk) transcripts reside first posteriorly and then anterodorsally, and sequentially establish
the anteroposterior and dorsoventral axes. bicoid (bcd) and oskar
(osk) transcripts localize to the anterior and posterior of the oocyte,
respectively, to pattern the anteroposterior body axis (Bullock, 2001).
The injection assay was used to investigate whether any maternal
transcripts that localize in the oocyte are recognized by the localization machinery of blastoderm embryos. Five such transcripts [bcd, grk, nanos (nos), osk and female sterile (1) K10 (K10)] were tested, and all accumulate in the apical cytoplasm after injection. With the exception of osk transcripts -- only a small proportion of which localize apically -- the efficiency of localization of these transcripts appears indistinguishable from that of pair-rule transcripts. Maternal transcripts also localize apically when zygotically expressed from endogenous transgenes. Preinjection with colcemid severely inhibits apical localization of the injected maternal transcripts, indicating that their localization in blastoderm embryos, like that of the pair-rule transcripts, is dependent on intact microtubules (Bullock, 2001).
The common aspect of maternal RNA localization measured in
these experiments is unlikely to be transport within the oocyte,
because the maternal transcripts tested are distinctly distributed
in late stage oocytes by means of different motors and accessory
factors. However, all the transcripts -- with the possible exception
of grk -- are synthesized in adjacent nurse cells and reach the
oocyte by transport along microtubules. To test whether this
process is analogous to apical localization in blastoderm embryos,
a bcd transcript was used containing a single nucleotide change
(4496G->U). This change prevents early oocyte-specific transport (stages
4-6) without disrupting later (stage 6 onwards) import of transcripts into the oocyte or their subsequent accumulation at the
anterior cortex. This mutation inhibits apical bcd localization in
blastoderm embryos, suggesting that transcripts localize in this injection assay through the same machinery
that transports transcripts into the early oocyte (Bullock, 2001).
These data suggest that components of the blastoderm localization machinery are also likely to function in RNA transport into the
early oocyte. Genetic screens for maternal mutations that affect
formation of the embryonic axis have identified egl and BicD as genes required for oocyte differentiation and for specific RNA
accumulation in the oocyte. However, their exact activities are
uncertain. BicD protein includes multiple heptad repeats, which may
mediate oligomerization and interactions with other proteins;
Egl includes a domain shared with 3'-5' exonucleases. During
oogenesis, these two proteins form complexes together and colocalize at the minus ends of microtubules. The integrity of the
microtubule cytoskeleton is defective in egl and BicD mutants, which has been proposed to explain subsequent defects in RNA
localization. Alternatively, Egl and BicD might act directly in
RNA transport. However, evidence that distinguishes between
these two possibilities is lacking (Bullock, 2001).
Whether Egl and BicD are present in early
embryos was examined. Both proteins are supplied maternally to the embryo.
They are noticeably enriched apical to the nuclei at blastoderm
stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the
proteins is present in the basal cytoplasm (Bullock, 2001).
Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of
protein/RNA co-transport. The complex may have an additional
role in anchoring transcripts at their destination. Alternatively,
maintenance of localized transcripts might not depend on an
independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).
A mutation in a novel gene, capulet (cap), was identified in a mosaic screen to isolate mutations that perturb actin organization in germline clones. Adenylate cyclase-associated proteins (CAPs) have been shown to inhibit actin polymerization in vitro, by sequestering monomeric actin. This actin-binding activity has been mapped to the carboxy-terminal region of CAP; however, a 'verprolin homology'-related domain has been identified in all CAPs, just carboxy-terminal of the polyproline-rich domain. In members of the verprolin/WASP family, this motif binds actin monomers in vitro, but catalyses actin polymerization in vivo. Therefore, in CAP homologues, this region of the protein may be used to facilitate actin binding. As CAP proteins have also been found associated with Abl tyrosine kinase and with adenylate cyclase, it is possible that CAP represents an intermediary in these signal transduction cascades, perhaps altering actin dynamics in response to extracellular cues (Baum, 2000).
The genetic screen also identified a mutation in the catalytic subunit of protein kinase A (PKA). Therefore, pka and cap mutant phenotypes in the Drosophila germline were compared. Like the cap mutant, pka germline clones lose nurse cell cortical actin, while simultaneously accumulating ectopic actin structures. In addition, the pka mutant phenotype is sensitive to the dosage of CAP, and actin defects are dramatically enhanced in pka;cap double germline clones. These data suggest that PKA and CAP functionally cooperate in the germline to control actin organization (Baum, 2000).
