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
Exu-Yps 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).
oskar mRNA localization at the oocyte posterior pole is essential for correct patterning of the Drosophila embryo. This study shows, at the ultrastructural level, that endogenous oskar ribonucleoprotein complexes (RNPs) assemble sequentially with initial recruitment of Hrp48 (Heterogeneous nuclear ribonucleoprotein at 27C) and the exon junction complex (EJC) to oskar transcripts in the nurse cell nuclei, and subsequent recruitment of Staufen and microtubule motors, following transport to the cytoplasm. oskar particles are non-membrane-bound structures that coalesce as they move from the oocyte anterior to the posterior pole. This analysis uncovers a role for the EJC component Barentsz in recruiting Tropomyosin II (TmII) to oskar particles in the ooplasm and reveals that TmII is required for kinesin binding to the RNPs. Finally, it was shown that both kinesin and dynein associate with oskar particles and are the primary microtubule motors responsible for transport of the RNPs within the oocyte (Trucco, 2009).
This study shows that osk mRNA is synthesized in the nurse cell nuclei, where it assembles into small particles comprising Hrp48 and the Berentz-containing EJC, which are recruited independently to the RNA. The particles are then exported into the nurse cell cytoplasm where, upon loading of Stau, whose association is partially EJC dependent, they recruit both dynein and kinesin and associate with MTs. Upon transport into the oocyte, the small particles transiently reside at the anterior where they are remodeled, in a process requiring Hrp48, Btz, and Stau. During this process, the osk RNPs presumably lose their association with the MTs that mediated their transport from the nurse cells into the oocyte and bind to oocyte MTs for their subsequent transport (Trucco, 2009).
From the anterior, small osk particles are transported by kinesin toward the center of the oocyte, where a mixed population of small and more abundant large particles are observed. It is likely that these large particles arise through coalescence of small particles, as a result of their concentration in the center. Once formed, large particles do not appear to shuttle back to the anterior, as they were not observed in this area. Although a transient accumulation of osk mRNA in the oocyte center has been described as an important step prior to posterior transport, a recent live-cell imaging study of transgenic tagged osk mRNA argued against its existence. A probable explanation for this discrepancy lies in the different stages of oogenesis at which the analyses were performed. A central enrichment of osk mRNA is indeed detected at stage 8, whereas the in vivo analysis was restricted to stage 9 oocytes. It is possible that the transient central enrichment of osk particles reflects the dynamic organization of the MTs in the oocyte at stage 8 (Trucco, 2009).
From the center, large osk particles are transported along MTs toward the posterior pole where they form aggregates. Although a detailed understanding of MT organization will require more sophisticated ultrastructural approaches, such as EM tomography, this study has revealed that MTs are present throughout the oocyte, including the posterior cytoplasm. Indeed, osk RNPs are associated with MTs even in this region, suggesting that osk mRNA is actively transported on MT from the center of the oocyte to the posterior pole (Trucco, 2009).
At the posterior pole, osk aggregates form a continuum interspersed among abundant endocytic tubules where Long Osk has been shown to bind, suggesting that these membranous structures are involved in anchoring the mRNA at the posterior pole. However, endogenous osk RNPs detected throughout the egg chamber are non-membrane-bound structures that are not associated with any specific intracellular organelle, excluding a direct involvement of membrane traffic in osk localization (Trucco, 2009).
This analysis has shown that the loading of the shuttling proteins Hrp48 and Btz on osk mRNA is independent yet that both are required to generate mature particles that can associate with motor proteins and MTs. In particular, EJC components enhance the association between osk RNPs and TmII, which in its turn promotes the loading or stable association of Khc on osk transport particles. Moreover, in hrp48 and EJC mutants, osk is present in small particles uniformly distributed in the oocyte. It is likely that in these mutants the bulk of the mRNA in the oocyte is transported by cytoplasmic flows, explaining its homogeneous distribution and its failure to coalesce into larger particles. This also explains the 5-fold decrease in the number of actively moving particles observed in EJC mutant oocytes. The current findings demonstrate that the nuclear history of the mRNA is critical for the loading of motor proteins to RNPs (Trucco, 2009).
Surprisingly, the association of osk particles with motors and MTs is better preserved in the nurse cells than in the oocytes of hrp48 and EJC mutants, suggesting that the RNPs are remodeled upon entry into the oocyte. Indeed, RNP remodeling in the oocyte is probable, as incoming RNPs must switch from dynein-dependent to kinesin-dependent transport, and presumably detach from the MTs mediating their nurse cell-to-oocyte transport, to oocyte MTs mediating their transport to the posterior pole (Trucco, 2009).
The ultrastructural approach of this study has also provided insight into the function of the cytoplasmic actin-binding protein TmII in osk mRNA transport. TmII associates with osk particles mainly in the ooplasm, consistent with the idea that the RNPs are remodeled upon entry into the oocyte. Surprisingly, in TmII mutant oocytes the association of Khc with osk particles is reduced, suggesting that TmII promotes the recruitment or stability of Khc on osk transport particles (Trucco, 2009).
Ultrastructural analysis shows that Staufen is required for efficient recruitment or stabilization of the MT motor proteins on osk transport particles, explaining the reduced frequency of movements observed during in vivo imaging. The residual motors associated with osk RNPs may mediate their binding to the dense MT network at the oocyte anterior but not support their efficient transport, leading to the observed retention of the mRNA in this region. Alternatively, Staufen may have a role in the switching of osk RNPs from MTs mediating their transport from the nurse cells into the oocyte to the oocyte MTs responsible for the final transport of the mRNA to the posterior pole. A failure to dissociate from nurse cell-to-oocyte MTs would result in retention of the mRNA at the oocyte anterior. A similar, but less pronounced accumulation of osk mRNA is observed in btz mutant oocytes, where the loading of Staufen is affected (Trucco, 2009).
In WT egg chambers nearly 90% of osk RNPs are associated with both Khc and Dhc in the nurse cell cytoplasm and throughout the oocyte. This can explain why Exu-GFP particles, which are thought to contain osk mRNA, show accelerated movement in the nurse cell cytoplasm of khc null mutants. Indeed, it is likely that kinesin, which is present on the same osk particles as dynein, counteracts the dynein-mediated transport of osk RNPs from the nurse cells to the oocyte (Trucco, 2009).
This analysis of khc and TmII mutants has shown that when osk particles fail to associate with Khc, they also fail to accumulate in the oocyte center at stage 8, undergo only partial coalescence, and mostly accumulate around the oocyte cortex at stage 9. This indicates that Khc is a key motor transporting osk to the posterior pole. Interestingly, however, in both khc and TmII mutants, osk RNPs retain their association with Dhc and MTs, suggesting that dynein links osk RNPs to MTs also in the oocyte. Experiments involving live-cell imaging, antibody injection, and MT depolymerization confirm this and show that Khc and Dhc are the primary MT motors actively transporting osk particles. This analysis further suggests that during a single minute, Khc is responsible for active movement of nearly 75% of the particles, whereas dynein mediates the movement of the remaining 25% of particles in the oocyte. It is therefore concluded that in khc mutant oocytes, Dhc most likely transports osk particles to the minus ends of MTs at the lateral cortex, and that in WT oocytes, the activity of dynein in osk transport is masked by that of kinesin. Consistent with this, the speed of osk particle transport is greater in dhc hypomorphic than in WT oocytes, and previous studies have proposed a role of dynein in restricting kinesin activity (Trucco, 2009).
In situ hybridization coupled with immuno-EM is now an established technique that has been successfully used in the present study to visualize the assembly of osk transport particles in the Drosophila oocytes and to reveal the function of the different osk RNP components in this process. As a mechanism for localized protein expression, RNA localization is most powerful when tightly coupled to translational control. Future ultrastructural analysis combined with live-cell imaging is bound to provide new insight into the relationship between the RNA transport and translational control machineries (Trucco, 2009).
oskar messenger RNA localization at the posterior pole of the Drosophila oocyte is essential for germline and abdomen formation in the future embryo. The nuclear shuttling proteins Y14/Tsunagi and Mago nashi are required for oskar mRNA localization, and they co-localize with oskar mRNA at the posterior pole of the oocyte. Their human homologues, Y14/RBM8 and Magoh, are core components of the exon-exon junction complex (EJC). The EJC is deposited on mRNAs in a splicing-dependent manner, 20-24 nucleotides upstream of exon-exon junctions, independently of the RNA sequence. This indicates a possible role of splicing in oskar mRNA localization, challenging the established notion that the oskar 3' untranslated region (3'UTR) is sufficient for this process. This study shows that splicing at the first exon-exon junction of oskar RNA is essential for oskar mRNA localization at the posterior pole. The issue of sufficiency of the oskar 3'UTR for posterior localization was revisited and it was shown that the localization of unrelated transcripts bearing the oskar 3'UTR is mediated by endogenous oskar mRNA. The results reveal an important new function for splicing: regulation of messenger ribonucleoprotein complex assembly and organization for mRNA cytoplasmic localization (Hachet, 2004).
To address the requirement of splicing for oskar mRNA localization, the effect of deleting oskar introns on mRNA localization was tested by a transgenic approach. oskar mRNA localization was evaluated in the oskA87/Df(3R)pXT103 background, in which no endogenous oskar RNA is produced. oskA87/Df(3R)pXT103 ovaries undergo an early arrest of oogenesis that can be rescued by transgenic oskar mRNA, providing a phenotypic confirmation of efficient transgene expression. Because the only source of oskar mRNA in these experiments is transgenic, each genotype is referred to by the name of the oskar transgene (Hachet, 2004).
The localization was tested of oskar mRNA produced from the intronless transgene oskδi(1,2,3), in which the three oskar introns i1, i2 and i3 were deleted. During early oogenesis, oskδi(1,2,3) mRNA is correctly transported from the nurse cells to the oocyte indicating that oskar introns are dispensable both for nuclear export and the early phase of oskar mRNA transport. A defect in oskδi(1,2,3) mRNA localization becomes evident during mid-oogenesis. At stage 8, oskδi(1,2,3) mRNA distribution seems diffuse compared with oskWT mRNA. At stage 9, whereas oskWT mRNA accumulates at the posterior with a transient accumulation at the anterior corners, oskδi(1,2,3) mRNA is distributed throughout the entire ooplasm. During late oogenesis, only a small amount of oskδi(1,2,3) mRNA is detected in an extended posterior crescent, most probably reflecting local anchoring of mRNA randomly distributed in the posterior area during mid-oogenesis. Consistent with the oskδi(1,2,3) mRNA localization defect was the finding that Staufen protein, a marker for oskar mRNA, is similarly mislocalized. Although oskar mRNA levels are similar in oskWT and oskδi(1,2,3) ovaries, oskδi(1,2,3) mRNA is poorly translated, presumably reflecting the requirement of localization for oskar mRNA translation. Finally, more than two-thirds of the embryos from eggs laid by oskδi(1,2,3) females fail to hatch and show a severe posterior group phenotype (Hachet, 2004).
oskar introns were systematically deleted to assess their relative contribution in oskar mRNA localization. The results show that, whereas the mRNAs produced from all i1-containing transgenes are localized (oskWT, oskδi(2,3), oskδi2 and oskδi3), those produced from all i1-deleted transgenes are mislocalized, although they accumulate correctly in the oocyte (oskδi(1,2,3), oskδi(1,3), oskδi(1,2) and oskδi1). Although the absence of i2 and i3 does not affect oskδi(2,3) mRNA localization, oskδi1 mRNA fails to localize at the posterior pole of stage 9 and 10 oocytes, although i2 and i3 are correctly spliced. Confirming the previously reported oskar mRNA-dependent localization of Y14, Y14 fails to localize in oskδi1 oocytes. The fact that oskδi(2,3) mRNA supports Y14 localization to the posterior shows that splicing of i1 is sufficient for the association of Y14 with oskar mRNA. Thus, these results reveal an unexpected and distinct role of i1, compared with i2 and i3, in oskar mRNA localization. They indicate either that i1 contains sequence-specific information or that splicing at the i1 position is essential for oskar mRNA localization (Hachet, 2004).
To discriminate between these two possibilities, the localization was tested of mRNAs produced by a transgene in which the i1 sequence was replaced by i3, called osk(i3 in i1). osk(i3 in i1) mRNA localizes at the posterior, showing that, although i3 is unable to promote oskar mRNA localization when located at its normal position, i3 can functionally substitute for i1 when placed in the i1 context, between exons I and II. Consistent with this was the observation that the EJC component Y14 is recruited by osk(i3 in i1) mRNA, as revealed by the localization of Y14 at the posterior pole, indicating that Y14 recruitment is independent of intron sequence. This demonstrates the importance of splicing at the first exon-exon junction of oskar mRNA, rather than a specific requirement for i1, for oskar mRNA localization. These results show that oskar RNA splicing and localization are mechanistically coupled (Hachet, 2004).
The demonstration that splicing is required for oskar mRNA localization seemingly contradicts previous work reporting that the oskar 3'UTR is sufficient for mRNA targeting to the posterior pole of the oocyte. This conclusion was based on several studies of lacZ-osk3'UTR hybrid RNAs, in which the intronless lacZ gene was fused to the oskar 3'UTR and the chimaeric mRNAs were observed to localize at the oocyte posterior. This apparent contradiction prompted the role of the 3'UTR in oskar mRNA localization to be revisited. The possibility was considered that the localization of lacZ-osk3'UTR mRNA might be influenced by endogenous oskar mRNA, which was present in all previous studies. A direct analysis of lacZ-osk3'UTR mRNA localization in oskar RNA-null oocytes is prevented by the early oogenesis arrest of the oskA87/Df(3R)pXT103 mutant. Therefore the localization of lacZ-osk3'UTR mRNAs was examined in oskWT and oskδi(1,2,3) oocytes, in which transgenic oskar mRNA supports oocyte development beyond the early stages. It was found that although lacZ-osk3'UTR mRNA localizes correctly in oskWT oocytes, it fails to accumulate at the posterior pole of oskδi(1,2,3) oocytes, as revealed by lacZ in situ hybridization. In addition, when placed in the Oskar protein-null background osk84/Df(3R)pXT103, in which the osk84 nonsense mRNA localizes correctly until stage 10 of oogenesis, lacZ-osk3'UTR mRNA also localizes at the posterior pole. Taken together, these results demonstrate that an endogenous source of oskar mRNA is required for lacZ-osk3'UTR localization at the posterior pole, and that this effect is independent of Oskar protein. Localization of the lacZ-osk3'UTR hybrid RNA to the posterior pole is therefore most probably due to its hitchhiking on endogenous oskar mRNA localization complexes, whose assembly involves splicing. These results indicate that the oskar 3'UTR promotes association of the RNA into higher-order oskar messenger ribonucleoprotein (mRNP) complexes. This idea is consistent with estimates indicating that oskar mRNA particles contain about 100 oskar mRNA molecules. The assembly of such multi-mRNP particles might be mediated by protein-protein interactions involving factors bound to the 3'UTR or by direct RNA-RNA interaction as occurs with bicoid mRNA (Hachet, 2004).
Although the oskar 3'UTR and associated factors are clearly important for oskar mRNA localization, because oskar transcripts lacking the 3'UTR fail to localize, the data show that oskar mRNA localization requires additional factors recruited to the mRNA upon splicing. Thus, information imparted to oskar RNA in the nucleus during pre-mRNA processing is crucial for the localization of oskar mRNA at the posterior pole of the oocyte cytoplasm. The fact that both splicing and the EJC components Y14 and Mago nashi are essential for oskar mRNA localization indicates that oskar RNA splicing and cytoplasmic localization are mechanistically coupled by the splicing-dependent deposition of the EJC. Unexpectedly, of the three oskar intron positions, only the first is strictly required and functional for oskar mRNA localization, although an EJC is presumably assembled at each oskar mRNA exon-exon junction. This indicates not only that an EJC landmark is required but also that its position is essential for oskar mRNA localization at the oocyte posterior. The importance of the splicing position, and thus of the EJC on oskar mRNA, suggests a structural role of the Y14-Mago nashi heterodimer and Barentsz, in assembly of the oskar mRNA localization complex. The first EJC landmark on oskar mRNA might have a pivotal function in mediating interactions between factors bound to different regions of oskar mRNA, including the 5' cap, the 3'UTR and potentially the coding region. It is proposed that the position of the first oskar EJC landmark is crucial in specifying the architecture of the oskar mRNA localization complex. This structural model could explain why the EJC is not always involved in cytoplasmic mRNA localization and why the transport of gurken and bicoid mRNAs, both of which are produced from intron-containing genes and are thus presumably imprinted with the EJC, seems to be independent of the EJC. The model also suggests that alternatively spliced mRNAs might be directed to different cytoplasmic locations, depending on the formation of alternative mRNP complex architectures (Hachet, 2004).
In humans, EJC imprinting allows the recognition of premature termination codons, triggering mRNA degradation by activation of the nonsense-mediated decay (NMD) pathway. In Drosophila, however, although NMD factors and EJC components are conserved, the recognition of premature termination codons depends neither on the EJC nor on intron position. The involvement of Y14, Magoh, eIF4AIII and Barentsz in NMD in humans and in oskar mRNA localization in Drosophila is striking and suggests the maintenance of an evolutionarily conserved complex with divergent functions. However, this does not exclude a possible involvement of splicing and the EJC in the cytoplasmic localization of some mRNAs in vertebrates. In particular, the localization of Barentsz in hippocampal neurons suggests that the use of these factors in cytoplasmic mRNA localization has been conserved in vertebrates. It will be of particular interest to determine whether the transport of other localized mRNAs is dependent on the EJC and to evaluate the relevance of the conservation of the EJC regarding mRNA cytoplasmic localization (Hachet, 2004).