In cap germline clones, F-actin accumulates in a highly polarized fashion within the egg chamber and oocyte. Thus, whether loss of CAP perturbs other aspects of normal polarity, including the asymmetric localization of mRNAs within the oocyte was investigated. The distributions of bicoid and oskar mRNAs, which localize to anterior and posterior poles of the oocyte, respectively, were examined. Although oskar mRNA is concentrated in one region of the oocyte in over 90% of egg chambers, oskar mRNA is mislocalized in 76% of stage 8-10 cap germline clone egg chambers. Moreover, in 28% of cases, oskar transcripts are localized to the anterior or lateral part of the oocyte. In addition, in 64% of stage-10 egg chambers that maintain correct overall polarity, oskar mRNA has a diffuse distribution and is not tightly focused at the posterior pole. The localization of bicoid transcripts was also examined. bicoid mRNA accumulates at an aberrant site in 65% of cap mutant egg chambers, and is localized to the posterior pole in 36% of stage 8-10 egg chambers. Thus, cap germline clones display two related mRNA polarity defects: (1) although oocytes are able to concentrate oskar and bicoid mRNAs locally within the oocyte, they appear unable to coordinate mRNA polarity with the morphological polarity of the egg chamber; (2) in the majority of egg chambers in which oskar mRNA is correctly transported to the posterior pole of the oocyte, oskar message is not tightly localized at the cortex (Baum, 2000).
It can be concluded that CAP is a major regulator of actin dynamics in Drosophila, and that CAP is likely to function to inhibit actin polymerization in vivo, as it does in vitro. A striking feature of the cap phenotype is the accumulation of actin filaments at polar sites within the egg chamber. This cannot be explained by differences in the monomeric actin pool in nurse cells versus the oocyte, as G-actin, as measured by DNaseI staining, is equally distributed within the egg chamber, as is profilin. Thus, CAP inhibits actin filament formation at specific cellular sites, possibly in response to signaling events (Baum, 2000).
In both yeast and multicellular eukaryotes, the actin cytoskeleton responds to cell signaling events. Therefore it is interesting to note that homologs of Drosophila CAP have been shown to interact physically with an Abl tyrosine kinase and adenylate cyclase. These latter proteins transduce extracellular cues, in a way that is not fully understood, to remodel the actin cytoskeleton within the growth cones of migrating neurons to facilitate axon guidance. Thus, CAP may constitute part of the machinery that reorganizes the actin cytoskeleton in response to these signals in neurons and in other polarized cells. Interestingly, the genetic screen also identified the catalytic subunit of protein kinase A (PKA), which acts downstream of adenylate cyclase, as a gene required for proper actin organization and oocyte polarity. Since yeast, Hydra and human CAPs have been shown to facilitate the activation of adenylate cyclase, CAP and PKA may be elements of a conserved signal transduction pathway. The phenotypic similarities shared by cap and pka germline clones suggest that CAP and PKA act together in the Drosophila female germline. Given this interaction, CAP could be a substrate for PKA, or could facilitate the activation of adenylate cyclase upstream of PKA. Alternatively, because a reduction in both CAP and PKA activity leads to a more severe phenotype, the two genes may act in parallel pathways. CAP and PKA are, however, unlikely to be essential components in a common signal transduction pathway in Drosophila because no evidence is found for related CAP and PKA functions in somatic tissues (Baum, 2000).
In existing mutants known to perturb the germline actin cytoskeleton, oocyte polarity is either unaffected or completely disrupted. Therefore, whether oocyte polarity is altered in the cap mutant was investigated by examining the localization of both bicoid and oskar mRNAs. When compared to other known mutants, cap germline clones exhibit novel mRNA polarity defects (although similar defects are exhibited by pka null germline clones). First, cap mutant oocytes are able to localize mRNAs to discrete areas within the oocyte, but the sites of mRNA deposition do not respect the existing morphological axes of the egg chamber. Second, in the majority of stage-10 egg chambers with the correct polarity, oskar mRNA is observed in a shallow gradient, as if diffusing away from the cortex at the posterior pole. Thus, CAP seems to be required, both to coordinate mRNA localization with the axial polarity of the egg chamber, and to tether mRNAs to the cortex. Because microtubules are thought to mediate the transport of mRNAs to opposite poles of the oocyte in the wild type, the defect in oocyte axial polarity in the cap mutant may result from defects in the underlying microtubule cytoskeleton. cap germline clones frequently contain a misoriented microtubule array, with plus ends focused at the anterior cortex. This altered microtubule polarity is therefore probably responsible for the mislocalization of oskar and bicoid mRNAs at early stages of oogenesis. At later stages, following disassembly of the polar microtubule array, an actin-based structure at the posterior pole of the Drosophila oocyte, dependent on CAP and tropomyosin, may act as a tether to hold oskar mRNA at the cortex (Baum, 2000).
The asymmetric localization of messenger RNA (mRNA) and protein determinants plays an important role in the establishment of complex body plans. In Drosophila oocytes, the anterior localization of Bicoid mRNA and the posterior localization of Oskar mRNA are key events in establishing the anterior-posterior axis. Although the mechanisms that drive Bicoid and Oskar localization have been elusive, oocyte microtubules are known to be essential. The plus end-directed microtubule motor kinesin I is required for the posterior localization of Oskar mRNA and an associated protein, Staufen, but not for the anterior-posterior localization of other asymmetric factors. Thus, a complex containing Oskar mRNA and Staufen may be transported along microtubules to the posterior pole by kinesin I (Brendza, 2000).
To determine if kinesin I is involved in oocyte patterning, mitotic recombination was used to generate mosaic female flies containing
clones of homozygous Khc null germ line stem cells. The
production of eggs and embryos by the mosaic females suggests that germ
line stem cells can proliferate and proceed through oogenesis without
kinesin I. However, embryogenesis fails, despite fertilization by
wild-type males. Most embryos arrest before blastoderm formation, but a few proceed into early gastrulation stages. This maternal lethal
effect is completely rescued by a wild-type Khc transgene. Thus, germ line expression of KHC is required for normal embryogenesis (Brendza, 2000).
Examination of embryos that reach the blastoderm stage reveals an
absence of pole cells, the germ line precursors. To assay for earlier
defects, the distributions of OSK and BCD mRNAs in Khc null oocytes were examined.
The localization of BCD mRNA is normal, concentrated at the anterior
during stages 8 to 10. In contrast, the localization of
OSK mRNA is defective. It normally
accumulates transiently at the anterior pole early in stage 8 and then
moves to the posterior pole. In
Khc null stage 8 to 10 egg chambers, OSK mRNA accumulates excessively at the anterior pole and is never concentrated at the posterior pole. This localization defect is completely rescued by a wild-type Khc transgene. Thus, although KHC is not required for anterior localization of either
BCD or OSK mRNAs, it is required for the posterior
localization of OSK. Perhaps kinesin I transports OSK mRNA along microtubules toward their plus ends and the posterior pole (Brendza, 2000).
Given that microtubule-disrupting drugs prevent the posterior
localization of OSK mRNA during oogenesis, the possibility was considered that the absence of KHC blocks OSK localization indirectly by
disturbing oocyte microtubules. The shift of the oocyte nucleus from
posterior to anterior poles during stage 6, which is microtubule-
dependent, appears normal in Khc
null oocytes. Furthermore, the anterior localization of the MTOC
component Centrosomin is normal (Brendza, 2000).
Microtubule organization was tested further by localizing a hybrid
protein composed of the motor domain of KHC fused to a reporter enzyme,
beta-galactosidase (beta-Gal). KHC::beta-Gal is thought to
localize in regions of cells with high concentrations of microtubule
plus ends. It is important to note that this chimeric protein does not rescue patterning defects in Khc null
oocytes. In wild-type stage 9 to 10a oocytes, KHC::beta-Gal
concentrates at the posterior pole. In Khc null oocytes, KHC::beta-Gal
concentrates at the posterior pole in most instances. This suggests that in most of the stage 9 to
10a null oocytes with detectable amounts of KHC::beta-Gal,
microtubule plus ends are concentrated at the posterior pole. This,
and the indications that microtubules in the anterior end are normal, suggests that OSK mRNA mislocalization in Khc
null oocytes is not due to a disruption of microtubule organization. In
Khc null oocytes, KHC::beta-Gal staining is often
not detected in oocytes, although it is visible in nurse cells.
Perhaps efficient transport of the hybrid protein from nurse cells to
oocyte requires the presence of native KHC (Brendza, 2000).
Posterior transport of OSK mRNA is thought to depend
on Staufen protein. Staufen is transiently localized at the anterior end of the oocyte during stage 8 where it may form a complex with OSK mRNA. If kinesin I transports such
OSK-Staufen complexes along microtubules to the posterior pole, then Staufen protein should be mislocalized in Khc
null oocytes. Immunostaining with anti-Staufen
confirms this prediction. In wild-type stage 8 to 10 oocytes, Staufen concentrates at the anterior end early, appears in
granules along the cortex, and then concentrates at the posterior end.