Bullock, S. L., Ringel, I., Ish-Horowicz, D. and Lukavsky, P. J. (2010). A'-form RNA helices are required for cytoplasmic mRNA transport in Drosophila. Nat Struct Mol Biol 17: 703-709. PubMed ID: 20473315
Ghosh, S., Marchand, V., Gaspar, I. and Ephrussi, A. (2012). Control of RNP motility and localization by a splicing-dependent structure in oskar mRNA. Nat Struct Mol Biol 19: 441-449. PubMed ID: 22426546
Hachet, O. and Ephrussi, A. (2004). Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization. Nature 428: 959-963. PubMed ID: 15118729
Sauliere, J., Haque, N., Harms, S., Barbosa, I., Blanchette, M. and Le Hir, H. (2010). The exon junction complex differentially marks spliced junctions. Nat Struct Mol Biol 17: 1269-1271. PubMed ID: 20818392
Zimyanin, V. L., Belaya, K., Pecreaux, J., Gilchrist, M. J., Clark, A., Davis, I. and St Johnston, D. (2008). In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization. Cell 134: 843-853. PubMed ID: 18775316
oskar RNA localization to the posterior pole of the Drosophila melanogaster oocyte requires splicing of the first intron and the exon junction complex (EJC) core proteins. The functional link between splicing, EJC deposition and oskar localization has been unclear. This study demonstrates that the EJC associates with oskar mRNA upon splicing in vitro and that Drosophila EJC deposition is constitutive and conserved. In vivo analysis reveals that splicing creates the spliced oskar localization element (SOLE), whose structural integrity is crucial for ribonucleoprotein motility and localization in the oocyte. Splicing thus has a dual role in oskar mRNA localization: assembling the SOLE and depositing the EJC required for mRNA transport. The SOLE complements the EJC in formation of a functional unit that, together with the oskar 3' UTR, maintains proper kinesin-based motility of oskar mRNPs and posterior mRNA targeting (Ghosh, 2012).
mRNA localization is an evolutionarily conserved process allowing spatial and temporal restriction of protein synthesis to specific sites within cells. Active transport of mRNAs involves recognition of cis-acting sequence elements in the transcript by trans-acting factors and formation of ribonucleoprotein (RNP) particles, for which translocation to their final destination is mediated by motor proteins and the cytoskeleton. In Drosophila, oskar (osk) localization to the posterior pole of the oocyte depends on microtubules (MTs) and kinesin heavy chain (KHC). The identity of the cis element(s) that mediate oskar mRNA posterior localization is largely unknown. Although a previous study indicated that posterior localization signals reside in the oskar 3′ untranslated region (UTR), subsequent analysis revealed that the 3′ UTR is not sufficient to target an mRNA to the posterior pole. This indicated that other features of oskar RNA have a crucial role in the localization process (Ghosh, 2012).
Consistent with this, splicing of oskar at the position of the first intron is essential for posterior localization of the mRNA (Hachet, 2004). In addition, Y14, Mago nashi (Mago), eIF4AIII and Barentsz proteins are required for oskar mRNA transport in the oocyte. Notably, the vertebrate homologs of Drosophila Y14, Mago, eIF4AIII and Barentsz constitute the core components of the EJC and have been shown to associate with mRNAs, approximately 20-24 nucleotides (nt) upstream of exon-exon junctions, upon splicing. Although the requirements of splicing and the EJC core proteins for oskar mRNA localization indicate that nuclear events have a role in determining the assembly of localization-competent oskar mRNPs, the splicing-dependent association of the Drosophila EJC proteins with mRNAs, including oskar, has not been demonstrated (Ghosh, 2012).
To gain insight into the roles of splicing and the EJC in mRNA transport, this study combined biochemical, genetic and live-imaging approaches. It was shown that the Drosophila EJC is assembled on mRNAs upon splicing in vitro. Splicing of the first intron in oskar results in EJC deposition and formation of the SOLE, which has a predicted stem-loop structure. Mutational analysis of the SOLE shows that its proximal stem is required for proper oskar RNP motility and, therefore, for mRNA localization at the posterior pole of the oocyte. Taken together, these results shed light on the significance of splicing at the position of the first intron in oskar RNA and highlight the functional interconnection between the SOLE and the juxtaposed EJC (Ghosh, 2012).
This study shows that the EJC is deposited on ftz and oskar mRNAs as a consequence of splicing in embryo nuclear extract and that it binds at the same position relative to exon-exon junctions as in mammals. Hence, EJC assembly seems to be a constitutive and conserved process among metazoans (Ghosh, 2012).
The discovery of the bipartite SOLE provides an explanation for the observation that splicing of oskar RNA at the first intron is essential for localization of the mRNA (Hachet, 2004). The SOLE is formed by the joining of the 18 and 10 intron-proximal nucleotides of the first and second exons, respectively, upon splicing. However, the role of splicing is not simply to join these exonic sequences: mRNAs produced from oskΔi(1) or oskΔi(1,2,3) transgenes, which do not require splicing for formation of the 28-nt SOLE, are mislocalized (Hachet, 2004). All four EJC core proteins are required for oskar localization, and EJC deposition on mRNAs ~20-24 nt upstream of the exon-exon junction is splicing dependent; hence, a second important function of splicing is to load the EJC. Thus, EJC assembly and SOLE formation upon splicing are closely linked, both in time and space, with the EJC bound immediately adjacent to the SOLE (Ghosh, 2012).
Indeed, the relative configurations of the EJC deposition site and the SOLE seem to be important, as oskΔi(1) mRNA - which contains the 28 contiguous SOLE nucleotides and so should bear EJC complexes on exons 2 and 3 after splicing - fails to localize. Furthermore, the transgenic oskar RNAs contain a vector-derived intron at their 5′ end; hence, after splicing in vivo, mRNAs produced from oskΔi(1) and the intronless oskΔi(1,2,3) transgenes should have an EJC bound ~635 nt upstream of the SOLE, and yet neither oskΔi(1) nor oskΔi(1,2,3) mRNA is localized (Ghosh, 2012).
The relationship between EJC binding and the SOLE is unclear. The experiments indicate that neither the sequence nor the structure of the SOLE influences splicing or EJC assembly at the first splice junction in vitro. Furthermore, EJC proteins present in nuclear extracts from Drosophila embryos and Kc cells were deposited on all in vitro-spliced mRNAs tested, irrespective of their sequence; therefore, all the factors required for Drosophila EJC deposition are present in or associated with the nucleus. Notably, a recent study involving genome-wide RNA immunoprecipitation from Drosophila S2 cell extracts suggests that EJC stability may depend on specific RNA sequences found in a subset of Drosophila RNAs that undergo nonsense-mediated decay (Saulière, 2010). Thus, it is possible that the SOLE regulates the association of the EJC with oskar, for example, by recruiting crucial cytoplasmic factors to selectively stabilize the complex on the mRNA in vivo. Conversely, the EJC or spliceosomal factors such as RNA helicases might be required for the SOLE to adopt and stably maintain its secondary structure, which is crucial for RNP motility and posterior localization of the mRNA. Finally, it is possible that a SOLE-binding trans-acting factor cooperates with the EJC for localization (Ghosh, 2012).
Analysis of the motion of both localizing and non-localizing oskar RNPs revealed no detectable difference in the directionality of their motion. It should be noted that, although the numerical values of the average net velocity vectors that were obtained were similar to those previously reported for localizing mRNAs, no statistically significant net posterior vector was detected for any RNPs, with the exception of oskPSLzc. A more detailed analysis will be necessary to resolve this discrepancy. The accumulation of runs within the vicinity of the posterior pole and the posterior-ward translocation of the center of the mass indicate that even the non-localizing mRNAs are displaced toward the posterior pole by a mechanism that remains to be understood (Ghosh, 2012).
Other motion parameters, however, were affected. The travel distance of individual non-localizing oskΔi(1,2,3) and oskPSLz RNPs was substantially shorter and the motile fraction was greatly reduced compared with the localizing oskΔi(2,3) RNPs. Combined, these reductions greatly affect transport efficiency, as has been reported in the case of mutants in the EJC core components (Zimyanin, 2008), and are probably the cause of oskar mRNA mislocalization. Despite the apparent nonzero motility of these mutant RNPs (~17% for oskΔi(1,2,3) and ~29% for oskPSLz), ultimately, a small amount of oskar mRNA accumulated at the posterior pole. A possible explanation for this is that, although the center of mass of the mRNA translocates toward the posterior, the growth of the oocyte along the AP axis is such that the increase in oocyte length exceeds the rate of displacement of the RNPs, suggesting that there is a dynamic threshold crucial for oskar mRNA localization (Ghosh, 2012).
Consistent with the rescuing effect of the compensatory mutation in oskPSLzc on oskar mRNA localization, a restoration was observed of the motion parameters of oskPSLzc*GFP particles to near-wild-type levels and a rate of posterior-ward translocation of the mRNA was similar to that of wild-type particles. Taken together, these observations indicate that the SOLE acts in the localization process by modulating the motility of oskar particles. The changes in transport efficiency of the non-localizing mRNAs are manifest in three different ways: (1) the fraction of motile particles, (2) the duration of the runs and (3) the speed of the runs. These defects might stem from compromised mechanoenzyme activity caused by a lack of associated motors, a failure to maintain the motors' activity or an improper balance of opposite polarity motors. Notably, the reduction observed in the average velocity is due to a relative increase in a slow-moving, short-displacing population of RNPs in oskPSLz and oskΔi(1,2,3) oocytes indicating functional defects in oskar transport. Because KHC is responsible for 70%-80% of the runs, the 60% decrease in the fraction of motile particles in the oskPSLz mutant indicates a severe abrogation of KHC function in oskar transport upon loss of SOLE structural integrity. In contrast, all SOLE mutant RNAs are enriched in the oocyte, indicating that they can associate with the Egl-BicD-dynein complex for their transport from the nurse cells to the oocyte. However, as it was not possible to assess whether their enrichment in the oocyte is quantitatively normal, it remains unclear whether integrity of the SOLE is a requirement of kinesin exclusively or of dynein-based transport of oskar RNPs as well. Analysis of oskar RNP motility with high-temporal resolution imaging may resolve the question of whether the defects observed in the SOLE mutants are of a structural or functional origin and help to elucidate the underlying mechanism(s) (Ghosh, 2012).
Many mRNA localization elements have been predicted to form secondary structures crucial for their function. The observation that SOLE activity depends on the secondary structure of the PS suggests that, as in the case of the K10 transport and localization sequence (TLS) (Bullock, 2010), the structure of the SOLE may be the crucial determinant recognized by the transport machinery. Consistent with this, the bipartite sequence constituting the SOLE stem is conserved across Drosophila species, including at wobble positions. Furthermore, the sequences flanking the first intron of Anopheles, Aedes and Culex oskar homologs, which show little sequence similarity with Drosophila oskar, are predicted to adopt a secondary structure that would be similarly positioned next to the presumed EJC deposition site in those mRNAs, which localize to the posterior pole plasm. To date, oskar is the only mRNA shown to depend on the EJC and a splicing-dependent localization signal for its localization. It will be of interest to look beyond oskar for similar splicing-dependent structures that might cooperate with the EJC in localization of other mRNAs. Alternative splicing has been shown to determine the inclusion of exons containing localization elements in a subset of stardust mRNA isoforms; it could similarly regulate mRNA localization by promoting formation of SOLE-like structures in specific tissues (Ghosh, 2012).
Most localized mRNAs, such as bicoid, gurken and pair-rule transcripts in Drosophila, and ASH1 mRNA in Saccharomyces cerevisiae, require a single type of motor—dynein or myosin, respectively—for localization. In contrast, oskar mRNA, which depends on dynein for its translocation from the nurse cells to the oocyte, switches to KHC within the oocyte for its transport to the posterior pole. This analysis shows that the EJC and SOLE become important for the KHC-dependent step of oskar transport, after dynein-dependent mRNA transport into the oocyte has occurred. Future work should determine whether the SOLE has a structural role in KHC recruitment to oskar RNPs or an important regulatory role in controlling the switch from a dynein to a KHC-dependent mode of transport (Ghosh, 2012).
This study suggests that the EJC and SOLE constitute a functional unit for posterior transport, and future dissection of their exact mechanistic relationship will require both biochemistry and genetics for a complete answer (Ghosh, 2012).
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 asymmetric mRNA localization during mid-oogenesis and for embryonic axis specification. The posterior determinant oskar mRNA is translationally silent until mid-oogenesis. Mutations in armitage (armi) and in three components of the RNAi pathway disrupt
oskar mRNA translational silencing, polarization of the
microtubule cytoskeleton, and posterior localization of
oskar mRNA. armitage encodes a homolog of SDE3, a
presumptive RNA helicase involved in posttranscriptional gene
silencing (RNAi) in Arabidopsis, and is required for RNAi in
Drosophila ovaries. Armitage forms an asymmetric network
associated with the polarized microtubule cytoskeleton and is
concentrated with translationally silent oskar mRNA in the oocyte. It is concluded that RNA silencing is essential for establishment of the cytoskeletal polarity that initiates embryonic axis specification and for translational control of oskar mRNA (Cook, 2004).
Thus armi is required for initial polarization of the
microtubule cytoskeleton and for temporal regulation of osk
mRNA translation. Armi is required to repress osk translation during early oogenesis, but does not alter osk mRNA levels. armi is also required for Stellate silencing
during spermatogenesis, which requires small homologous miRNAs and the RNAi components Spn-E and Aub. Armi is also
required for RNAi and efficient RISC assembly in ovary extracts (Tomari, 2004). These findings strongly suggest that
the RNAi system is required for an early step in the axis
specification pathway (Cook, 2004).
Consistent with this
hypothesis, the RNAi components Spn-E, Aub, and Mael are also
required for osk mRNA silencing and for polarization of the
microtubule cytoskeleton during early oogenesis. Aub enhances osk translation during mid-oogenesis. However, aub disrupts
posterior localization of osk mRNA during mid-oogenesis, and
posterior localization is required for efficient osk
translation. It is therefore speculated that the reduced Osk protein
levels during later stages of oogenesis are secondary to defects in
posterior patterning during early oogenesis, when Aub and other RNA
silencing components are required to establish the microtubule
asymmetries required to specify the posterior pole (Cook, 2004).
While the RNA silencing system represses osk during stages 1 to 6, other studies indicate that products of the bruno/arrest,
cup, and Bicaudal-C genes are essential for
osk silencing during stages 6 to 8. The role of Bicaudal-C in osk silencing is unclear. However, Bruno binds to three sites in the osk 3' UTR, called Bruno response elements (BREs), and
deletion of the BREs leads to osk translation during stages
7 and 8 and severe patterning defects. Recent data show that Cup associates with both Bruno and the 5'-cap binding factor eIF4E (Nakamura, 2004). This suggests that Cup and Bruno may
function together to repress osk translation by sequestering
the 5' end of osk mRNA, thereby blocking translation
initiation. Bruno and Cup do not appear to play a role in osk mRNA translational repression during stages 1-6, when the RNAi pathway is required for silencing. The biological reason for this two-step translational control mechanism is unclear, but may be linked to changing functions for the RNAi system during oogenesis (Cook, 2004).
All of the RNAi mutations examined
in this study produce nearly identical defects in microtubule
polarization and osk silencing during early oogenesis and
posterior and D/V patterning during mid-oogenesis. The early defects
in osk silencing and microtubule organization appear to
reflect independent functions for the RNAi system, since mutations in
osk do not suppress the cytoskeletal defects in
armi mutants, and forced premature expression of Osk protein
from a transgene does not induce changes in microtubule organization. The defects in anterior-posterior polarization of the
microtubule cytoskeleton during early oogenesis, by contrast, may
directly lead to the posterior and D/V patterning defects observed in
mid-oogenesis. Microtubule-dependent posterior
localization of grk mRNA in early oogenesis is thought to
facilitate Grk signaling from the oocyte to the posterior follicle
cells: this initiates a chain of signaling events that trigger
microtubule reorganization and mRNA localization during mid-oogenesis. Thus, defects in microtubule
polarization associated with RNAi mutations are likely the primary
cause of later defects in axial patterning (Cook, 2004).
The spectrum of defects observed in RNAi mutants
suggests that the RNA silencing machinery targets multiple processes
during early oogenesis. The endogenous miRNAs that mediate RNA
silencing are predicted to bind complementary sequences in the
3' UTRs of numerous target transcripts, suggesting that they
may coordinate translational control of gene cassettes during complex
biological processes (Stark, 2003). A computational screen for miRNA
targets has identified osk mRNA, kinesin heavy chain mRNA,
and transcripts encoding several other cytoskeletal proteins involved
in oogenesis as targets for the miR-280 miRNA (Stark, 2003). It is interesting to note that
kinesin, like the RNAi components, is required for posterior and D/V
axis specification. This motor also drives ooplasmic streaming during late oogenesis, and mutations in mael lead to premature ooplasmic streaming, which could reflect overexpression of kinesin due to defects in silencing. These observations raise the possibility that the RNAi system, through miR-280 and other miRNAs, coordinates axis specification by silencing osk mRNA and simultaneously regulating genes involved in microtubule function (Cook, 2004).
Armi is asymmetrically localized during early
oogenesis, when it is concentrated in the oocyte with osk
mRNA. This finding raises the possibility that asymmetric Armi
localization plays a role establishing developmental asymmetries
during early oogeneis. Tomari (2004) has shown that Armi is required for mRNA target cleavage and RISC maturation in vitro. Armi could therefore
promote local RISC assembly and thus increase the efficiency of
osk mRNA silencing. This could play a role in regulating
other transcripts that accumulate in the oocyte. Local increases in
RISC activation could also lead to oocyte-specific silencing of
transcripts that are uniformly distributed within the oocyte-nurse
cell complex. The molecular, genetic, and cytological tools available
in Drosophila should allow direct tests of these
possibilities (Cook, 2004).
The PAR-1 kinase is required for the posterior localization
of the germline determinants in C. elegans and Drosophila,
and localizes to the posterior of the zygote and the oocyte
in each case. Drosophila PAR-1 is also
required much earlier in oogenesis for the selection of one
cell in a germline cyst to become the oocyte. Although the
initial steps in oocyte determination are delayed, three
markers for oocyte identity, the synaptonemal complex, the
centrosomes and Orb protein, still become restricted to one
cell in mutant clones. However, the centrosomes and Orb
protein fail to translocate from the anterior to the posterior
cortex of the presumptive oocyte in region 3 of the
germarium, and the cell exits meiosis and becomes a nurse
cell. Furthermore, markers for the minus ends of the
microtubules also fail to move from the anterior to the
posterior of the oocyte in mutant clones. Thus, PAR-1 is
required for the maintenance of oocyte identity, and plays
a role in microtubule-dependent localization within the
oocyte at two stages of oogenesis.
PAR-1 localizes on the fusome, and provides a link between
the asymmetry of the fusome and the selection of the
oocyte (Huynh, 2001).
The re-localization of cytoplasmic markers in region 3
reveals the earliest known A-P polarity within the oocyte. It is very intriguing that PAR-1 is required both for this polarization, and the later polarization of the
oocyte at stage 9. This raises the question of whether the two
are linked. For example, it is possible that the altered
organization of the microtubules and mislocalization of Oskar
mRNA at stage 9 in par-1 hypomorphs is a consequence of
earlier problems in the polarization of the oocyte in the
germarium. This seems unlikely, however, for several reasons.
(1) The localization of all known markers for oocyte polarity
changes during stage 7, suggesting that the early polarization
of the oocyte is erased when it re-polarizes in response to a
signal from the posterior follicle cells. (2) In the vast
majority of egg chambers that are mutant for par-1
hypomorphs, Orb protein moves normally from the anterior to
the posterior of the oocyte, and is maintained there as it is in
wild type. Furthermore, the Oskar mRNA localization defect of
these mutants can be rescued by a par-1 transgene that is only
expressed after the cysts have left the germarium. The view that PAR-1 plays a role in the anterior-posterior polarization of the oocyte at two stages of oogenesis is therefore favored. It will be interesting to determine whether these processes are related in other ways, and whether PAR-1 serves a common function in
oocyte determination and Oskar mRNA localization (Huynh, 2001).
Par-1 kinase is critical for polarization of the Drosophila oocyte and the one-cell C. elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Drosophila Par-1 is shown to phosphorylate the posterior determinant Oskar (Osk). It has been demonstrated genetically that Par-1 is required for accumulation of Osk protein. In cell-free extracts Osk protein is intrinsically unstable and Osk is stabilized after phosphorylation by Par-1. The data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk (Riechmann, 2002).
The Drosophila anterior-posterior axis is established at stage 7 of oogenesis when the posterior follicle cells signal to polarize the oocyte microtubule cytoskeleton. This requires the conserved PAR-1 kinase, which can be detected at the posterior of the oocyte in immunostainings from stage 9. However, this localization depends on Oskar localization, which requires the earlier PAR-1-dependent microtubule reorganization, indicating that Oskar-associated PAR-1 cannot establish oocyte polarity. This study analyzed the function of the different PAR-1 isoforms; only PAR-1 N1 isoforms can completely rescue the oocyte polarity phenotype. Furthermore, PAR-1 N1 is recruited to the posterior cortex of the oocyte at stage 7 in response to the polarizing follicle cell signal, and this requires actin, but not microtubules. This suggests that posterior PAR-1 N1 polarizes the microtubule cytoskeleton. PAR-1 N1 localization is mediated by a cortical targeting domain and a conserved anterior-lateral exclusion signal in its C-terminal linker domain. PAR-1 is also required for the polarization of the C. elegans zygote and is recruited to the posterior cortex in an actin-dependent manner. These results therefore identify a molecular parallel between axis formation in Drosophila and C. elegans and make Drosophila PAR-1 N1 the earliest known marker for the polarization of the oocyte (Doerflinger, 2006).
The par-1 gene encodes multiple isoforms that belong to three families (N1, N2, and N3) that differ in their N-terminal domains, raising the possibility that an isoform that is not detected in antibody stainings polarizes the microtubule cytoskeleton. To test this possibility, transgenic flies were generated expressing N-terminally tagged GFP fusions of the major PAR-1 isoforms under the control of the maternally expressed alpha4-tubulin promoter. Their ability was analyzed to rescue the polarity defect of a viable transheterozygous combination of par-1 alleles, in which oskar mRNA is partially or completely mislocalized to the center of the oocyte in 99% of stage 9-10 egg chambers. Expression of either GFP-PAR-1 N1S or N1L fully rescues the posterior localization of oskar mRNA, whereas GFP-PAR-1 N2S and GFP-PAR-1 N3L rescue only partially (30% and 40% mislocalization, respectively). PAR-1 N1S is the major isoform expressed during oogenesis and is strongly reduced in the par-1 hypomorphs that give a polarity phenotype 2 and 3. These results suggest that PAR-1 N1S plays the principal role in the repolarization of the microtubule cytoskeleton, although the N2 and N3 isoforms can partially compensate for a partial loss of N1 when overexpressed (Doerflinger, 2006).
Abdomen and germ cell development of the Drosophila embryo requires proper localization of Oskar mRNA to the posterior pole of the developing oocyte. Oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, a novel interaction type of genetic screen was developed by which 14 dominant enhancers were isolated of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. The detailed analysis of two allelic modifiers is described that identify Drosophila Rab11, a gene encoding small monomeric GTPase. Mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of Oskar mRNA, whereas localization of Gurken and Bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. The ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. These results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in Oskar RNA localization (Jankovics, 2001).
Whatever the role of the Drosophila Rab11 gene in the Osk localization pathway, it must be indirect and act in the reorientation of the oocyte microtubule network, as seen by the Tau:GFP microtubule visualization and by mislocalization of the Kinesin:ß-galactosidase fusion protein to the center in the Rab11 mutants. Central mislocalization of the Kinesin:ß-galactosidase protein has been observed in mutants that impair any step of the reciprocal signaling events between the oocyte and the posterior follicle cells. Given that the best-characterized role of the Rab11 proteins is the targeting of recycling endosomes or trans-Golgi vesicles to the plasma membrane, it seemed to be plausible that Rab11 mutants would exert their phenotypes by blocking signaling events between the oocyte and the follicular cells. However, two types of evidence suggest that both the oocyte-to-follicle cells and the follicle cells-to-oocyte signals are functional in the Rab 11 mutants: (1) absence of expression of an enhancer trap in follicle cells at the posterior cap indicates that the posterior polar follicle cell fate is properly adopted; (2) a focus of microtubules at the posterior pole was never observed by Tau:GFP labeling, indicating that posterior MTOC disassembles. Consistently, mislocalization of the Bcd mRNA to the posterior pole was never observed, indicating again that the back signaling from the posterior polar follicle cells is received, and the MTOC and the minus ends of the microtubules disappear from the posterior pole. However, working with hypomorphic allele combinations, the possibility that mislocalization of Bcd mRNA was not detected because of its relative insensitivity to the microtubule reorientation cannot be excluded. The analysis of the hypomorphic phenotypes also supports this interpretation. An intermediate Osk mislocalization phenotype occurs in Rab11 mutants, when Osk mRNA is detected in the center and simultaneously at the normal posterior position in the same oocyte. This indicates that in such mutant oocytes the MTOC does indeed disappear and the minus ends of the microtubules are replaced by plus ends at the posterior. Rab11 phenotypes are reminiscent of that of par1. In par1 mutant oocytes, the posterior MTOC also disappears but central osk mRNA localization is observed. It is therefore concluded that even though the posterior MTOC normally disassembles, the reorientation of the oocyte microtubule network is incomplete in Rab11 mutants. It is proposed that instead of having reverse polarity when the microtubules nucleated predominantly from the anterior, in Rab11 mutants only a subset of microtubules, which are nucleated over the entire cortex of the oocyte, is intact driving Kinesin:ß-galactosidase motor protein and Osk mRNA to the center of the oocyte. In Rab11 mutants, Osk mRNA mislocalization phenotype is not fully penetrant and transient, and by stage 10-11, egg chambers exhibit wild-type Osk mRNA localization. It is suggested that the Rab11-dependent localization pathway for Osk RNA itself is redundant and the recovery observed in later stages is due to an alternative, Rab11-independent Osk localization mechanism when cytoplasmic streaming, which begins at stage 10, directs the Osk mRNA to the posterior pole. These results indicate that posterior MTOC breakdown may not be sufficient for reorientation of the microtubule network during stages 6-8 of the Drosophila oocyte; rather, the reorientation process depends on other factors too, like internal membrane functions. The precise mechanism by which Rab11 contributes to the microtubule reorientation is still unclear. A similar phenotype, characterized by Osk mislocalization to the center of the oocyte, is observed in Ter94 mutant ovaries. Ter94 encodes an AAA type ATPase that is also responsible for internal membrane trafficking, namely, for homotypic fusion of endoplasmic reticulum vesicles. Rab11 and Ter94 phenotypes reveal that the internal membranous structures and the cytoskeleton of the Drosophila oocyte have a functional connection to conduct cytoplasmic mRNA localization (Jankovics, 2001).
The Drosophila embryonic body plan is specified by asymmetries that arise in the oocyte during oogenesis. These
asymmetries are apparent in the subcellular distribution of key mRNAs and proteins and in the organization of the
microtubule cytoskeleton. Evidence suggests that the Drosophila oocyte also contains important asymmetries in its
membrane trafficking pathways. Specifically, alpha-adaptin and Rab11, which function critically
in the endocytic pathways of animal cells, are localized to neighboring compartments at the posterior pole of stage 8-10
oocytes. Rab11 and alpha-adaptin localization occurs in the absence of a polarized microtubule cytoskeleton, i.e. in grk null mutants, but is
later reinforced and/or refined by Osk, the localization of which is microtubule dependent. Analyses of germline clones of a rab11 partial
loss-of-function mutation reveal a requirement for Rab11 in endocytic recycling and in the organization of posterior membrane compartments. Such
analyses also reveal a requirement for Rab11 in the organization of microtubule plus ends and osk mRNA localization and translation. It is proposed
that microtubule plus ends and, possibly, translation factors for osk mRNA are anchored to posterior membrane compartments that are defined by
Rab11-mediated trafficking and reinforced by Rab11-Osk interactions (Dollar, 2002).
The best evidence that the plasma membrane of the oocyte and membrane trafficking pathways are polarized in the Drosophila oocyte comes from the expression pattern of human transferrin receptor (Htr) in transgenic flies. During stages 8-10, the posterior pole of the oocyte becomes enriched with Htr, both along the plasma membrane and in vesicles. Htr rapidly disappears from vesicles upon inhibition of endocytosis, indicating that Htr is actively internalized and recycled in Drosophila oocytes as it is in many other examined cells. Together with the observation that Htr is restricted to the posterior plasma membrane, its active internalization and recycling strongly suggests that the membrane recycling pathway of the oocyte is polarized towards the posterior pole (Dollar, 2002 and references therein).
To investigate the role of Rab11 in the organization of the posterior pole of the oocyte, the distribution of osk mRNA and protein was examined in rab11P2148 GLCs. In wild-type oocytes, osk mRNA is transported to the posterior pole during stages 8 and 9, coincident with the polarization of the microtubule cytoskeleton. During transport, and in the initial hours following transport, osk mRNA is seen as a large ball. By the end of stage 9, the ball resolves into a thin cap along the posterior cortex, which persists through the end of oogenesis. The nature of the transition from the ball to the cap is not clear, but coincides with the activation of osk translation. The cap structure is much more resistant to disruption with colchicine than is the ball and appears, then, to represent the binding of the mRNA to an anchor, which might be Osk. rab11P2148 germ-line clones (GLCs) are defective in the transport of osk mRNA to the posterior pole, and in its subsequent translation and anchoring. The transport defect is temporal in nature. Thus, while most osk transcripts reach the posterior pole of wild-type oocytes during stage 8, only a small fraction of transcripts reach the posterior pole of rab11 oocytes during stage 8. Typically, the lagging transcripts are aggregated into a mass near the center of the cell, possibly representing stalled transport at an intermediate step. Although most osk transcripts eventually reach the posterior pole of rab11 oocytes, two observations suggest that they are never anchored: (1) no Osk protein was ever detected in rab11 oocytes; (2) the osk transcripts of rab11P2148 GLCs never formed the characteristic cap at the posterior pole, but instead remained as a ball. Moreover, during late stages of oogenesis (e.g. when microtubules are bundled along the entire egg cortex), the ball of osk mRNA appears to drift away from the posterior pole and is often fragmented into several smaller balls. It is concluded that Rab11 is required for the efficient transport of osk mRNA to the posterior pole of the oocyte and for its subsequent translation and anchoring (Dollar, 2002).
Rab11 and alpha-adaptin are localized to the posterior pole of mid-stage oocytes and such localization does not require a polarized microtubule cytoskeleton or grk signaling. A reduction of rab11 activity in the oocyte alters the subcellular distribution of Rab11, alpha-adaptin and internalized transferrin. These alterations indicate that Rab11 is required for endocytic recycling and to organize posterior membrane compartments. A reduction of rab11 activity in the oocyte also causes defects in Kin:ß-gal localization, osk mRNA transport, and osk mRNA translation and anchoring. These latter defects suggest that the posterior membrane compartments established by Rab11 organize microtubule plus ends and, possibly, the translation factors and/or anchors for osk mRNA (Dollar, 2002).
The expression pattern of Htr in transgenic flies shows clearly that the oocyte establishes a posterior plasma membrane domain (PMD). Further evidence for such a domain comes from the finding that Rab11 localizes to the posterior pole of wild-type oocytes. Interestingly, the PMD is established independently of microtubule polarity or grk signaling, since Rab11 is localized normally in grk null mutants. Given the rapid rate at which Htr is internalized and recycled, it is likely that the maintenance, if not also the initial specification, of the PMD requires polarized endocytic recycling directed towards the posterior pole. The data presented indicate that Rab11 is responsible for such recycling: Rab11 is localized to the posterior pole of the oocyte and is required for the recycling of internalized transferrin (the ligand for transferrin receptor) to the plasma membrane of cultured oocytes. Independent evidence that Rab11 mediates polarized endocytic recycling comes from studies with vertebrates, where Rab11 recycles internalized molecules to the apical surface of polarized epithelial cells (Dollar, 2002).
What polarizes endocytic recycling to the posterior pole of the oocyte? The polarization of the endocytic pathways of other cells is triggered by Rho GTPase family members (i.e. Rho, Cdc42 and Rac), which are activated at specific regions of the cell cortex by a variety of intrinsic and extrinsic cues. Rho GTPases have also been strongly implicated in the polarization of exocytosis. Specifically, they have been shown to recruit the 'exocyst' to specific sites of the plasma membrane. The exocyst is a conserved complex of proteins to which vesicles of the secretory pathway fuse. Thus, through local activation of Rho GTPases, secretory vesicles are targeted to specific regions of the plasma membrane. By analogy, the Rho GTPases could localize Rab11 and polarize receptor recycling through local recruitment of an exocyst-like complex for Rab11-containing vesicles. Because Drosophila Rho GTPases are required for progression through early oogenesis, the analysis of their role in Rab11 localization and other aspects of oocyte polarization must await the identification of conditional mutants (Dollar, 2002).
Kinesin:ß-gal expression studies indicate that microtubule plus ends are not sharply focused onto the posterior pole of the oocyte in rab11P2148 mutant oocytes. The simplest interpretation of this finding is that microtubule plus ends are attached to the PMD, or to a neighboring membrane domain whose identity is established and/or maintained by Rab11. In wild-type oocytes, this domain is tightly defined such that Kin:ß-gal is concentrated at the posterior tip of the oocyte, while in rab11P2148 mutant oocytes, the domain is poorly defined and the Kin:ß-gal expression pattern is expanded. The slight enrichment of Kin:ß-gal at the posterior tip of rab11P2148 oocytes could reflect partial Rab11 activity and/or the polarizing activities of membrane trafficking pathways that may not rely on Rab11 (e.g. the secretory pathway), which targets newly synthesized molecules from the Golgi to the plasma membrane. Recent studies have identified two types of protein-protein interactions (CLIP-CLASP and APC-EB1) responsible for the stable association of microtubule plus ends with membranes. While the CLIP, CLASP, APC and EB1 protein families are all well-represented in the Drosophila genome, their role in the establishment of oocyte polarity has not yet been investigated (Dollar, 2002).
Apart from Rab11, the only protein known to play a specific role in microtubule plus end organization in Drosophila oocytes is Par-1, a kinase, whose suspected targets include the microtubule associated protein Tau. In strong par-1 mutants, microtubule plus ends, as revealed by Kin:ß-gal expression patterns, are not enriched at the posterior pole of the oocyte, but instead are concentrated tightly, forming a dot at the center of the cell. In weak par-1 mutants, a small amount of Kin:ß-gal is also found at the posterior pole. This small amount of Kin:ß-gal is always tightly localized to the cell tip, suggesting that Par-1 is not required for the specification of the PMD, but rather only for the efficient movement of already focused microtubule plus ends from the cell center to the PMD. Consistent with the idea that microtubule plus ends initially focus to a sharp point at the center of the cell and then move to the posterior pole, Kin:ß-gal and oskar mRNA show transient concentration at the center of the cell in wild-type oocytes. How Par-1 might promote the movement of microtubule plus ends from the cell center to the posterior pole is not clear. One possibility is that it promotes attachment of microtubule plus ends to a structure that is then moved to the posterior pole. Alternatively, Par-1 might stimulate a burst of microtubule growth, forcing growth toward the posterior end of the cell (Dollar, 2002).
The observation that osk mRNA transport to the posterior pole is delayed in rab11P2148 mutant oocytes suggests that Rab11 might also have a role in the movement of microtubule plus ends from the cell center to the posterior pole, and therefore, that such movement is membrane dependent. For example, microtubule plus ends could become attached to membrane compartments or vesicles at the cell center, and the vesicles may then be targeted to the posterior pole in a Rab11-dependent manner. Because osk mRNA arrives at the posterior pole as a fairly well-defined ball in rab11P2148 oocytes, Rab11 does not appear to be required for focusing microtubule plus ends at the cell center, but rather only for their timely movement and attachment to the posterior membrane domain (Dollar, 2002).
Although most osk transcripts are eventually transported to the posterior pole in rab11P2148 oocytes, they are not translated. Since Osk is required to anchor oskar mRNA at the posterior pole, the lack of oskar translation in rab11P2148 GLCs could explain the inability of the oskar mRNA ball to resolve into the thin posterior crescent. The nature of the osk translation block in rab11P2148 oocytes is not clear. One possibility is that key osk translation factors are localized to the posterior membrane domain established by Rab11. In rab11P2148 oocytes, this domain may be too poorly defined to support assembly of such factors into an active translation complex (Dollar, 2002).