Granular Staufen distribution was detected in most oocytes
observed. This is consistent with the hypothesis that Staufen and
OSK mRNA form complexes at the anterior cortex that are
transported to the posterior pole. In Khc mutant oocytes, Staufen protein overaccumulates in the anterior end during stage 8, is
not detected in granules, and does not concentrate at the posterior pole. Normal Staufen distribution patterns are
restored in Khc null oocytes by the addition of a wild-type
Khc transgene (Brendza, 2000).
Thus, KHC, the force-generating component of the plus
end-directed microtubule motor kinesin I, is required for the
posterior localization of both OSK mRNA and Staufen protein.
The participation of kinesin I in this mRNA motility process could be
direct. It might attach specifically to osk-Staufen
complexes at the anterior pole and transport them toward the posterior
pole. However, initial tests for coimmunoprecipitation of KHC and
Staufen from Drosophila ovary cytosol have not revealed any
robust association, so perhaps the linkage is less direct. It is
generally accepted that kinesin I transports membranous organelles
toward microtubule plus ends. Thus, OSK and
Staufen could localize to the posterior pole by virtue of association
with mitochondria or other organelles carried by kinesin I. An
alternative to these models is derived from the effect of a loss of KHC
on the particulate staining pattern of Staufen. Before stages
7 to 8, while microtubules are still oriented with their plus ends toward the anterior, kinesin I might deliver, to the cortex, materials necessary for the assembly of transport-competent
OSK-Staufen complexes. Thus, the lack of visible Staufen
particles in Khc null oocytes may indicate that their
assembly or persistence depends on kinesin I activity. New studies,
using green fluorescent protein tags to follow the localization
dynamics of OSK mRNA, Staufen, and organelles, may
distinguish between these models and provide further insight into the
mechanisms that drive the movements of maternal determinants for early
developmental patterning (Brendza, 2000).
Microtubules and the Kinesin heavy chain (the force-generating component of the plus end-directed microtubule motor Kinesin I) are required for the localization of oskar mRNA to the posterior pole of the Drosophila oocyte, an essential step in the determination of the anteroposterior axis. The Kinesin heavy chain is also
required for the posterior localization of Dynein, and for all cytoplasmic movements within the oocyte.
Furthermore, the KHC localizes transiently to the posterior pole in an oskar mRNA-independent manner.
Surprisingly, cytoplasmic streaming still occurs in kinesin light chain null mutants, and both oskar mRNA and Dynein localize to the posterior pole. Thus, the Kinesin heavy chain can function independently of the light chain in the oocyte, indicating that it associates with its cargoes by a novel
mechanism (Palacios, 2002).
To determine whether kinesin functions in the same step of oskar
mRNA localization as the other proteins required for this process, the distribution of the RNA in germline clones of a null allele of the Kinesin
heavy chain, Khc27 were compared to barentsz, staufen and
mago nashi mutants. Although no oskar mRNA reaches the
posterior of the stage 9 oocyte in Khc27, there is a clear
difference in the distribution of the mRNA from that observed in the other
mutants, such as barentsz. In the latter, oskar mRNA remains
tightly localized at the anterior cortex, whereas it is found throughout the
anterior half of the oocyte in Khc27 mutant clones. Fluorescent in situ hybridization was performed to examine the distribution of
oskar mRNA in the Khc mutant in more detail, using confocal
microscopy. This reveals an anterior-to-posterior gradient of mRNA with an
enrichment along the lateral cortex. Consistent with this, antibody staining for Staufen protein shows a distribution identical to oskar mRNA. These results suggest that the Khc mutant blocks oskar mRNA localization
after it has been released from the anterior cortex, whereas all of the other
factors are required for this release (Palacios, 2002).
In an attempt to understand the mechanism for oskar mRNA transport
to the posterior, the movement of a GFP-Staufen fusion protein was analyzed in
living oocytes. Although this fusion protein localizes to the posterior with
oskar mRNA and rescues the oskar mRNA localization defect of
a staufen null mutant, movements
that unambiguously correspond to posterior transport have not been resolved. One possible explanation
for this failure is that most of the fluorescent GFP-Staufen particles do not
contain oskar mRNA, which is expressed at much lower levels than the
fusion protein. Thus, the relevant oskar mRNA/GFP-Staufen complexes
may be too rare or too weakly fluorescent to follow in time-lapse films.
Although it was not possible to determine how GFP-Staufen reaches the
posterior, the results do reveal several important features of this process
that are relevant to the discussion of the models for the mechanism of
oskar mRNA localization (Palacios, 2002).
One model proposes that cytoplasmic flows circulate oskar mRNA
around the oocyte, so that it can then be efficiently trapped at the posterior
by a pre-localized cortical anchor.