A genetic screen was carried out in Drosophila to identify mutations that disrupt the localization of Oskar mRNA during
oogenesis. Based on the hypothesis that some cytoskeletal components that are required during the mitotic divisions will
also be required for Oskar mRNA localization during oogenesis, a screen was carried out for
P-element insertions in genes that slow down the blastoderm mitotic divisions. A secondary genetic screen was used to
generate female germ-line clones of these potential cell division cycle genes and to identify those that cause the
mislocalization of Oskar mRNA. Mutations were identified in ter94 that disrupt the localization of Oskar mRNA to the
posterior pole of the oocyte. Ter94 (see Eyes closed, the enzyme cofactor of Ter94) is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in
homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog,
ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. A test was carried out to see whether other membrane
biosynthesis genes are required for localizing Oskar mRNA during oogenesis. Ovaries that are mutant for
syntaxin-1a, rop, and synaptotagmin are also defective in Oskar mRNA localization during oogenesis (Ruden, 2000).
In order to identify new genes required for OSK mRNA
localization, OSK localization defects in egg
chambers were sought in mutants for cell division cycle (CDC)
genes that had been isolated in a 'mitotic delay-dependent
survival' (MDDS) genetic screen. The
rationale for this is that many cytoskeletal proteins required
for mitotic divisions may also be required for mRNA
localization. The advantage of studying the function of
CDC genes during oogenesis, in which all of the mitotic
divisions occur in region 1 of the germarium, is that later in
oogenesis one can analyze the biological functions of the
CDC genes independent of their mitotic functions. For
example, Klp38B, a chromatin-binding
kinesin-like-protein isolated in the MDDS genetic screen,
is required not only for chromosome segregation during the
meiotic and mitotic divisions, but also for the proper
development of the oocyte, possibly by localizing mRNA or
protein in the oocyte (Ruden, 2000 and references therein).
Based on the phenotypes of syx-1a, ter94, rop and
syt mutant egg chambers, a three-step genetic pathway is proposed for
the role of membrane fusion proteins on OSK mRNA localization
during oogenesis. (1) Syx-1a is required in
stage 1 egg chambers to get OSK mRNA to the oocyte. Syx was originally identified as a Drosophila homolog
of a human tSNARE that is required for synaptic vesicle
fusion in neurons. Interestingly, Syx5
in humans has recently been shown to be required for
TERA-mediated (the human Ter94 ortholog) assembly of
Golgi cisternae from mitotic Golgi fragments in vitro
(Rabouille, 1998). (2) Ter94 is required to localize
OSK mRNA within the oocyte. It is
speculated that OSK mRNA might be transported in membranous
particles because both the endoplasmic reticulum and OSK mRNA form particulate complexes in ter94-mutant egg chambers.
(3) The final step in OSK mRNA localization is anchoring the
mRNA to the posterior pole of the oocyte. It is proposed that
Rop and Syt are required for this process because rop and syt mutant egg chambers have poorly
formed cytoplasmic membranous structure in the oocytes,
and, possibly as a result, OSK mRNA fails to remain localized
at the posterior pole. Rop is a Drosophila
homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18
proteins and is a negative regulator of neurotransmitter
release in vivo (Schulze, 1994). Syt controls and
modulates synaptic vesicle fusion in a Ca2+ dependent
manner (Littleton, 1993). It is concluded that
many synaptic vesicle fusion proteins also function during
other cellular processes such as OSK mRNA localization
during oogenesis (Ruden, 2000).
licorne codes for a MAP kinase kinase exciting the p38 pathway in Drosophila. licorne mutant embryos are defined, for the purpose of this study, as hemipterous;licorne double mutants engineered to express a hemipterous transgene (see Licorne Effects of Mutation for more information about this genotype). lic mutant embryos show a segmentation phenotype that is
reminiscent of the one produced by mutations in the maternal
posterior-group genes, including oskar, vasa, and
nanos. Most of the posterior-group genes can provoke both abdominal
segmentation defects and a loss of germ cells, a dual defect that is
due to the common localization of the posterior and germ cell
determinants in the posterior germ plasm.
Like several posterior-group genes, lic embryos lack or have a
strongly reduced number of pole cells, as shown using Vasa and Nanos as
markers. In most lic mutant embryos, Vasa
protein fails to be accumulated at the posterior pole, although in some cases weak staining is observed.
It is concluded that lic has a role in abdominal segmentation, proper Vasa protein
and Nanos mRNA localization at the posterior pole, and
formation of the pole cells. These results suggest that lic
also has a role in germ plasm assembly (Suzanne, 1999).
The assembly of the germ plasm takes place during oogenesis and
proceeds in several steps leading to the successive posterior localization of many different components (for review, see Rongo,1996). A pivotal step in this process is the
localization of the OSK mRNA to the posterior pole of the
oocyte in stage 8-9 egg chambers, which is the basis for the
recruitment and assembly of downstream components like Vasa and Nanos.
In lic germ-line clones, both OSK mRNA expression and early posterior localization appear normal until stage 8 of oogenesis. However, in stage 9 and older egg chambers, the
OSK mRNA is mislocalized, diffusing in the whole oocyte in a
gradient from the posterior to the anterior pole. In later stages, OSK transcripts are
barely detectable, indicating that diffusion proceeds continuously in
mutant egg chambers. A similar phenotype is observed in some
osk missense mutants, suggesting a role for Osk
protein in the anchoring of its own mRNA at the posterior pole.
Staining of lic mutant egg chambers using an anti-Osk antibody
did not allowed detection of any reduction in Osk protein accumulation,
indicating that lic affects OSK mRNA
localization independent of Osk translation (Suzanne, 1999).
In some lic egg chambers, the mislocalized OSK
mRNAs also seem to partly accumulate in a more central position, reminiscent of the position of OSK
transcripts in mutants that have not reorganized the microtubules, as in
EGFR pathway mutants. This result suggests that lic
oocytes are not completely repolarized. However, no defect in the positioning of the nucleus, or in the
localization of a kinesin-lacZ microtubule-associated motor
protein fusion is observed, suggesting that
OSK mRNA mislocalization is a more sensitive assay and lic
defects are weak. The correct localization of osk RNA at stage 8 and its later
diffusion indicate that lic affects the maintenance of
OSK mRNA asymmetric localization in the oocyte (anchoring)
rather than the mechanism of localization per se, most likely as a
result of incomplete polarization along the AP axis (Suzanne, 1999).
The follicle cell monolayer that encircles each developing
Drosophila oocyte contributes actively to egg development
and patterning, and also represents a model stem cell-derived
epithelium. Mutations in the receptor-like transmembrane tyrosine phosphatase Lar have been identified that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an
indicator of oocyte anterior-posterior polarity. Alterations
in actin filament organization correlate with these defects.
Actin filaments in the basal follicle cell domain normally
become polarized during stage 6 around the anterior-posterior
axis defined by the polar cells (follicle cells lie at the anterior and
posterior poles of ovarian egg chambers beginning at stage 3), but mutations in
Lar frequently disrupt polar cell differentiation and actin
polarization. Lar function is only needed in somatic cells,
and (for Oskar localization) its action is autonomous to
posterior follicle cells. Polarity signals may be laid down by
these cells within the extracellular matrix (ECM), possibly
in the distribution of the candidate Lar ligand Laminin A,
and read out at the time Oskar is localized in a Lar-dependent
manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. Lar acts somatically early in oogenesis, during follicle
formation, and it is postulated that Lar functions in germarium
intercyst cells that are required for polar cell specification
and differentiation. These studies suggest that positional
information can be stored transiently in the ECM. A major
function of Lar may be to transduce such signals (Frydman, 2001).
A clue to the mechanism of Lar action comes from studies on its role in Oskar localization. Posterior follicle cells
must express Lar to ensure that Oskar is localized properly at
the oocyte posterior. When posterior follicle cells lack the ECM component Laminin A (LanA), Oskar localization is usually disrupted. These studies suggest
that LanA and ECM mediate the posterior follicle cell-oocyte
signal. As Lar has been reported to bind to the laminin-nidogen
complex, Lar might act as the LanA
receptor in this pathway. However, it remains less clear how a signal initiated by an interaction between LanA in the ECM and Lar on a posterior
follicle cell would be transduced into the oocyte. Some LanA-containing ECM
resides between the apical surface of the posterior follicle cells
and the oocyte, and it has been proposed LanA interacts directly with the oocyte
surface. An alternative model is proposed. LanA was observed only on the basal side of the follicle cells, and LanA clones induce round eggs. These
observations and the follicle cell autonomous requirement of
Lar for Oskar localization argue that the LanA signal is
received by Lar on the basal surface of the follicle cells and
leads to some change in the receiving cells that is transduced
to the oocyte. This could be via a secondary signal, or by
changes in the structural or adhesive properties of the cells that
can locally affect the oocyte surface with which they come into
contact. Lar mutation does not affect the apical basal polarity of
follicle cells, because the apical-basal asymmetry of actin staining is maintained and multiple-layered follicle cells are never observed (Frydman, 2001).
Polar cells are likely to play a key role in polarizing
actin in stage 5-6 follicle cells. Several observations support the idea that polar cells organize the actin planar polarity. Actin polarity focuses
around both the anterior and posterior polar cell pairs, and
spreads from the poles towards the equator of the follicle.
Additionally, ectopic polar cells induced by Hedgehog (Hh) expression
sometimes have actin polarizing activity. These findings
suggest that polar cells send a signal that orients the actin
alignment in circumferential direction. In follicles where actin
fails to become aligned, the polar cell signal may have been
blocked or reduced, despite the presence of morphologically
recognizable polar cells. Not all the ectopic polar cells induced
by Hh expression affect the actin alignment of nearby follicle
cells, supporting the idea that polar cells can express
differentiation markers, but still be incompetent as polarizing
centers. However, Lar was not required in polar cells, because
follicles with normally oriented actin are observed, despite the
presence of a Lar mutant polar cell pair (Frydman, 2001).
The autonomy of the effects of Lar on Oskar localization
contrasts with its apparently non-autonomous action on follicle
cell planar polarity at stages 6-8. There was no relationship
between actin alignment and the Lar genotype of particular
somatic cells in stage 7 or later follicles. This observation can
be rationalized by postulating that Lar acts on a subset of cells
that is required for polar cell specification and differentiation.
Lar is required in region 2b of the germarium, a time when
polar cells are not yet fully specified, and in its absence somatic
cell behavior and follicle formation is compromised. It is
proposed that the intercyst cells interact with the polar
cell precursors in a Lar-dependent manner. Intercyst cells
mostly become main body follicle cells and do not remain a
recognizable subpopulation; hence, it is not possible to infer their genotype from later stage egg chambers and compare it with the state of actin polarization. A correlation was noted between mutant intercyst cells and pinching defects. Thus, a relationship may exist between the intercyst cell genotype and actin polarization that cannot be followed with existing markers (Frydman, 2001).
One possibility is that Lar acts in a similar manner in
intercyst cells and in posterior follicle cells. The germarium
contains peripheral somatic cells whose basal actin fibers are
aligned perpendicular to the AP axis. Integrin is aligned in a similar manner, suggesting that the basement membrane is correspondingly organized. As budding proceeds, Lar may be required to interpret polarity information from the basement membrane as part of the process that partitions cells into main body and polar
cells. Alternatively, Lar may act in a different manner within these intercyst cells to assist in polar cell specification and differentiation (Frydman, 2001).
The Lar requirement for polar cell determination provides
an explanation for another interesting fact. The phenotypic
effects of Lar mutations observed are very similar to weak
mutations in Notch or other Notch-pathway genes. Notch
mutants, like those in Lar, cause the production of extra polar
cells, interfere with egg chamber budding and disrupt the
anterior-posterior axis of the oocyte.
Notch signaling is required for polar cell specification. Mutations in
either Lar or Notch may cause similar disruptions in polar cell
differentiation and hence similar downstream effects on egg
chamber development and patterning (Frydman, 2001).
These experiments suggest a novel function for the ECM during
follicle cell development -- the storage of patterning
information for later use. Somatic cells maintain an ECM that
surrounds the germarium; when a follicle buds off, it contains
a portion of this ECM in the basement membranes of its
component cells. These studies emphasize that this ECM may
be a repository of polarity information that is used at critical
times when polarization and cell specification are taking
place. During follicle budding, the AP axis of the new
chamber is correlated with the differentiation of two pairs of
polar cells at each terminus. An interaction between posterior
follicle cells and the oocyte ensures that the germline AP axis
will correspond to the somatic axis. It is suggested that at
about the same time, somatic cell interactions ensure that
exactly four correctly positioned polar cells differentiate per
follicle. This requires Lar-dependent readouts from the same
ECM that the interacting cells contributed to polarizing. In
stage 8, posterior follicle cells are likewise guided to maintain
localized Oskar over an appropriately sized polar region. In
both cases, cells that helped synthesize an ordered ECM, later
use it in a Lar-dependent manner for additional and possibly
more refined patterning. The interactions studied may
serve as a model for the roles of the ECM and of Lar signaling
in the development of other epidermal and neural cells (Frydman, 2001).
In Drosophila, dorsoventral polarity is established by the asymmetric positioning of the oocyte nucleus. In egg chambers mutant for cap 'n' collar, the oocyte nucleus migrates correctly from a posterior to an anterior-dorsal position, where it remains during stage 9 of oogenesis. However, at the end of stage 9, the nucleus leaves its anterior position and migrates towards the posterior pole. The mislocalization of the nucleus is accompanied by changes in the microtubule network and a failure to maintain Bicoid and Oskar mRNAs at the anterior and posterior poles, respectively. Gurken mRNA associates with the oocyte nucleus in cap 'n' collar mutants and initially the local secretion of Gurken protein activates the Drosophila EGF receptor in the overlying dorsal follicle cells. However, despite the presence of spatially correct Grk signaling during stage 9, eggs laid by cap 'n' collar females lack dorsoventral polarity. cap 'n' collar mutants, therefore, allow for the study of the influence of Grk signal duration on DV patterning in the follicular epithelium (Guichet, 2001).
In cnc mutant egg chambers, nuclear movement occurs
normally. The nucleus remains cortically localized even
after its posterior displacement. Since interference with
components of the dynactin complex leads to the dissociation
of the nucleus from the cortex, it is believed that the dynactin complex is not
affected by the loss of cnc function.
However, the polarization of the microtubule network is
aberrant in stage 10A cnc oocytes. Higher numbers of
microtubules accumulate in the posterior region of the
oocyte at the expense of the anterior cortical ring, which
dominates the microtubule network of wild-type stage-9 to
-10A egg chambers. This second microtubule reorganization
could either be the cause of or result from the
late displacement of the nucleus. In the first case, cnc
would be required for a process that stabilizes and maintains
the microtubule polarity after stage 8. Prolonged
signaling from posterior follicle cells might be necessary to
suppress the reestablishment of microtubule organizing centers (MTOCs) at the posterior pole. The reception of such a signal or its transmission to
the cytoplasm might be impaired in the absence of cnc
function. In this model, the reassembly of MTOCs in
posterior regions would lead to the redistribution of free
tubulin and consequently weaken anterior MTOCs. The
nucleus would subsequently migrate towards these ectopic
posterior MTOCs. BCD mRNA also would become mislocalized
since it is known to move, like the nucleus, towards
the minus ends of microtubules, i.e., towards the MTOCs (Guichet, 2001).
In the other scenario, cnc would be required specifically
for oocyte nucleus anchoring at the anterior cortex. Anterior
anchoring might be necessary since there is a massive
influx of cytoplasm from the nurse cells to the anterior pole
of the oocyte during egg chamber growth. If the nucleus is
not properly anchored, these transport processes might
dislodge the nucleus from the anterior pole. Why would this
mispositioning of the nucleus lead to the reorganization of
the microtubule network? Such microtubule reorganizations
have not been described in other mutant backgrounds
where the nucleus does not reach the anterior cortex, such
as grk, cni, mago, and DLis-1. It has
been shown that the nucleus gets encaged by microtubules
when it arrives at the anterior pole in wild-type oocytes,
indicating that the anteriorly localized nucleus acquires a
microtubule-nucleating activity. This activity might remain associated with the mispositioned nucleus in cnc egg chambers and might subsequently
cause the increased microtubule density in the
posterior half of the cnc oocytes (Guichet, 2001).
In both scenarios, the mislocalization of OSK mRNA remains
somehow enigmatic. OSK should not localize to the same
region to which BCD is transported. However, normal OSK
transport from the anterior to the posterior might just be
blocked by the mispositioned nucleus and its associated
microtubules. Thus OSK might be trapped in the vicinity of
the ectopic nucleus on its way to the posterior pole (Guichet, 2001).
Embryonic germ cell formation and abdomen development in Drosophila requires localization and site specific translation of oskar mRNA in the posterior part of the oocyte. Targeting of oskar function to the posterior pole of the oocyte needs a large set of proteins and RNAs, encoded by posterior group genes. Consequently, mutations in the posterior group genes can result in embryos without abdomens and/or germ cells. During a systematic hobo-mediated mutant isolation screen, poirot, a novel posterior group gene, was identified owing to its germ cell-less phenotype. poirot bears the Flybase designation of parcas. The lack of poirot activity dramatically decreases Osk protein levels, without affecting the oskar mRNA distribution. In poirot mutant oocytes, delocalized Osk protein is observed, indicating that wild-type poirot has a role in the anchoring process of the Osk protein at the posterior pole. Furthermore, poirot acts in an isoform-specific manner, only the short OSK isoform is affected, while the long Osk isoform remains at wild-type levels in poirot mutants (Sinka, 2002).
Conceptual translation of prt cDNA predicted a 477 amino acid protein that shows extensive homology to human and mouse Sab proteins. Sequence analysis revealed that the 257 amino acid N-terminal part of Prt shows significant
homology to the Sab protein family. Prt shares 48% identity and 66% similarity to the human, and 45% identity and 63% similarity to murine Sab
proteins, while the C-terminal region did not show any significant homology to known proteins. Sequence comparisons also identified a predicted C.
elegans K03E6.7 (38% identity and 56% similarity) and a Drosophila CG14408 (38% identity and 54% similarity) homolog of the 252 amino acid
N-terminal part of the Prt protein. The N-terminal region of human Sab contains an unconventional SH3-binding domain, which preferentially binds to
the SH3 motif of Bruton's Tyrosine kinase (Btk) protein. Interestingly, the 32 amino acids long SH3-binding domain of human Sab shows 48% identity and 73% similarity to the corresponding part of Prt, indicating that the two proteins may have conserved functions (Sinka, 2002 and references therein).