Indeed, this mechanism would account for the failure to detect any directed
transport of GFP-Staufen to the posterior pole. The observation that the KHC
is required for all cytoplasmic flows in the oocyte also supports this model,
since it provides an explanation for why the KHC is required to localize
oskar mRNA. However, several other considerations make this mechanism
unlikely. (1) The cytoplasmic flows are much weaker at the posterior of the
oocyte than elsewhere, presumably because there are fewer microtubules in this
region, and many oocytes show little or no cytoplasmic movement near the
posterior pole. It is therefore hard to imagine how cytoplasmic flows could
efficiently deliver the mRNA to a posterior anchor. (2) The hypothetical
anchor would have to localize to the posterior before oskar mRNA and
in an oskar mRNA independent manner, and no proteins that meet these
criteria have been identified so far. Indeed, the only proteins that fulfil
the second criterion are the KHC and the components of the dynein/dynactin
complex. (3)oskar mRNA localizes to the center of the oocyte in
mutants that alter the organization of the microtubule cytoskeleton, such as
gurken, pka and par-1, and it is hard to reconcile this with
trapping by a cortical anchor, since there is no plasma membrane or cortical
cytoskeleton in this region. The localization of oskar mRNA still correlates
with the position of microtubule plus ends in these mutants, because
Kin-ßGal forms a dot in the center of the oocyte with the mRNA, and this
is more consistent with the model in which oskar mRNA is transported
along microtubules towards the posterior pole. Finally, the KHC accumulates at
the posterior during the stages when oskar mRNA and DHC are
localized, strongly suggesting that KHC plays a direct role in transporting
them there (Palacios, 2002).
Another model for oskar mRNA localization proposes that the KHC
functions to transport the RNA away from the minus ends of the microtubules at
the anterior and lateral cortex towards the plus ends in the interior of the
oocyte, and that the lack of microtubules at the posterior somehow allows the
mRNA to accumulate at this pole. Two aspects of the data do not fit this cortical exclusion model. (1) No oskar mRNA or Staufen was seen at the posterior of the oocyte in Khc germline clones, regardless of whether fluorescent or wholemount in situ hybridization or antibody staining was performed. This observation seems incompatible with a model in which kinesin removes oskar mRNA from the anterior and lateral cortex, but is not required for its localization to the posterior pole. (2) The demonstration that endogenous kinesin localizes to the posterior cortex, like kinesin-ßGal, provides further evidence that the plus ends of the microtubules are enriched in this region, and strongly suggests that kinesin mediates transport to this pole. These localizations are not visible until stage 9, however, which is when oskar mRNA starts to accumulate at the posterior. Thus, conflicting results can be resolved by proposing that the plus ends lie in the middle of the oocyte at stage 8, when a kinesin-dependent accumulation of oskar mRNA in the central dot is seen, and that microtubules are only recruited to the posterior at stage 9, coincident with the onset of oskar mRNA localization (Palacios, 2002).
In light of the posterior localization of endogenous kinesin, it is thought
most likely that this motor does transport oskar mRNA to the
posterior of the oocyte, even though this movement has not yet been seen.
The link between the KHC and the oskar mRNA localization complex need
not be direct, however. The KHC probably transports something else to the
posterior of the oocyte, in addition to oskar mRNA and dynein. This is thought to be so
because mutants that abolish either oskar mRNA localization (such as
staufen and barentsz) or DHC localization
(Dhc64C6-6/Dhc64C6-12) have no effect on the
posterior localization of the KHC, even though the motor activity of the KHC
is thought to require binding to a cargo. The KHC is also required for
cytoplasmic streaming, and presumably induces these flows by moving a large
structure, such as a vesicle or organelle, along microtubules. This structure
should therefore accumulate at the posterior of the oocyte during stage 9,
because this is where the microtubule plus ends and the KHC itself localize.
Thus, oskar mRNA and dynein could reach the posterior at stage 9 by
hitch-hiking on the large cargo that drives streaming. This proposal is
consistent with several other observations: (1) the fact that cytoplasmic
streaming, oskar mRNA localization and dynein localization all share
the very unusual property of being light chain independent suggests that they
all depend on a single KHC-mediated transport process, which could be the
transport of the cargo that induces streaming to the posterior; (2) it has
been shown in a number of other systems that plus and minus end directed
microtubule motors, such as kinesin and dynein, are found on the same
organelles; (3) if dynein and oskar mRNA interact with the
kinesin cargo independently of each other, this would explain why both their
posterior localizations require the KHC, but do not require each other, and
finally, (4) there is already evidence that links oskar mRNA localization
with vesicle trafficking, since mutants in rab11, a small GTPase
implicated in the regulation of endocytic vesicle recycling, disrupt the
posterior localization of oskar mRNA. Furthermore, Rab11 itself localizes to the posterior
of the oocyte. The effect of Rab11 on oskar mRNA localization may be
indirect, however, since these mutants also disrupt the organization of the
microtubule cytoskeleton (Palacios, 2002).
If the hitch-hiking model for oskar mRNA localization is correct,
Staufen, Barentsz, Mago nashi and Y14 would be required to couple the mRNA to
the vesicle or organelle that is transported by kinesin. In this context, it
is interesting to note that mammalian Staufen homologs have been shown to
associate with the endoplasmic reticulum. The localization of Vg1 mRNA to the vegetal pole of Xenopus oocytes requires the RNA-binding protein VERA/Vg1 RBP, which co-fractionates with markers for the endoplasmic reticulum, and this has led to the suggestion that Vg1 mRNA is transported in association with ER vesicles. Thus, hitchhiking on vesicles may represent a general mechanism for mRNA transport (Palacios, 2002).
Mass movements of cytoplasm, known as cytoplasmic streaming, occur in some
large eukaryotic cells. In Drosophila oocytes there are two forms of
microtubule-based streaming. Slow, poorly ordered streaming occurs during
stages 8-10A, while pattern formation determinants such as oskar mRNA
are being localized and anchored at specific sites on the cortex. Then fast
well-ordered streaming begins during stage 10B, just before nurse cell
cytoplasm is dumped into the oocyte. The plus-end-directed
microtubule motor kinesin-1 is required for all streaming and is
constitutively capable of driving fast streaming. Khc mutations
reduce the velocity of kinesin-1 transport in vitro, block streaming, yet
still support posterior localization of oskar mRNA -- this suggests that
streaming is not essential for the oskar localization mechanism.
Inhibitory antibodies indicated that the minus-end-directed motor dynein is
required to prevent premature fast streaming, suggesting that slow streaming
is the product of a novel dynein-kinesin competition. Since F-actin and some
associated proteins are also required to prevent premature fast streaming, these
observations support a model in which the actin cytoskeleton triggers the
shift from slow to fast streaming by inhibiting dynein. This allows a
cooperative self-amplifying loop of plus-end-directed organelle motion and
parallel microtubule orientation that drives vigorous streaming currents and
thorough mixing of oocyte and nurse-cell cytoplasm (Serbus, 2005).
is likely that fast streaming is not absolutely
essential (Serbus, 2005).
The Khc allelic series also allowed exploration of a role for slow
ooplasmic streaming in determinant mRNA localization. The null allele
Khc27 prevents streaming: it blocks oskar mRNA
accumulation at the posterior pole and it blocks gurken mRNA
localization to the anterodorsal corner.
However, the hypomorphic alleles Khc17 and
Khc23, which prevented most slow streaming, support both
oskar and gurken localization. Thus, although localization of both determinants
requires Khc, it does not require slow streaming (Serbus, 2005).
It has been suggested that posterior oskar localization during
stages 7-10a proceeds via two phases. (1) oskar RNPs are driven by kinesin-1
away from microtubule minus ends at the anterior and lateral cortex, which
leads to a transient concentration of oskar in the central region of
the oocyte. (2) Then diffusion or other random forces, coupled with a dearth of
minus ends at the posterior cortex, facilitates encounters of oskar
RNPs with posterior anchors. Tests of Khc17 and
Khc23, which slow the ATPase activity and velocity of Khc
in vitro, show a delay in the central accumulation of oskar,
consistent with slowed kinesin-1-driven transport away from the anterolateral
cortex. Strikingly, Khc17 and Khc23
allow that central accumulation to persist through later stages, as if the
shift to posterior anchors is also slowed.
This correlation between slowed motor mechanochemistry and slowed
oskar localization supports the hypothesis that kinesin-1 links to
and transports oskar RNPs in both phases of localization (Serbus, 2005).
If microtubules are poorly ordered during oskar localization, as
suggested by GFP-tubulin imaging and by studies of fixed oocytes, how
could kinesin-1 accomplish such directed posterior transport? There may be a
special subset of microtubules, with plus-ends oriented directly toward the
posterior pole, that are difficult to distinguish among a mass of randomly
oriented microtubules. However, given that the period of oskar
localization spans at least 10 hours, and that the distance from the oocyte
center to the posterior pole is only 25-40 µm, such perfectly oriented
transport tracks should not be necessary. With microtubule minus ends most
abundant at the anterior cortex and least abundant at the posterior cortex,
plus ends should be somewhat biased toward the posterior. If kinesin-1 binds
an oskar RNP and transports it to a plus end, then binds a
neighboring microtubule and runs to its plus end, and so forth, it would
accomplish a biased random walk away from the anterolateral cortex that would
concentrate oskar RNPs near posterior anchors. This highlights a
central question about the mechanism of localization. What is the degree of
directional bias for oskar RNP transport? Advances in osk
RNP imaging that allow single particle tracking will be needed to obtain clear
answers to that question (Serbus, 2005).