The biochemical function of Sab, the human homolog of the prt gene has been revealed by in vitro and in vivo assays. The Sab protein preferentially binds to the Bruton's tyrosine kinase protein and negatively regulates its function. Because, theoretically, the absence of a negative regulator can be suppressed by decreasing the level of the regulated component, tests were performed to see whether hypomorphic Btk alleles can suppress prtgs mutation. This study showed that Prt negatively regulates Drosophila Btk, because the prt null mutant phenotype is manifested only if Btk activity is present. Drosophila Btk29A itself has no loss-of-function grandchildless phenotype. Btk29A activity is not required for germline formation (Sinka, 2002).
In prtgs mutants, only the short Osk isoform level is reduced, while the long isoform remains at wild-type levels. This may explain the normal osk mRNA distribution, since the long Osk isoform can maintain its own mRNA at the posterior pole. However, the isoform specificity of the prtgs allele reveals that the two Osk isoforms may use independent posterior anchoring mechanisms or can be subjected to different post-translational regulation processes during their anchoring. However, genetic and molecular evidence suggests that some residual short Osk must still be present in prtgs null mutants, because the complete loss of the short Osk isoform would not only result in grandchildless but rather a complete abdomen and germ cell-less phenotype. This result also demonstrates that prt function in Osk anchoring must be redundant (Sinka, 2002).
Subcellular localization of Prt is reconcilable with osk regulatory function, because at the subcortical region their localization overlaps. This colocalization potentiates the functional interaction. Some osk translational regulatory proteins, which form a large ribonucleoprotein (RNP) complex, demonstrate a similar expression pattern during their transport to oocytes to that of Prt. Biochemical evidence exists that this RNP contains Exu and at least seven other proteins, in addition to osk and bicoid mRNAs. Exu, Orb and Me31 B, three elements of the above RNP complex, as well as Cup, an RNP independent protein with similar localization pattern, are not colocalized with Prt in nurse cells. Consequently Prt aggregates, in spite of their similar subcellular localization, have an independent transport system from either the Exu or the Cup complexes (Sinka, 2002).
The genetic interaction found between prt and Btk29A indicates that the Prt regulatory function is evolutionarily conserved. This is especially interesting, since the Drosophila BTK homolog is not required in germ cell formation. The Drosophila genome contains a single BTK homolog Btk29A, which encodes two protein species. Btk29A has several pleiotropic functions, such as male fertility and ring canal formation. It is expressed in the adult head, the larval immune system, the male and female gonads, and several other tissues. However, germline clone analysis of the two hypomorhic alleles has failed to reveal a role in germ cell formation. Based on the structural conservation of the Sab and Prt proteins, it is anticipated that Prt might also exhibit a negative regulatory function similarly to Sab. According to this hypothesis, when Prt, the presumed negative regulator, is absent, an ectopic BTK activity would interfere with the normal function of the posterior gene hierarchy. Being normally suppressed, loss of function of such a negatively regulated gene would not be expected to cause any posterior phenotype. Indeed, the suppression of prtgs phenotypes by hypomorphic Btk29A alleles indicates that in Drosophila, Prt also negatively regulates BTK29A (Sinka, 2002).
Therefore, it is proposed that, in wild-type oocytes, Prt inactivates the BTK protein in the subcortical region. In prtgs mutants, however, unregulated BTK interferes either with the localization of subcortical cellular components in the oocyte, or it modifies the phosphorylation pattern of the short Osk protein itself. The latter is a less feasible explanation, because in prtgs mutants no increased levels of phosphorylated short Osk are found that would be the result of extra kinase activity: instead, the level of 57 kDa phosphorylated short Osk isoform is also significantly reduced. It is suggested that uncontrolled BTK kinase activity modifies the anchoring capability of short Osk directly or indirectly to the subcortical region, resulting in delocalization and degradation of both phosphorylated and unphosphorylated short Osk proteins. Because only the phosphorylated short Osk isoform was detected in prtgs mutants, it is supposed that either this protein is more stable or it is better anchored to the posterior pole, compared with the unphosphorylated form. The weakly anchored Osk protein is displaced by the cytoplasmic streaming and loses its pole plasm organising activity. Future research should be directed toward determining the precise biochemical function of Prt and elucidating the anchoring mechanism of Osk protein (Sinka, 2002).
In Drosophila, development of the embryonic germ cells depends on posterior transport and site-specific translation of oskar (osk) mRNA and on interdependent anchoring of the osk mRNA and protein within the posterior subcortical region of the oocyte. Transport of the osk mRNA is mediated by microtubules, while anchoring of the osk gene products at the posterior pole of the oocyte is suggested to be microfilament dependent. To date, only a single actin binding protein (TropomyosinII) has been identified with a putative role in osk mRNA and protein anchoring. Mutations in the Drosophila Moesin-like (Moe) gene, which encodes another actin binding protein, result in delocalization of osk mRNA and protein from the posterior subcortical region and, as a consequence, in the failure of embryonic germ cell development. In moe mutant oocytes, the subcortical actin network is detached from the cell membrane, while the polarized microtubule cytoskeleton is unaffected. Colocalization of ectopic actin and Osk protein in moe mutants suggests that the actin cytoskeleton anchors Osk protein to the subcortical cytoplasmic area of the Drosophila oocyte (Jankovics, 2002).
Moe is the Drosophila member of the ERM protein family, which contains three vertebrate members: ezrin, radixin, and moesin. ERM proteins have a C-terminal actin binding domain and an N-terminal FERM domain, which is responsible for interaction with several membrane proteins. Based on their structure and predominant subcortical distribution, ERM proteins have been suggested to function as crosslinkers between the cell membrane and the actin cytoskeleton. ERM proteins have been demonstrated to take part in several biological processes, such as cell-cell adhesion, maintenance of cell shape, cell motility, and internal membrane trafficking. Despite their distinct expression pattern, the vertebrate ERM protein family members show functional redundancy. The Drosophila genome, however, contains only a single ERM homolog: Moesin-like. Drosophila is a valuable model organism for studying ERM functions, since the ERM homolog is not genetically redundant (Jankovics, 2002).
In a screen for P element-induced maternal effect germ cell-less mutations, a recessive allele of the moe gene was identified. The moeGT193 allele was isolated by making use of a newly designed EGFP-GT mutator P element. EGFP-GT was made by replacing the Gal4 marker gene of a dual-tagging gene trap element, pGT1, with the EGFP marker gene. A collection of 200 viable, X-chromosomal EGFP-GT insertions was generated by the attached-X technique and was screened for maternal effect germ cell-less phenotype by hand dissection of adult test animals. The moeGT193 allele was identified as a weak maternal effect germ cell-less allele. Subsequent complementation analyses with P element-induced mutations obtained from the Bloomington Stock Center revealed the existence of five additional insertional alleles of moe. Combinations of different moe alleles resulted in germ cell-less phenotypes with a penetrance of 1%-61%. For further analyses, the moeEP1652/moeG0415 and moeEP1652/Df(1)KA14 combinations were chosen; these resulted in 60% and 61% germ cell-less phenotypes, respectively. Hereafter, the phenotype of these combinations will be referred to as the moe mutant phenotype (Jankovics, 2002).
Complementation analyses revealed several additional pleiotropic moe phenotypes such as lethality, female sterility, and imperfect eye and wing development. Western blot analysis of moe mutant ovaries revealed a reduction in Moe protein levels, while precise excisions of P elements from moe alleles restored wild-type protein levels and germ cell development. These results demonstrate that the maternal effect germ cell-less phenotype is a direct consequence of P element insertions in the moe gene (Jankovics, 2002).
Since ERM proteins have been suggested as functioning as crosslinkers between the actin network and the cell membrane, the organization of actin filaments was examined in moe oocytes. Instead of a tight, wild-type subcortical localization, in moe mutants the actin network seems to be detached from the cortex. It intrudes into the cytoplasm of the oocyte either at the posterior pole or at more lateral regions. Interestingly, mislocalized subcortical actin is also found in Schizosaccharomyces pombe after being transformed with truncated Drosophila moe cDNA. In this heterologous transformation experiment, actin abnormalities were also coupled with cell shape changes. This raises the possibility that observed actin abnormalities in Drosophila oocytes may be the result of abnormally shaped oocytes. To rule out this possibility, the cell and vitelline membranes were visualized by making use of fluorescently labeled lectins (Lycopersicon esculentum and Datura stramonium lectins, respectively) and immunostained for DE-cadherin, a transmembrane protein that is present in all cell membranes of egg primordia. In developing mutant eggs, however, normal distribution of the lectins and DE-cadherin stainings are detected. Furthermore, by simultaneous visualization of the cell membrane-specific lectin and actin, it has been shown that, at actin intrusion sites, the cell membrane is normal. These results demonstrate that, in moe mutants, the shape of the oocyte is normal and the observed actin abnormality is due to detachment of subcortical actin from the cell membrane. It is concluded, therefore, that one of the functions of moe in developing oocytes is to crosslink the subcortical actin network and the cell membrane (Jankovics, 2002).
Embryonic germ cell formation is initiated during oogenesis by posterior localization of a highly specialized cytoplasmic region, the pole plasm. Since the assembly of the pole plasm depends on the anterior-to-posterior transport and subsequent anchoring of osk mRNA, the distribution of osk mRNA was examined in moe oocytes. Instead of the characteristic wild-type posterior localization, several different types of abnormal osk mRNA distribution were found in the mutants. Most frequently, the mislocalized osk mRNA appears in a scattered pattern concentrated near the posterior pole. Mislocalization of osk mRNA to the central regions of oocytes was also observed. In some stage-10 oocytes, ectopic osk mRNA was found to be localized tightly to the lateral cell cortex. In order to gain insight into the time course of osk mRNA localization in mutants, the penetrance of these phenotypes was measured and compared in stages 9 and 10. A slight, but convincing, decrease of the wild-type osk mRNA localization was observed in stage 10, and this decrease suggests that moe mutations do not interfere with the early posterior transport of osk mRNA but rather with its anchoring to the posterior pole. Another pole plasm component, the Staufen (Stau) protein, which always colocalizes with osk mRNA, consistently shows a distribution identical to that of osk mRNA in mutant oocytes (Jankovics, 2002).
Since correct localization of osk mRNA requires proper functioning of both the oocyte and follicle cells, and moe is expressed predominantly in the follicle cells, moe mutant germline clones were generated to identify the cell type in which moe mutations exert their effect. In developing eggs composed of the homozygous germline and heterozygous follicle cells for moeG0415, moeG0404, moeG0067, and moeG0323 mutations, osk mRNA mislocalization phenotypes were found to be identical to those of moeEP1652/DmoeG0415and moeEP1652/Df(1)KA14 mutant oocytes. This result demonstrates that moe is required in the germ cells for posterior localization of osk mRNA (Jankovics, 2002).
To investigate whether moe mutations affect other localized determinants in the Drosophila oocyte, the distributions of gurken (grk) and bicoid (bcd) mRNAs were examined. In mutant oocytes, normal grk and bcd mRNA localization was found, which demonstrates that the moe mutant phenotype is osk specific. Similar to moe mutations, par-1 and Rab11 mutant alleles cause abnormal osk mRNA localization and normal distribution of the bcd and grk mRNAs. In these mutants, a plus end microtubule marker molecule, Kin:β-Gal, is localized to the central region of the oocyte, while the minus end-specific Nod:β-Gal remains at the anterior pole. In contrast to these mutants, however, normal Nod:β-Gal and Kin:β-Gal localization was observed in moe oocytes. Furthermore, a normal arrangement of the microtubule network was revealed both by immunostaining and by direct in vivo visualization of the microtubules by using Tubulin:GFP or Tau:GFP fusion proteins. By a simultaneous visualization of STAU protein and microtubule plus ends, Stau mislocalization was shown not to be a consequence of an abnormal microtubule network. In moe mutant oocytes, the plus ends of the microtubules normally point to the posterior pole, as demonstrated by the correct localization of Kin:β-Gal. In contrast, however, Stau is mislocalized, suggesting again that the mislocalization of osk mRNA is not a consequence of abnormal transport; rather, it appears to be caused by abnormal anchoring (Jankovics, 2002).
In order to further investigate the role of Moe in osk regulation, the level and distribution of Osk protein was examined in moe oocytes. In mutant ovaries, a reduced level of Osk was found by Western analysis. Consistent with this observation, no Osk was detected by immunostaining in the majority of the mutant oocytes, while, in the remaining cases, the pattern of the mislocalized Osk protein was found in a spatial and temporal distribution similar to that of osk mRNA. Osk was mostly found in a scattered pattern at the posterior region of the oocytes. In the majority of moe oocytes in which Osk was delocalized, the actin network appears to be attached normally to the cell membrane, at least as far as can be visualized with light microscopy. This indicates that moe might have a role in Osk anchoring, which is separable from its actin-cell membrane crosslinking function (Jankovics, 2002).
In the rare cases when detachment of actin from the cell membrane occurs at the posterior pole, it was observed that Osk protein colocalizes with the ectopic actin network. This ectopic colocalization of the subcortical actin and Osk protein in moe mutants reveals that the actin network has Osk anchoring capacity and strongly suggests that the actin network anchors Osk at the normal place, at the posterior subcortical region, too. In moe mutants, the abnormal osk mRNA and protein localization phenotype is observed when osk products are tightly localized to lateral segments of the subcortical actin. These results confirm a model that proposes that the focused posterior localization of osk is not defined by a special segment of the posterior subcortical actin; rather, it is determined by the microtubule-based posterior transport of osk mRNA to this region (Jankovics, 2002).
Thus, partial loss of moe activity weakens the ability of the actin network to anchor osk mRNA and protein, resulting in different degrees of their delocalization. moe has actin-cell membrane crosslinking activity in the developing oocyte, and evidence is presented that factors other than actin define the site of osk mRNA and protein localization (Jankovics, 2002).
The Drosophila antero-posterior axis is established during oogenesis, where the localized activity of Oskar determines the posterior pole and recruits Nanos and Vasa. To assay the role of Moe in posterior pole formation, Moe germline mutant clones were generated. Females with a Moe mutant germline are sterile and do not lay eggs. In wild-type oocytes, Oskar accumulates in a crescent that is tightly localized to the posterior of the oocyte cortex. By contrast, in Moe oocytes, Oskar is found at more anterior locations and forms abnormal fibers, apparently loosely bound to the cortex. Localization of the Oskar protein is linked to the polarized accumulation of oskar mRNA. oskar is normally transcribed in Moe oocytes and until stage-8, its localization is indistinguishable from that in wild-type oocytes. However, the posterior localization of oskar mRNAs that starts from stage-9 is affected in Moe mutants. Whereas oskar mRNAs are restricted to the posterior of wild-type oocytes, a diffuse signal is seen throughout the oocyte cytoplasm (ooplasm) in stage-10 Moe oocytes. In some cases, a weak accumulation of oskar mRNAs is observed at the posterior pole, indicating that Moe mutations do not completely abrogate the transport of oskar mRNAs. In contrast, gurken and bicoid mRNAs localize normally at an antero-dorsal position and the anterior margin, respectively. This indicates that Moe mutations do not alter the formation of the dorso-ventral axis or the anterior pole. Localization of Oskar depends on Staufen, an RNA-binding protein that is required for the transport of oskar mRNA. Compared with wild-type oocytes, the posterior accumulation of Staufen is reduced in Moe oocytes; Staufen is not firmly localized to the posterior and a diffuse staining is observed in the ooplasm (Polesello, 2002).
The establishment of oocyte polarity and Oskar localization depends on microtubule organization. To determine whether Moe mutations affect microtubules, the movements of yolk granules were analyzed in living egg chambers. In wild-type oocytes, granules move erratically up to stage-10a and eventually flow rapidly in the coordinated cytoplasmic streaming. These movements are blocked by microtubule-depolymerizing drugs, and mutations affecting microtubule organization cause premature streaming. No sign of premature cytoplasmic streaming was observed in Dmoe106 germline clones, although typical coordinated movements of granules are observed at stage-10b-11. This indicates that Moe mutations do not disrupt the microtubule cytoskeleton. Microtubule polarity was analyzed using Nod-ßgalactosidase, which localizes to the minus-end of microtubules. As in the wild-type oocytes, Nod-ßgalactosidase accumulates in the anterior of the mature Moe oocyte. Finally, a lacZ fusion to Kinesin, a plus-end-directed motor that is required for the localization of posterior determinants, was examined. From late-stage-8 onwards, Kinesin-ßgalactosidase displays a polarized distribution in wild-type oocytes. It accumulates transiently (stage 9-10) at the posterior extremity of the oocyte, where it colocalizes with Staufen. In DmoeX5 germline clones, Kinesin-ßgalactosidase localizes normally in stage-9 oocytes. At stage 10a, the staining, although reduced, was still detected at the posterior end, confirming the normal polarity of microtubules in DmoeX5 oocytes. However, Staufen does not completely colocalize with Kinesin-ßgalactosidase in DmoeX5 oocytes; Staufen forms ectopic clumps and displays diffuse staining in the ooplasm. This further supports the conclusion that the mislocalization of posterior determinants observed in Moe oocytes does not result from the disruption of early microtubule nucleation and polarity. Taken together, these results indicate that Moesin is specifically required for the localization of posterior determinants and suggest a critical role of Moe in the functional organization of the oocyte cortex (Polesello, 2002).
To analyse the role of Moe in the organization of the oocyte cortex, the localization of a green fluorescent protein (GFP)-tagged Moesin protein, which fully rescues DmoePG26 mutant phenotypes, was examined. From the early stages of oogenesis until the development of mature egg chambers, Moesin-GFP is present in the oocyte cytosol and accumulates at the cortex, where it colocalizes with F-actin. This membrane-associated fraction corresponds to activated Moesin, because it is recognised by an antibody specific for activated ERM. The consequences of Moe mutations on the cortical actin cytoskeleton were analzed. In the wild-type oocyte, microfilaments accumulate in the cortex and form bundles parallel to the membrane. In oocytes from DmoeX5 germline clones, microfilaments appear loosely bound to the posterior and lateral cortex. In stronger Dmoe alleles, in addition to filaments loosely attached to the membrane, packs of F-actin are also found in the ooplasm. This raises the possibility that these actin defects are responsible for the altered posterior determinant distribution. As observed at earlier stages, cortical F-actin has an abnormal fibrous appearance in stage-10 Dmoe oocytes, and clumps of F-actin are also observed in the ooplasm. Interestingly, Oskar associates with these abnormal actin structures, colocalizing with both lateral F-actin fibres and ooplasmic clumps. These data show that Oskar closely interacts with F-actin and strongly support the conclusion that the mislocalization of Oskar observed in Dmoe oocytes results from defects of the actin cytoskeleton (Polesello, 2002).