Recently, several other factors have been identified that are required for
prevention of premature fast streaming. Mutations in Maelstrom
(Mael), Orb and Spindle-E (Spn-E) allow
premature fast streaming and parallel microtubule arrays during stages 8-10A. Orb, a
CPEB homolog, is required for osk translation, spn-E is
an RNA helicase, and Mael is a modifier of Vasa, which
is another RNA helicase. Perhaps these proteins control expression of actin
regulators or other factors needed to prevent premature activation of a dynein
inhibitory signal. Future work aimed at identifying the regulatory mechanisms
that control kinesin in oocytes should be an important focus in understanding
the slow-fast streaming transition and also for the broader issue of how the
functions of the actin and microtubule cytoskeletons are integrated (Serbus, 2005 and references therein).
The swallow (sww) gene encodes a novel protein whose function
in oogenesis is not well understood, and the observation that it is required for the localization
of two anteriorly positioned RNAs, Bicoid and Hu-li tai shao (hts), provides an
opportunity for a comparative study of the role sww
plays in RNA localization. Further, the reported differences
between HTS and BCD RNA localization raise several
questions: To what extent are the sww-mediated localizations
of the two RNAs similar or different? Do the localizations
of HTS and BCD RNAs share other molecular
and biochemical requirements? Are there other RNAs
that exhibit a dependence on sww for proper localization
in the oocyte (Pokrywka, 2000)?
A detailed characterization
of the phenotypes associated with each of eight sww alleles was initiated
as a means of investigating the role of sww in oogenic
patterning. Several previously
unreported RNA localization defects have been observed.
Although BCD RNA localization is often lost completely in
sww oocytes, in a high proportion of cases, BCD RNA is localized
inappropriately along the periphery of the mature
oocyte. In several sww mutants, a portion of the BCD mRNA
population becomes concentrated at the posterior pole of
the oocyte during late oogenesis. Several sww mutations
also result in oskar RNA localization defects, consistent
with a global role for sww in cytoskeletal regulation or organization.
A detailed temporal and spatial analysis of HTS
RNA localization in sww mutants and in drug-treated ovaries
reveals many similarities to BCD RNA localization, and
implies the two independent localization events are accomplished
by the same mechanism (Pokrywka, 2000).
In the Drosophila ovary, membrane skeletal proteins such as the adducin-like Hts protein(s), Spectrin, and Ankyrin are found in the spectrosome, an organelle in germline stem cells (GSC) and their differentiated daughter cells (cystoblasts). These proteins are also components of the fusome, a cytoplasmic structure that spans the cystoblast's progeny that develop to form a germline cyst consisting of 15 nurse cells and an oocyte. Spectrosomes and fusomes are associated with one pole of spindles during mitosis and are implicated in cyst formation and oocyte differentiation. The asymmetric behavior of the spectrosome persists throughout the cell cycle of GSC. Eliminating the spectrosome by the htsl mutation leads to randomized spindle orientation, suggesting that the spectrosome anchors the spindle to ensure the asymmetry of GSC division; eliminating the fusome in developing cysts results in defective spindles and randomized spindle orientation as well as asynchronous and reduced cystocyte divisions. These observations suggest that fusomes are required for the proper formation and asymmetric orientation of mitotic spindles. Moreover, they reinforce the notion that fusomes are required for the four synchronous divisions of the cystoblast leading to cyst formation. In htsl cysts that lack fusomes and fail to incorporate an hts gene product(s) into ring canals following cyst formation, polarized microtubule networks do not form, the dynamics of cytoplasmic dynein are disrupted, and Oskar and Orb RNAs fail to be transported to the future oocyte. These observations support the proposed role of fusomes and ring canals in organizing a polarized microtubule-based transport system for RNA localization that leads to oocyte differentiation (Deng, 1997).
Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, the origin of oocyte and germ plasm-associated mitochondria was studied during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton since it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. These findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support the hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles (Cox, 2003).
The Balbiani bodies in many species contain structures resembling germinal
granules. In Xenopus, these granules are found in a region containing
specific RNAs that are also destined to be localized in the egg and
incorporated in germ cells. Consequently, the Balbiani body has been proposed
to function as a messenger transport organizer (METRO) that organizes and
mediates the delivery of RNAs and germinal granules to the vegetal pole of the
egg. Specific elements have been mapped in the 3' UTR of the Xcat2 mRNA that are sufficient for localization to the Balbiani body or
to the germinal granules themselves (Cox, 2003).
The Drosophila Balbiani body may play a
related role. oskar RNA, a key component that is capable of inducing
germ plasm formation, is associated with the posterior segment of the
Balbiani body in early stage 1 oocytes, much as Xcat2 is localized in
the Xenopus Balbiani body. A few hours later, towards the end of
stage 1, osk RNA moves to the oocyte posterior along with the other
Balbiani-associated RNAs and proteins that have been studied, presumably in response to the shift in microtubule polarity that occurs at this time. Thus, at least some molecules that participate in germ plasm assembly associate with the
Balbiani body in early Xenopus and Drosophila oocytes (Cox, 2003).
Drosophila RNAs that become associated with the
Balbiani body, like organelles, first interact with the fusome during early
stages of cyst development. However, there are significant differences in
these fusome interactions with RNAs and organelles that probably reflect different molecular mechanisms
of delivery to the Balbiani body. Organelles associate next to the fusome
along much of its length and subsequently move toward the center, in concert
with microtubule minus ends. By contrast, the RNAs associate with one or a few
cells at the center of the fusome from the earliest stages they could be
detected, and are located within it, as well as nearby. These observations
suggest that localized RNAs may read the fusome polarity directly, and need
not rely on changes in microtubule organizing activity to get to the oocyte or
be stabilized within it (Cox, 2003).
Drosophila oocytes develop within cysts containing 16 cells that are interconnected by cytoplasmic bridges. Although the cysts are syncytial, the 16 cells differentiate to form a single oocyte and 15 nurse cells, and several mRNAs that are synthesized in the nurse cells accumulate specifically in the oocyte. Shortly after formation of the 16 cell cysts, a prominent microtubule organizing center (MTOC) is established within the syncytial cytoplasm, and at the time the oocyte is determined, a single microtubule cytoskeleton connects the oocyte with the remaining 15 cells of each cyst. Recessive mutations at the Bicaudal-D and egalitarian loci, which block oocyte differentiation, disrupt formation and maintenance of this polarized microtubule cytoskeleton. Microtubule assembly-inhibitors phenocopy these mutations, and prevent oocyte-specific accumulation of Oskar, Cyclin B and 65F mRNAs. Formation of the polarized microtubule cytoskeleton is required for oocyte differentiation. This structure mediates the asymmetric accumulation of mRNAs within the syncytial cysts (Theurkauf, 1993).
To determine whether Egalitarian and Bicaudal D directly affect the extent to which OSK mRNA mislocalizes, the distribution of OSK mRNA was examined in BicD-Dominant mutants. Reducing the amount of egl wild-type product decreases ectopic localization of osk to the anterior and increasing the amount of egl wild-type product enhances the mislocalization of OSK to the anterior. Because the effect of BicD-Dominant mutants depends on egl wild type function, it is concluded that egl and BicD act in the same pathway and that the two function in concert to control OSK mRNA localization. It is also thought that Egl and BicD have a role in dorsoventral polarity, as mutation of the two genes reduce the level of GURKEN mRNA. Localization of GUR is also known to require an intact microtubule cytoskeleton (Mach, 1997).
The Ovarian tumor (OTU) protein is required for the correct distribution of the Pumilio and Oskar mRNAs, while the Bic-D, K10 and Staufen mRNAs are localised in wild type fashion in otu mutants. A region of homology exists between the carboxy-terminal part of the OTU protein and the mammalian microtubule associated proteins. The more severe the mutation in this region of homology, the more disturbed mRNA distribution is observed in otu mutants (Tirronen, 1995).
Microtubule polarity has been implicated as the basis for polarized localization of morphogenetic determinants that specify the anterior-posterior axis in Drosophila oocytes. A mutation affecting Protein Kinase A (PKA) acts in the germ line to disrupt both microtubule distribution and RNA localization along this axis. In normal oocytes, the site of microtubule nucleation shifts from posterior to anterior immediately prior to polarized localization of Bicoid and Oskar mRNAs. In PKA-deficient oocytes, posterior microtubules are present during this transition: OskarmRNA fails to accumulate at the posterior, and BicoidmRNA accumulates at both ends of the oocyte. Similar RNA mislocalization patterns reported for Notch and Delta mutants (Ruohola, 1994) suggest that PKA transduces a signal for microtubule reorganization that is sent by posteriorly located follicle cells (Lane, 1994).
Polarization of the microtubule cytoskeleton during early oogenesis is required to specify the posterior of the Drosophila oocyte, which is essential for asymmetr