Nevertheless, oocyte polarity depends on a signal from the posterior follicle cells, and Dmerlin, which is structurally similar to Moesin, is required in the posterior follicle cells to ensure oocyte polarity. Thus, whether Dmoe activity is also required in posterior follicle cells to organize oocyte polarity was analyzed. When only follicle cells are mutant for Dmoe, neither alteration of the oocyte F-actin organization, nor mislocalization of Oskar was observed. Altogether, these results show that Dmoe is required cell-autonomously in the germline, to anchor actin microfilaments to the oocyte cortex and to localize posterior determinants (Polesello, 2002).
Phosphorylation of a threonine residue located in the actin-binding domain has been shown to regulate ERM activity in mammalian cells. To assay the role of this conserved residue for Drosophila Moesin function, transgenes carrying a Thr 559 mutation that either prevents (Thr-Ala; TA) or mimics (Thr-Asp; TD) phosphorylation of ERM33 were generated. These Moesin variants were fused to GFP and expressed specifically in the germline. Unlike wild-type Moesin-GFP, the entire pool of phosphomimetically mutated protein is directed to the membrane, where it colocalizes with F-actin. Although slightly enriched in the cytoplasm, the non-phosphorylatable form of Moesin associates with the oocyte membrane, where it accumulates ectopically at the anterior. Expression of Moesin-TA also results in the formation of F-actin clumps in the ooplasm. These results show that Thr 559 is involved in the regulation of the subcellular distribution of Moesin and identify its importance for the organization of the oocyte actin cytoskeleton. Consistently, neither Moesin-TD nor Moesin-TA can substitute for wild-type Moesin in rescue assays. In addition, whereas overexpression of MoesinWT-GFP does not alter the distribution of Oskar, the posterior localization of Oskar is either undetectable or strongly reduced in oocytes that express Moesin-TA. By contrast, expression of Moesin-TD results in a marked over-accumulation of Oskar at the posterior end of the oocyte. These data show that Moesin must be properly regulated to allow the normal localization of Oskar at the posterior end of the oocyte (Polesello, 2002).
Although the oocyte constitutes an invaluable system for studying cell polarity, the peculiar structure of its cytoskeleton prevents analysis at the level of microfilaments. Therefore the role of Moe in developing nurse cells was examined. At stage 9-10, the cellular face in contact with follicle cells presents a dense network of microfilaments that is organized in a mesh-like structure. Moesin accumulates strongly at the cortex of stage 9-10 nurse cells and colocalizes with this F-actin mesh. In DmoeX5 germline clones, the regular arrangement of the cortical actin network is lost. Instead, microfilaments are localized in a few aggregates that radiate from the cell surface, forming abnormal structures that resemble flowers. In DmoePL106 clones, F-actin is often absent from the cortex and concentrated at the cell perimeter. Although defects are more pronounced in the cortical region, F-actin also accumulates in abnormal structures at the point of contact between nurse cells. However, Moe mutations affect neither actin cables nor the actin-rich ring canals. These data show that Moe is specifically required in nurse cells for the organization of the cortical actin network, where Moesin strongly accumulates during stage 9-10. Therefore, this constitutes a suitable system to analyse the effects of Moesin Thr 559 substitutions on microfilament organization. In nurse cells expressing Moesin-TD, the mesh-like structure disappears and F-actin is concentrated in dense spots in the cortex and lateral membranes. Overall cell shape is altered and nurse cells become abnormally round. Expression of Moesin-TA also affects the organization of cortical microfilaments, which are enriched in 'actin-flowers' reminiscent of those observed in weak Moe mutants. Interestingly, Moesin-TA associates with the membrane, where it colocalizes with F-actin at the periphery of these aberrant structures. Individual microfilaments, which display a dotted-line staining characteristic of actin cables, are observed at the centre of the mutant structure. Altogether, these results provide the first genetic evidence that the regulated activity of Moe is required for the organization of a specific subset of microfilaments that are asymmetrically localized in the cell (Polesello, 2002).
Transport, translation, and anchoring of osk mRNA and proteins are essential for posterior patterning of Drosophila embryos. Homer and Bifocal act redundantly to promote posterior anchoring of the osk gene products. Disruption of actin microfilaments, which causes delocalization of Bifocal but not Homer from the oocyte cortex, severely disrupts anchoring of osk gene products only when Homer (not Bifocal) is absent. The data suggest that two processes, one requiring Bifocal and an intact F-actin cytoskeleton and a second requiring Homer but independent of intact F-actin, may act redundantly to mediate posterior anchoring of the osk gene products (Babu, 2004).
Both Bif and Hom show asymmetric localization at the apical cortex of embryonic neuroblasts, indicating that these F-actin binding proteins may be involved in neuroblast asymmetric divisions. However, animals lacking both the maternal and zygotic components of either gene are fertile, viable, and show no obvious defects in embryonic CNS development. This prompted construction of double mutants of bif and hom. However, although double homozygous mutant females are viable, they show defects in oogenesis, with the vast majority of the eggs produced remaining unfertilized, as judged by the lack of staining in eggs using an antibody directed against the sperm tail. In the few fertilized embryos that do undergo development, the numbers of Vasa positive germ cells are drastically reduced, suggesting possible defects in the function or localization of posterior determinants during oogenesis (Babu, 2004).
Analyses of bif and hom single mutants as well as double mutant oocytes indicate that the two genes act in a redundant manner for the correct anchoring of posteriorly but not anteriorly localized molecules. In stage 10 oocytes, posterior group molecules, including oskar (osk) RNA, the two isoforms of Osk proteins, Staufen (Stau), and a fusion protein in which ß-galactosidase has been fused to the N-terminal extension of the long form of the Osk protein (referred to as Osk-ßGal, used as a marker for the long form of the Osk protein, which has been shown to have a role in the posterior cortical maintenance of Osk), are all localized as tight posterior cortical crescents in wild-type oocytes. In most double mutant oocytes, these molecules, when detectable, are present largely at the posterior region; however in contrast to wild-type oocytes, they show a diffuse distribution that extends into regions of the posterior cytoplasm distinctly interior to the posterior cortex. In about 30% of the cases, Osk or Stau protein cannot be detected. The defects seen in the oocytes of double mutants are essentially absent in the single mutant oocytes. These findings indicate that whereas bif and hom are individually dispensable, together they are required for the localization of the posterior components of the oocytes. These defects in localization are specific for the posterior group molecules, because the anterior/dorsal localization of Gurken and anterior localization of bicoid RNA are unaffected (Babu, 2004).
Not surprisingly, staining with anti-Bif and anti-Hom antibodies indicates that both proteins are expressed in the oocyte. Bif localizes to the oocyte cortex in a manner very similar to that seen for F-actin. The staining seen with the anti-Hom antibody is highly punctated and, although localization is cortically enriched, Hom is also present in the cytoplasm. The cortical staining seen in wild-type oocytes (and nurse cells) is absent in mutant oocytes stained with the corresponding antibodies, confirming the specificity of both antibodies and that the mutant alleles do not produce detectable amounts of protein. Since homLL17 is a complete deletion of the coding region and bifR47 removes a significant portion of the coding region, they are both likely to be null alleles (Babu, 2004).
Several observations indicate that the defect in posterior localization of the osk gene products is due to defective anchoring and not transport or translation of osk RNA. Osk RNA and Osk proteins as well as Stau are localized normally in stage 9 double mutant oocytes. Consistent with this, both the F-actin and microtubule cytoskeletons in the double mutants are indistinguishable from those in the wild-type oocytes. Not only does the polarity of the microtubules appear normal, as assayed using a kinesin heavy chain (khc) ß-Gal marker, the cytoskeleton-dependent cytoplasmic streaming is also absent in stage 9 oocytes and occurs normally in stage 10 double mutant oocytes, as is seen with wild-type oocytes. Taken together, these observations indicate that the double mutant oocytes retain, at a gross level, normal cytoskeletal structure. They can transport osk mRNA to the posterior cortex, and translate it appropriately, but do not maintain the posterior anchoring of the osk gene products (Babu, 2004).
An intact F-actin cytoskeleton is thought to be required for asymmetric protein localization in several contexts. In the oocyte, loss or reduction of the actin binding proteins moesin and tropomyosin have been shown to affect the posterior anchoring of Osk in the oocyte and the embryo, respectively. To assess the requirement for an intact microfilament cytoskeleton for the anchoring of osk RNA and proteins in the oocyte, the localization of these molecules was examined in wild-type oocytes treated with an actin depolymerizing drug, Latrunculin A (Lat A). Following treatment with 20 µm Lat A, cortical F-actin in the oocytes was largely undetectable with phalloidin, yet, unexpectedly, both Osk proteins (short and Osk-ßGal) and osk RNA remain localized to the posterior cortex of the great majority of wild-type oocytes from stage 9-10B. This was seen even when the oocytes were overstained for the osk RNA: in around 17% of the Lat A-treated wild-type oocytes, mild defects in anchoring are observed. osk RNA and proteins show a diffuse localization at the posterior cortex. However, in no cases were they seen concentrated in the cytoplasm or had they become delocalized or undetectable (as seen in the hom/bif double mutant oocytes) as would be expected if an intact F-actin cytoskeleton were to be an absolute requirement for the normal anchoring of Osk. These mild effects on protein localization in the oocyte are in distinct contrast to those seen in embryonic neuroblasts where severe and high-penetrance defects in asymmetric protein localization are observed following disruption of microfilaments. These observations suggest that the role of microfilaments in the anchoring of proteins to the cortex may differ in the different cellular contexts (Babu, 2004).
Additional experiments were performed to ascertain whether the mild effects on Osk posterior anchoring following disruption of microfilaments are peculiar to Lat A treatment. In fact, the posterior cortical localization of Osk remained in the great majority of oocytes even after treatment with cytochalasin D (CD), Lat A followed by CD, CD followed by Lat A, and up to 100 µM Lat A. These results are consistent with previous reports of CD disruption of F-actin, for example. However, they indicate that an intact F-actin cytoskeleton, although required for the normal posterior anchoring of Osk in a small proportion of oocytes, is probably not the only factor involved in normal anchoring of Osk to the posterior cortex. There are at least two possible explanations for these observations. First, a small amount of residual F-actin might remain even after sequential treatment with Lat A and CD, which is sufficient to anchor Osk normally in a small fraction of the drug-treated oocytes. Alternatively, there may be other factors besides an intact F-actin cytoskeleton, which can, in parallel, contribute toward the posterior anchoring of osk RNA and proteins (Babu, 2004).
The requirement for intact microfilaments on Hom and Bif localization was assessed. Both Bif and Hom localize to the cortex (and in the case of Hom also the cytoplasm) of wild-type oocytes. Following depolymerization of F-actin with Lat A, such that cortical F-actin becomes undetectable in the oocyte, Hom localization appears unchanged from the wild-type pattern in all oocytes, but Bif becomes highly diffuse with essentially no detectable enrichment at the cortex. This appears to be an effect on Bif localization and not stability, because the levels of the protein are not reduced as judged by Western blot analysis. Treating oocytes with colchicine, which disrupts the microtubules, did not affect either Hom or Bif localization in the oocyte. These findings raise the possibility that bif function might be dependent on intact F-actin; however, Hom localization is Lat A-insensitive, suggesting that its function in the oocyte may not require intact microfilaments. However, the possibility cannot be excluded that Lat A treatment allows for the retention of a small amount of the F-actin cytoskeleton, and this is stabilized in some way by Hom (Babu, 2004).
These data raise the possibility that there might be two processes, one that is microfilament-dependent and requires Bif, and another which is not dependent on intact microfilaments and requires Hom. Either process is sufficient to anchor the osk gene products to the posterior cortex of the great majority of the oocytes. One prediction of this hypothesis is that hom should be necessary to anchor posterior components in the absence of intact F-actin. Indeed, when hom single mutant oocytes were treated with Lat A, a large amount of cytoplasmic Osk was found at stage 9 and 10 near the posterior pole, and there was a large reduction in the Osk-ßGal signal. This could indicate that the loss of the longer Osk isoform may be the primary defect seen in Lat A-treated hom mutants; this longer Osk isoform is known to be essential for osk RNA and protein anchoring. The defects induced by depolymerizing F-actin in hom oocytes are similar to but more severe than those seen in bif;hom double mutant oocytes. This is probably due to the fact that F-actin disruption also leads to premature streaming in stage 9 and enhanced streaming in stage 10 oocytes, thus accentuating the effects of the loss of Osk anchoring at the posterior cortex (Babu, 2004).
The above results demonstrate that disruption of F-actin in the absence of hom function disrupts anchoring of the osk gene products. Similar results were obtained when hom mutants were treated with just CD or treated successively with Lat A and CD or vice versa. CD does not cause loss of F-actin as seen with phalloidin staining, and causes changes in the cortical F-actin as well as causing some of the F-actin to be seen in the cytoplasm. This latter effect is not seen with Lat A. This could be attributed to the difference in the mechanism of action between CD and Lat A. However, despite this difference between CD and Lat A, the effects of these drugs singly or in combination on Osk posterior anchoring are similar, causing mild defects in Osk posterior anchoring in only one-fifth of the treated wild-type oocytes and severe defects in the great majority of treated hom oocytes (Babu, 2004).
A second prediction is that disruption of microfilaments in the absence of bif should not affect anchoring of Osk. Indeed, most wild-type and bif single mutant oocytes treated with Lat A show largely wild-type anchoring of the Osk gene products, similar to Lat A treatment of wild-type oocytes. These results are consistent with the notion that Bif functions in an F-actin-dependent manner in maintaining Osk to the posterior of the oocyte (Babu, 2004).
If there are two independent mechanisms that act redundantly for normal Osk anchoring at the posterior cortex, then it would follow that the bif;hom double mutants in the absence of an intact F-actin cytoskeleton would show a phenotype similar to that of hom single mutants treated with Lat A, and not a more severe phenotype. This is indeed the case that is observed on testing osk RNA and Osk proteins in Lat A-treated double mutant oocytes (Babu, 2004).
In light of the finding that Hom posterior cortical localization remains unchanged following F-actin disruption, its ability to localize Osk to the posterior may be because it forms a complex with Osk. Co-immunoprecipitation experiments, using Drosophila ovarian extracts, indicate that Hom and Osk form a complex in vivo. Further, the stability of this complex is not dependent on an intact F-actin cytoskeleton (Babu, 2004).
The maintenance of Osk at the posterior of the oocyte may be mediated by two distinct mechanisms, either of which is sufficient, at least for the great majority of the oocytes. One mechanism does not require an intact F-actin cytoskeleton, and Hom seems to be an important player in this process. Hom can complex with Osk, and the stability of this complex is not dependent on an intact F-actin cytoskeleton. The second mechanism requires an intact F-actin cytoskeleton. Bif seems to be required for this mechanism. Overexpression of Bif can promote actin polymerization in cultured cells. Since it is also known that F-actin forms a complex with Bif in Drosophila embryonic lysates and that Bif binds directly to F-actin filaments in vitro, it is possible that Bif acts to stabilize actin filaments. In this scenario its absence may cause subtle changes in the F-actin cytoskeleton that may affect its capacity to anchor molecules at the cortex when hom is absent. In contrast, hom can function and is required to anchor Osk in the absence of an intact F-actin cytoskeleton or in the absence of bif function. Only when both mechanisms are disrupted in the oocyte, either through the simultaneous disruption of both hom and bif, or when F-actin is disrupted in the absence of hom, do the osk gene products fail to remain anchored to the posterior cortex (Babu, 2004).
Recent studies showed that Drosophila moesin is essential to link the cortical F-actin to the oocyte cell membrane. When moesin function is compromised, cortical F-actin can detach from the cell membrane and "fall" into the oocyte cytoplasm, and this results in the mislocalization of Osk. The effects of loss of moesin function on the localization of both Bif and Hom were examined; in these mutant oocytes, where the cortical F-actin detaches from the mutant oocyte cell membrane, components of both of the proposed anchoring pathways, Hom and Bif, also detach. These observations are consistent both with the Osk mislocalization phenotype seen in moesin mutant oocytes and with the model. It will be interesting to identify additional molecules involved in these separate pathways and to elucidate the mechanisms that are required to localize Hom to the posterior cortex of the oocyte in the absence of an intact actin cytoskeleton (Babu, 2004).
valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. vls encodes a divergent WD domain protein and the three available EMS-induced point mutations cause premature stop codons in the vls ORF. A null allele was identified that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules (Cavey, 2005).
Because all aspects of the vls mutant phenotype observed in embryos, including abdominal segment deletions, lack of pole cells, gastrulation defects and weak ventralization are rescued completely by a vls transgene and not even partially by a chk2 transgene, it is concluded that vls alone has a developmental requirement. Furthermore, chk2 function is only clearly required upon activation of cell cycle checkpoints. The vls phenotypes are reminiscent of a collapse of pole plasm assembly that seems to occur around stage 10 of oogenesis in vlsnull mutants. vas is crucial for the pole plasm to assemble properly and recruit the mRNAs and proteins required for pole cell specification and abdominal patterning. Genetic evidence implicates vas in the translational activation of several targets during oogenesis, including osk, grk and, in particular, nos at the posterior pole of the embryo. Vasa levels directly correlate with pole plasm activity, pole cell formation being more vulnerable to decreased Vasa levels than is abdominal patterning. Immunostaining for Vasa has been reported to show indistinguishable Vasa accumulation at the posterior pole of vls mutant and wild-type oocytes, and young embryos. These studies, performed with the homo- and hemi-zygous EMS mutants, showed a loss of posterior localization in the embryos from vls mothers sometime between fertilization and pole cell formation. The current study used vas-eGFP transgenes to assess the posterior localization of Vasa in vlsnull and hemizygous EMS alleles in detail. Maximal localization was still very weak and was found in oocytes and embryos from vlsEMS mothers. In vlsnull mutants a nearly complete failure to localize Vas-eGFP at the posterior pole was observed. This failure coincides with the collapse of the pole plasm and is probably the cause for the various embryonic phenotypes mentioned above. Consistent with this, the observed Vasa localization defects parallel the severity of the phenotypes of these vls alleles. The weak accumulation of Vasa at the posterior of vlsPG65 hemizygous oocytes gives rise to a grandchildless phenotype, whereas the almost complete absence of Vasa from the posterior of vlsnull oocytes results in a fully penetrant maternal-effect lethal phenotype (Cavey, 2005).
vls is thus required during oogenesis for the localization (transport or anchoring) of Vasa to the posterior cortex of the oocyte. The fact that Vls is not specifically enriched at the posterior may suggest that it acts to modify or transport pole plasm components before they reach the posterior pole. Preliminary experiments also failed to produce evidence that Vls and Vasa are part of the same protein complex. This suggests that the mode of action of vls on Vasa localization is transient or indirect. The fact that osk mRNA and protein are initially correctly localized implies that oocyte polarity is normal in vls mutants and that vls is not required for osk mRNA localization. Levels of Osk protein isoforms are then reduced in later stages and Western analysis reveals a much more drastic decrease of overall Osk levels than immunostaining does for both types of vls alleles. This suggests that most of the drop in Osk levels occurs during the late stages of oogenesis, when the vitelline membrane prevents antibody staining for oocyte Osk. Therefore, it seems that shortly after initiating pole plasm assembly, Osk fails to be maintained at the posterior of vls mutants and progressively disappears, concurrent with a complete collapse of the pole plasm (Cavey, 2005).
Several lines of evidence implicate the Short Osk isoform in directly anchoring Vas. Short Osk interacts strongly with Vasa in the two-hybrid system and recruits Vasa when ectopically localized in the oocyte. Because Vas-eGFP mis-localization patterns in stage 10 oocytes are indistinguishable in vls and osk54 mutants, vls could act directly at the level of Osk accumulation (e.g. in stimulating translation of osk), which is necessary for anchoring Vasa at the posterior pole. In contrast, it is also possible that vls acts primarily on Vasa protein localization. Because Vasa also seems to act in a positive feedback loop back on Osk protein accumulation, the lack of Vasa localization in vls mutants would then also preclude maintenance of posterior accumulation of Osk protein. In vls mutants, Osk levels appear to decrease just slightly after Vasa should have localized to the posterior pole, thus it appears that the failure to localize Vasa could be the cause of the pole plasm collapse in vls mutants. To investigate these issues further, Osk levels in vas and tud mutants were compared with those in vls mutants by Western analysis where a more significant drop was detected than by immunostaining. This analysis revealed generally stronger phenotypes for vls than for vas and tud mutants. A comparable decrease of Short Osk levels was observed on Western blots of vls, vas and tud mutant extracts, but with slight differences in the extent of reduction of the hyper- and hypo-phosphorylated forms, both of which are more severely affected in vls mutants. In addition, a clear reduction of Long Osk levels was observed in vls, a minor reduction in tud, but none in vas mutant extracts. However, this analysis is complicated by the fact that the vas and tud alleles that are useful and available, respectively, for these experiments, are not nulls. Their residual activity may therefore maintain Osk at the posterior for a longer period of time. These data are thus consistent with the idea that vls acts on either pathway target, Vasa or Osk, in a process which could involve additional intermediates that remain to be identified (Cavey, 2005).
The nuclear-cytoplasmic shuttling heterogeneous nuclear RNA-binding protein (hnRNP) Squid is required during Drosophila melanogaster oogenesis, where it plays a critical role in the regulation of the TGFalpha-like molecule Gurken (Grk). Three Sqd isoforms have been described, SqdA, S and B, and two of these, SqdA and SqdS, differentially function in grk mRNA nuclear export, cytoplasmic transport and translational control during oogenesis. Sqd is also required for the regulation of oskar mRNA, functioning in the cytoplasmic localization of the osk transcript. In oocytes from sqd females, osk mRNA is not efficiently localized to the posterior pole, but rather accumulates at the anterior cortex. Furthermore, anterior patterning defects observed in embryos from sqd females expressing only the SqdS protein isoform suggest that Sqd may also play a role in the translational regulation of the mislocalized osk mRNA. These findings provide additional support for models of mRNA regulation in which cytoplasmic events, such as localization and translational regulation, are coupled. These results also place Sqd among an emerging class of proteins, including such other members as Bruno (Bru) and Hrb27C/Hrp48, that function in multiple aspects of both grk and osk mRNA regulation during Drosophila oogenesis (Norvell, 2005).
The Drosophila body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. This study investigated the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. Exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. In vivo, Rab6 is found in a complex with Bicaudal-D, and Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila (Coutelis, 2007).
During polarized exocytosis, secretory vesicles emerging from the TGN are
targeted via molecular motors and cytoskeletal tracks to the plasma membrane,
where they are tethered. Subsequently, their fusion with the plasma membrane
permits the secretion of the vesicle contents, as well as the incorporation of
vesicular lipids and proteins into the plasma membrane, allowing membrane
growth and the establishment of specific domains. The exocyst complex plays a
crucial role in the incorporation of particular membranes and membrane
proteins at specific sites or in active domains of the plasma membrane.
Consistent with this, Drosophila sec5 mutant egg chambers display mislocalization of other exocyst components, cytoplasmic clusters of actin and a loss of plasma membranes. Thus, Sec5 protein is at the core of the exocyst complex in Drosophila, as is the case in yeast and in mammals (Coutelis, 2007).
Both sec5 null (sec5E10) and strongly affected
rab6D23D egg chambers display actin and general
organization defects, and arrest development during early oogenesis. Similarly, sec5 hypomorphic (sec5E13) and rab6D23D egg chambers that develop past stage 7 display phenotypes ranging from wild type to a loss
of nurse cell cortical actin and the concomitant presence of ring canal
clusters in the nurse cell cytoplasm. The striking parallel between the rab6 and
sec5 phenotypes, together with the finding that a loss of Rab6
affects Sec5 localization, suggests that the varying degrees of membrane loss
observed in rab6D23D egg chambers reflects the relative
reduction of exocyst-complex function in the egg chamber. Thus, during
Drosophila oogenesis, Rab6 promotes Sec5 localization and therefore
appears to be important for exocyst-complex organization and function.
However, consequent to loss of rab6 function, a striking difference was observed between nurse cells and oocyte in the severity of plasma membrane collapse and Sec5 mislocalization. It is hypothesized that the oocyte acts as a major source of membrane in rab6D23D egg chambers and/or that multiple exocytic pathways cooperate within the germline cyst to promote cyst development (Coutelis, 2007).
Differences in membrane content between the oocyte and the nurse cells, as
well as between the individual nurse cells, are observed as early as the
germarium stage in wild-type egg chambers. The fusome, a membranous
Spectrin-rich structure derived from the spectrosome, which itself is a
precursor organelle present in the germline stem cells, grows asymmetrically
through the ring canals during the divisions of the germline cyst, linking
each cystocyte. It is thought that the oocyte is the four-ring-canal cell
that retains the greater part of fusome during the first division. Furthermore, a Drosophila Balbiani body has recently been discovered, which, together with the fusome, organizes the specific enrichment of organelles in the oocyte throughout oogenesis. It is therefore possible that, in rab6 clones, in which the fusome
appears normal, such a mechanism of enrichment of organelles
in the oocyte concomitantly ensures that the concentration in the oocyte of
any perduring Rab6 protein, thus privileging the growth of the plasma membrane
of the oocyte over that of the nurse cells. Supporting this notion is the
observation that GFP-tagged Rab6 expressed in the germline is enriched in the
oocyte from the early stages of oogenesis (germarium region 2) onwards. Together, the combined actions of a residual Rab6-dependent and of additional Rab6-independent pathways might also permit most rab6D23D oocytes to maintain sufficient
vesicular trafficking to develop past stage 7 (Coutelis, 2007).
The stereotypic organization of affected rab6D23D egg
chambers at mid-oogenesis is striking. The oocyte is connected to open syncytia via its four ring canals, suggesting that the membranes linking nurse cells and oocyte are the most resistant. Furthermore, the
growth of the remaining membranes indicates that additional vesicular material
is delivered and incorporated into these plasma membranes. This suggests that,
in these rab6D23D egg chambers, sustained vesicle trafficking in the oocyte causes new membrane addition to the oocyte plasma membrane. It is hypothesized that, due to the continuity of the plasma membrane defining the cyst, the oocyte acts as a source of membrane that spreads by lateral diffusion throughout the plasma membrane of the cyst, allowing its growth (Coutelis, 2007).
It appears that Rab6-independent exocytic pathways also contribute to the
delivery of vesicular material to the plasma membrane in the
Drosophila egg chamber. Indeed, Syx1A is detected on the remaining
plasma membrane of both rab6-null and sec5 egg
chambers, supporting the existence of a Rab6- and Sec5-independent
exocytic pathway mediating protein export. This selective loss of Sec5 from
nurse cell membranes in rab6 open syncytia, together with the known
functions of the exocyst, suggest a simple explanation for the defects caused by a lack of Rab6 function in oogenesis. It is hypothesized that Rab6-dependent and -independent pathways might differ qualitatively in the proteins whose traffic they mediate, or
quantitatively in their relative contributions to the delivery of the same
cargo between nurse cells and oocyte. These differences may account for the
observed differential requirement for Rab6 in the localization of Sec5 in
nurse cell, versus oocyte, plasma membranes (Coutelis, 2007).
Our analysis has revealed two separate functions of Rab6: one is a general
role in the organization and growth of the egg chamber, and the other is its
specialized role in MT cytoskeleton polarization and oskar mRNA
localization. This second function appears specific to Rab6 because, in sec5 mutant egg chambers, Staufen localization is normal and the MT cytoskeleton is correctly organized. Only oskar mRNA, and not Oskar protein, is ectopically detected in
rab6D23D egg chambers. This suggests an
impairment of oskar mRNA localization, rather than a defect in its
anchoring, in which case Oskar protein would be detected with the detached RNA.
Defects in oskar mRNA localization, which relies on MT polarity,
could be due to a failure in the focusing of the MT cytoskeleton that is
observed in rab6 egg chambers (Coutelis, 2007).
In Drosophila and mammalian cells, BicD is known to regulate MT
organization. At mid-oogenesis, Rab6 and BicD cooperation could direct MT
organization and/or promote the vesicular transport necessary for oocyte
polarization and oskar mRNA localization. Given the implication of
membrane trafficking in the asymmetric localization of mRNAs, it also possible
that polarized membrane transport along the oocyte MT network directs
oskar mRNA to the posterior of the oocyte, by hitch-hiking along
trafficking vesicles (Coutelis, 2007).
In MDCK cells, definition of apical and basolateral plasma membrane domains
is required during polarization for the arrangement of MT along an
apical-basal axis. Vesicular trafficking is crucial to establish, specify and
maintain these membrane domains. By analogy, at stage 7, the polarizing signal from the posterior follicular cells to the Drosophila oocyte that causes repolarization of the MT cytoskeleton might do so by inducing the definition of anterior-lateral and
posterior membrane domains. It is therefore possible that, in
rab6D23D oocytes, as in epithelia, defects in vesicular
trafficking and TGN sorting underlie the observed defects in MT-network
organization. Consistent with this idea, a mispolarized MT cytoskeleton is
also observed in oocytes lacking Rab11. Thus,
vesicular trafficking and the specification of membrane domains may be
required for repolarization of the MT network and for the localization of
molecular determinants in the Drosophila oocyte at mid-oogenesis (Coutelis, 2007).
A mutant, maelstrom (mael), is described that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, Bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell (Clegg, 1997).
In Drosophila, the dorsal-ventral polarity of the egg chamber depends on the localization of the oocyte nucleus and the GurkenmRNA to the dorsal-anterior corner of the oocyte. Gurken protein presumably acts as a ligand for the Drosophila EGF receptor (torpedo/DER) expressed in the somatic follicle cells surrounding the oocyte. cornichon is a gene required in the germline for dorsal-ventral signaling. cornichon, gurken, and torpedo function in an earlier signaling event involving Gurken ligand and follicular EGF-Receptor. The earlier signaling event establishes the anterior-posterior polarity of the egg by establishing anterior follicle cell fate. Apparently anterior follicle cells signal back to the oocyte, since mutations in all three genes prevent the formation of a correctly polarized oocyte microtubule cytoskeleton required for proper localization of the anterior and posterior determinants Bicoid and Oskar and for the asymmetric positioning of the oocyte nucleus. In a subsequent signaling activity, the Gurken/EGF-R system determines dorsal-ventral polarity (Roth, 1995).
Unlike other bicaudal mutants, Oskar mRNA is localized correctly to the posterior pole of the oocyte in bullwinkle mutants at stage 10. By early embryogenesis, however, some Oskar mRNA is mislocalized to the anterior pole. Consistent with the mislocalization of Oskar mRNA, a fraction of the VASA protein and Nanos mRNA are also mislocalized to the anterior pole of bullwinkle embryos. Mislocalization of Nanos mRNA to the anterior is dependent on functional VASA protein. Although the mirror-image segmentation defects appear to result from the action of the posterior group genes, germ cells are not formed at the anterior pole. The bicaudal phenotype is also germ-line dependent for bullwinkle. It seems that Bullwinkle interacts with the cytoskeleton and extracellular matrix and is necessary for gene product localization and cell migration during oogenesis after stage 10a (Rittenhouse, 1995).
The mago nashi locus was sequenced and found to encode a 147 amino acid protein with no similarity to proteins of known or suspected function. Nonsense mutations in mago nashi, as well as a deletion of the 5' coding sequences, result in zygotic lethality. The original mago nashi allele disrupts the localization of Oskar mRNA and Staufen protein to the posterior pole of the oocyte during oogenesis; anterior localization of Bicoid mRNA is unaffected by the mutation. These results demonstrate that mago nashi encodes an essential product necessary for the localization of germ plasm components to the posterior pole of the oocyte (Newmark, 1994).
The D-elg gene encodes an ETS domain transcription factor that functions in Drosophila oogenesis. D-elg belongs to a small group of genes that are required for the formation of both the anterior-posterior and dorsoventral axes of the egg chamber. During oogenesis in D-elg mutant females, the spatial localization of Oskar and Gurken mRNAs in the oocyte is disrupted and a follicle cell enhancer trap marker identifies dorsoventral polarity defects. Specialized follicle cells, called border cells, fail to migrate from their anterior location to a position adjacent to the developing oocyte (Gajewski, 1995).
Although Bicaudal-C (Bic-C) is required for correct targeting of the migrating anterior follicle cells and for specifying anterior position, its primary action is oocyte based. BIC-C mRNA is expressed only in germline cells. BIC-C RNA is localized to the oocyte in early oogenesis, and later concentrates at its anterior cortex. The BIC-C protein includes five KH domains similar to those found in the human fragile-X protein FMR1. Alteration of a highly conserved KH domain codon by mutation abrogates in vivo Bic-C function. These results suggest roles for the BIC-C protein in localizing oocyte mRNAs and indicate that the effects on follicule cell migration is indirect (Mahone, 1995).
The female sterile (3) homeless gene of Drosophila is required for anterior-posterior and dorsoventral axis formation during oogenesis. Previtellogenic transport to the oocyte is not affected. Transport and localization of bicoid and oskar messages during vitellogenic stages are strongly disrupted. Examination of the microtubule structure with anti-alpha-Tubulin antibodies reveals aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork (Gillespie, 1995).
In both Drosophila and mammals, IkappaB kinases (IKKs)
regulate the activity of Rel/NF-kappaB transcription
factors by targeting their inhibitory partner proteins, IkappaBs, for
degradation. Mutations were identified in ik2, the gene that
encodes one of two Drosophila IKKs; the gene is
essential for viability. During oogenesis, ik2 is required in
an NF-kappaB-independent process that is essential for the localization
of oskar and gurken mRNAs; as a result, females that
lack ik2 in the germline produce embryos that are both
bicaudal and ventralized. The abnormal RNA localization in ik2
mutant oocytes can be attributed to defects in the organization of
microtubule minus-ends. In addition, both mutant oocytes and mutant
escaper adults have abnormalities in the organization of the actin
cytoskeleton. These data suggest that this IkappaB kinase has an NF-kappaB-independent role in mRNA localization and helps to link microtubule minus-ends to the oocyte cortex, a novel function of the IKK family (Shapiro, 2006).
Protein kinases of the IkappaB kinase (IKK) family are known for their roles in innate immune response signaling pathways in both mammals and Drosophila. Mammalian IKKs all have roles in immune responses, but have a variety of targets. and IKKβ were identified in a protein complex that phosphorylates IkappaB and targets it for degradation, thereby allowing the nuclear localization and activation of NF-kappaB transcription factors. Gene targeting experiments in the mouse demonstrated that IKKβ, but not IKKalpha, is required for NF-kappaB activation by pro-inflammatory stimuli through receptors such as TLR4. IKKα activates the Rel/p52 transcription factor, because it activates proteolytic processing of the p100 precursor of p52 in an IkappaB-independent process. IKKepsilon and TANK binding kinase 1 (TBK1) are required to phosphorylate and activate the transcription factor Interferon regulatory factor 3 (IRF3) in response to viral infection. In addition to these immune response functions, IKKα has an NF-kappaB-independent role in epidermal differentiation and limb development (Shapiro, 2006 and references therein).
Dorsoventral patterning of the Drosophila embryo relies on the
activation of Dorsal, a Rel-family transcription factor, by a
signaling pathway that is homologous to mammalian TLR pathways. In response to activation of the receptor Toll, Cactus (the Drosophila IkappaB) is degraded, which allows Dorsal to move to embryonic nuclei and activate genes, such as twist, that are required for specification of ventral cell
types. Phosphorylation of Cactus is required for its degradation, but the responsible kinase has not been identified. The
Drosophila genome encodes two members of the IKK family.
DmIkkβ (ird5 - FlyBase) is essential for the response
to bacterial infection. DmIkkβ is required for proteolytic processing and activation of Relish, a p100-like Rel/ankyrin-repeat protein, like the role of mammalian IKKα in the activation of p100. The function of the second
Drosophila protein kinase of the IKK family, ik2
(IkappaB kinase-like 2), has not been characterized, but it was
a good candidate to control the phosphorylation and degradation of
Cactus (Shapiro, 2006).
To test whether ik2 encodes a Cactus kinase, the phenotypes caused by loss of ik2 function were characterized. This study presents data showing that Ik2 is essential for dorsoventral and
anteroposterior embryonic patterning of the Drosophila embryo.
However, Ik2 does not act as a Cactus kinase, but exerts its effects
on embryonic patterning through the localization of specific mRNAs
during oogenesis. The data indicate that Drosophila Ik2
regulates RNA localization through regulation of the cytoskeleton and
define a novel function for this protein family (Shapiro, 2006).
A saturation mutagenesis experiment had identified
lethal complementation groups in polytene chromosome region 38E, the region that includes the ik2 gene. Missense mutations were identified in the ik2 kinase domain in all five alleles of the l(2)38Ea complementation group. A sixth allele, ik2alice, identified in a genetic mosaic screen for maternal effect mutations, had a
missense mutation in the C-terminal end of the kinase domain. All six
ik2 alleles caused recessive lethality, and the majority of
the mutants die as first instar larvae. At low temperature and in
uncrowded culture conditions, rare escaper adults (<1%) were
observed, but they died shortly after eclosion (Shapiro, 2006).
If ik2 encoded the Cactus kinase, embryos
produced by females that lack ik2 would not be able to degrade
Cactus, so Dorsal would not enter embryonic nuclei to activate genes
required for ventral cell fate specification and the embryos would
be dorsalized. Because ik2 mutations were lethal,
FRT/FLP recombination combined with the ovoD
dominant female-sterile mutation were used to generate mutant clones in the
female germline. More than
95% of the embryos laid by ik2alice and
ik21 mutant females did not hatch; however, larval
cuticle preparations showed that none of the embryos were
dorsalized. Instead, the majority of embryos produced by
ik2alice and ik21 mutants had a
bicaudal phenotype, ranging from headless embryos to embryos with a duplicated abdomen in place of the head and thorax.
In addition to this anteroposterior patterning defect, a large number
of embryos from both ik2alice and ik21
germline clones had expanded ventral cuticular structures, the
opposite of the expected phenotype. Some embryos were both
ventralized and bicaudal, with expanded ventral denticle bands and
filzkörper (a posterior structure) in both the tail and the anterior of the embryo. Both ik2 alleles produced bicaudal and ventralized embryos, but 89% of the embryos produced by ik2alice mutant females were bicaudal with no apparent dorsoventral abnormalities, whereas only 47% of embryos produced by ik21 mutant females were bicaudal, and the remainder of the embryos appeared to be too ventralized to score for ectopic posterior cuticular structures. A similar range of phenotypes was observed in embryos produced by ik22, ik23 and ik25 females with mutant germline clones (Shapiro, 2006).
Contrary to the prediction based on its sequence, ik2 does not act as a
Cactus/IkappaB kinase in the Drosophila embryo. The embryonic
ventralization caused by loss of ik2 is the opposite of the
phenotype predicted for a Cactus kinase, and all effects of
ik2 on the dorsoventral pattern of the embryo can be explained
by a loss of activity of the Grk/Egfr pathway during oogenesis.
Embryos that lack maternal activity of both Drosophila IKKs,
Ik2 and DmIkkβ, are ventralized and are indistinguishable from
ik2 single mutants, which rules out the
possibility that the Drosophila IKKs act in both the Grk/Egfr
and Toll pathways, and indicates that an unidentified kinase of
another family is required to target Cactus for degradation.
Additional experiments will be required to test whether ik2
plays other roles in the immune response (Shapiro, 2006).
It was found that instead of playing a role in Cactus degradation,
Drosophila ik2 is required for the localization of specific
mRNAs during oogenesis. Both the actin and microtubule cytoskeletons
are disrupted in ik2 mutants, and defects in microtubule-based
transport are sufficient to account for the defects in mRNA
localization seen in ik2 mutants. Because Drosophila
ik2 is specifically required for organization of the oocyte cytoskeleton, the results raise the possibility that some of the NF-kappaB-independent roles of the mammalian IKKs may act through the cytoskeleton (Shapiro, 2006).
The embryonic patterning defects caused by the loss of ik2
function are due to the failure to transport all osk mRNA to
the posterior pole of ik2 mutant oocytes, which leads to
bicaudal embryos, and failure to localize grk mRNA to the
dorsal anterior of the oocyte, which leads to ventralized embryos.
Loss of ik2 has a milder effect on bcd mRNA
localization; bcd is correctly localized in most oocytes, but is not tightly restricted to the anterior pole in a minority of cases (Shapiro, 2006).
Many lines of evidence indicate that osk localization to the
posterior pole depends on kinesin and gurken localization to
the dorsoanterior corner depends on dynein. However,
the kinesin and dynein motors in the oocyte are interdependent. For
example, posterior localization of dynein and the anterodorsal localization of gurken are both disrupted in Khc mutants, and
hypomorphic Dhc mutants have a reduced amount of Khc-β-gal at the posterior pole. Both motor systems are at least partially functional in ik2 mutants: most oskar is localized to the posterior pole of the
oocyte (a kinesin-dependent process) and grk mRNA is localized
anteriorly (a dynein-dependent process) (Shapiro, 2006).
Several lines of evidence suggest that the RNA localization defects seen in ik2 oocytes are associated with defects in a subset of dynein-mediated, minus-end-directed transport processes. The movement of grk mRNA to the dorsoanterior corner of the oocyte depends on two sequential dynein-based movements: grk mRNA moves first to the anterior of the oocyte along microtubules with plus-ends at the posterior pole and minus-ends at the anterior, and then moves dorsally on microtubules with minus-ends that form a cage around the oocyte nucleus. The dorsal movement of grk mRNA is specifically blocked in ik2 mutants, which would be consistent with a failure in this
dynein-based movement. Restriction of bcd mRNA to the anterior
margin of the oocyte, which is disrupted in some ik2 oocytes,
depends on the swallow gene product, which binds dynein light
chain. Overexpression of dynamitin disrupts dynein function and causes
changes to the localization of grk and bcd mRNA that are similar to the phenotype of ik2 oocytes. In
addition, BicD mutations produce a maternal effect phenotype
similar to that of ik2. BicD is part of a protein complex with
dynein light chain in early oocytes, neuroblasts and the early embryo, and has
been proposed to link cargo to microtubules in both Drosophila
and mammalian cells (Shapiro, 2006).
Although this evidence links ik2 to a dynein transport system, the most penetrant phenotype in ik2 mutants is osk
mislocalization and subsequent production of bicaudal embryos, a
kinesin-dependent process. However, loss of ik2 function, like
the BicD gain-of-function mutations, does not eliminate
kinesin function, because the majority of osk mRNA accumulates
at the posterior pole. Because the kinesin and dynein motors in the
oocyte are interdependent, osk mRNA mislocalization could be caused by a decreased kinesin activity that is secondary to dynein disruption (Shapiro, 2006).
In addition to defects in minus-end-directed transport, the organization of the microtubules is also perturbed in ik2 oocytes. The plus-ends of microtubules are localized correctly to the posterior pole of the oocyte. However, there are abnormal aggregates of microtubules around the oocyte nucleus, where a population of microtubule minus-ends is normally anchored, and the
microtubule minus-end marker, Nod-β-gal, is not localized at the
anterior of the oocyte. These defects suggest that abnormal
organization of microtubule minus-ends during mid-oogenesis could be
the basis of the defect in minus-end-directed transport (Shapiro, 2006).
The adult bristles and ovaries of ik2 mutants also displayed
abnormalities in the actin cytoskeleton. The bristle defects are
nearly identical to those caused by mutations in actin-associated
proteins, or to bristles that were treated with F-actin-inhibitors.
Bristles contain a central core of microtubules, but mutations in
the dynein heavy chain gene Dhc64C or treatment with drugs
that disrupt microtubule dynamics do not cause bristle
phenotypes like the thick, branched bristles seen in ik2
mutants. The actin cytoskeleton of the oocyte is also disrupted in
ik2 mutants, with ectopic sites of actin polymerization in the
ooplasm. These actin defects are distinct from those caused by
mutations that affect nurse cell ring canal actin, which suggests that actin organization is not globally disrupted in ik2 mutants and that the actin defects are restricted to the oocyte cortex (Shapiro, 2006).
Recent data have defined two sets of microtubules in the oocyte that are both nucleated from minus-ends at the centrosome associated with the oocyte nucleus; one set remains associated with the oocyte nucleus, whereas the remaining microtubules shift their minus-ends from the oocyte to the cortex. It was
suggested that translocation of the minus-ends of the latter set of
microtubules to the cortex could depend on actin and motor proteins. The current
data suggest that anchoring of microtubule minus-ends to the
oocyte cortex depends upon an Ik2-dependent interaction of
microtubule minus-ends with the F-actin network, analogous to the
interaction of microtubule plus-ends with the actin cytoskeleton
through microtubule tip proteins (Shapiro, 2006).
The phenotypes of ik2 in the ovary and adult bristles are very
similar to those caused by mutations in spn-F. Like ik2 mutations, null mutations in spn-F affect the
localization of osk and grk mRNAs during oogenesis, and
cause bicaudal and ventralized embryos. spn-F mutant oocytes
have ectopic sites of F-actin polymerization, and spn-F
bristles are similar to ik2 mutant bristles. Ik2 and Spn-F
have been shown to interact in a yeast two-hybrid screen, which
suggests that these proteins can form a complex. Spn-F associates
specifically with microtubule minus-ends. It is therefore proposed that Ik2 and Spn-F act together to regulate interactions between the minus-ends of microtubules and the actin-rich cortex (Shapiro, 2006).
mRNA export from the nucleus requires the RNA helicase UAP56 (Helicase at 25E) and involves remodeling of ribonucleo-protein complexes in the nucleus. This study shows that UAP56 is required for bulk mRNA export from the nurse cell nuclei that supply most of the material to the growing Drosophila oocyte and for the organization of chromatin in the oocyte nucleus. Loss of UAP56 function leads to patterning defects that identify uap56 as a spindle-class gene similar to the RNA helicase Vasa. UAP56 is required for the localization of gurken, bicoid and oskar mRNA as well as post-translational modification of Osk protein. By injecting grk RNA into the oocyte cytoplasm, this study shows that UAP56 plays a role in cytoplasmic mRNA localization. It is proposed that UAP56 has two independent functions in the remodeling of ribonucleo-protein complexes. The first is in the nucleus for mRNA export of most transcripts from the nucleus. The second is in the cytoplasm for remodeling the transacting factors that decorate mRNA and dictate its cytoplasmic destination (Meignin, 2008).
UAP56 is a conserved member of the DExH/D RNA helicase superfamily implicated in many aspects of RNA metabolism including general mRNA export from the nucleus. In human cells, UAP56 is preferentially associated with spliced mRNA and has a major role in bulk mRNA export (Gatfield, 2001). Furthermore, Xenopus UAP56 and its yeast homologue Sub2p are thought to be required co-transcriptionally for the recruitment of the mRNA export factor Aly/REF (see Drosophila Aly) to mRNA. In yeast, the THO complex, which functions in transcription elongation, interacts with mRNA export factors to form the TREX (TRanscription/EXport) complex, linking transcription and export. The human THO complex becomes associated with spliced mRNA during the course of splicing. In Drosophila cells, UAP56 has been shown to be essential for the export of both spliced and intronless poly(A)+ mRNAs. Drosophila uap56 is an essential gene that was first identified as an enhancer of position effect variegation and encodes a nuclear protein named Hel25E/UAP56. UAP56 is proposed to promote an open chromatin structure by unwinding or releasing the mRNA from the site of transcription. It is also thought to be involved in regulating the spread of heterochromatin (Meignin, 2008).
During Drosophila oogenesis, the antero-posterior and dorso-ventral axes of the future embryo are specified through the cytoplasmic localization and translational regulation of a large number of specific transcripts. The most extensively studied mRNAs are gurken (grk) that encodes a TGFβ signal, bicoid (bcd) that encodes the anterior morphogen, and oskar (osk), which specifies posterior structures and the future germ line. All transcripts in the oocyte are thought to be transcribed in the nurse cell nuclei and transported through actin-rich ring canals into the oocyte, where they are selectively localized by transport on microtubules (MTs) by molecular motors. Some of these mRNAs are then localized within the oocyte cytoplasm. During their complex path of localization, these transcripts are thought to be present in large RNP complexes that contain a variety of RNA binding proteins. The composition of these complexes is thought to vary during the different steps of the biosynthesis, export and localization of the transcripts, and RNA helicases play essential roles in remodeling the RNPs during RNA processing, transport, localization, anchoring and translation (Meignin, 2008).
RNA helicases are also likely to be involved in remodeling a diverse range of trans-acting factors required for mRNA localization. These include cytoplasmic determinants and motor cofactors such as BicaudalD (BicD) and Egalitarian (Egl) as well as nuclear components such as hnRNPs or splicing factors. Such trans-acting factors are thought to dictate which molecular motor the transcripts associate with and therefore their cytoplasmic destination. For example, in the Drosophila oocyte, oskar (osk) mRNA requires Kinesin 1 to transport it to the plus ends of MTs while grk mRNA requires cytoplasmic Dynein (Dynein) to transport it to the minus ends of MTs (Meignin, 2008).
In some cases, factors recruited to the RNA in the nucleus remain with the RNA and function in the cytoplasm. For example, the nuclear exon-exon junction components (EJC) Mago nashi, Y14 and eIF4AIII are recruited by osk transcripts in the nucleus and then play a role in its cytoplasmic localization. Once at its final destination at the posterior pole, osk mRNA is translated using two distinct initiation codons, resulting in two different proteins. The short form of Osk promotes the assembly of polar granules by recruiting Vasa, a member of DExH/D-box family of putative RNA helicases at the posterior pole of the oocyte. Vasa is also required for promoting translation of grk through an interaction with the translation factor eIF5B/dIF2. In contrast, grk transcripts are able to recruit in the cytoplasm all the factors required for their localization (Meignin, 2008).
In Drosophila, at least 12 genes are members of the DExH/D RNA helicase superfamily. One of the best described, vasa, was originally identified as a member of the posterior class of maternal effect genes. Vasa is required for the translation of osk and nos mRNAs during the assembly of the pole plasm and for the localization and translation of grk mRNA during oogenesis. Other RNA helicases play a role during Drosophila oogenesis; belle (bel), hel25E or uap56, spindle E (spn-E) and eIF-4AIII. In addition, the small repeat-associated siRNAs (rasiRNAs) pathway, containing spn-E, aubergine and armitage, is required for axis specification. In many of these cases, the intracellular localization and translational regulation of bcd, osk and grk mRNA are affected to varying degrees (Meignin, 2008).
This study tested whether the RNA helicase UAP56 is required in the cytoplasm for mRNA localization and post-translational modification in addition to its well-studied roles in the nucleus in splicing and mRNA export. By creating new alleles of the gene, it was shown that uap56 is a spindle-class gene. uap56 mutants have strong Dorso-ventral egg shell defects caused by a mislocalization of grk mRNA and its incorrect translational regulation. grk mRNA injected into the oocyte cytoplasm of uap56 mutants fails to localize correctly, suggesting that the uap56 phenotype is due to a lack of a factor required in the cytoplasm for mRNA localization. It was also shown that UAP56 plays a role in osk and bcd mRNA localization and Osk post-translational modification. Thus UAP56 plays multiple roles in mRNA localization and post-translational modification in the oocyte cytoplasm. It is proposed that UAP56 is required for remodeling of cytoplasmic RNP complexes required for mRNA localization and post-translational modification (Meignin, 2008).
UAP56 is an RNA helicase that has been shown to play important roles in mRNA metabolism in the nucleus. In Drosophila, UAP56 is a component of the Exon Junction Complex (EJC) (Gatfield, 2001) and is required in tissue culture cells for bulk mRNA export from the nucleus. Using new mutations in the gene, this study has shown that UAP56 is also required for bulk mRNA export during Drosophila oogenesis. UAP56 also has a novel and unexpected role in mRNA localization and post-translational modification in the cytoplasm since uap56 mutants have defects in grk, osk and bcd mRNA localization and Osk post-translational modification. uap56 mutants show strong dorso-ventral egg shell defects due to disruption in cytoplasmic transport of grk mRNA, in addition to other phenotypes that display phenotypes that define uap56 as a spindle-class gene, like the RNA helicase and posterior group gene vasa. Therefore, these data have uncovered a new cytoplasmic role for UAP56 in mRNA transport and post-translational modification. It is proposed that UAP56 is required in the oocyte cytoplasm to remodel RNP complexes involved in mRNA localization and post-translational modification (Meignin, 2008).
The analysis of new alleles of the uap56 gene has revealed that UAP56 is required for general nuclear mRNA export from the nurse cell nuclei. This explains why it was impossible to study the uap56 null mutants during oogenesis since a total block in mRNA export causes cell lethality. The current observations are in good agreement with the previous work showing that UAP56 is essential for bulk mRNA export in Drosophila tissue culture cells and other model systems. In wild-type, the intracellular distribution of bulk polyadenylated RNAs reveals a cytoplasmic localization with an accumulation in the Nuage. In contrast, this distribution is disrupted in uap56 mutants. During Drosophila oogenesis, the Nuage is characterized by electron dense germ line specific structures that form around the nurse cell nuclei. It is suggested that the Nuage someway facilitates the assembly of mRNP particles that mediate the transport, translational regulation and storage of specific transcripts. The Nuage contains Vasa, Maelstrom, Aubergine and Belle, all thought to be required for axis specification in the oocyte. Most components of the Nuage are also localized at the posterior pole of the Drosophila oocyte. This study found that poly(A)+ RNA is present at high levels in particles in the Nuage and that UAP56 is involved in the formation of the Nuage since Vasa protein is absent from the Nuage in uap56 mutants. However, UAP56 is not specifically localized in the Nuage nor at the posterior pole, so UAP56 is neither a posterior group gene nor a component of the Nuage. These observations are interpreted as indicating that UAP56 is required upstream of Nuage and pole plasm formation. It is proposed that UAP56 is required to facilitate the correct assembly of different mRNAs, including grk and osk with the appropriate RNA binding proteins in RNPs, before, during and possibly after export from the nurse cell nuclei. This assembly is crucial for the downstream events responsible for the transport and assembly of the RNPs into the Nuage (Meignin, 2008).
Interestingly, a small fraction of poly(A)+ RNA was found in the oocyte nucleus, an observation that could be explained in two possible ways. Either a sub-population of RNA is transcribed in the oocyte nucleus or a sub-population of poly(A)+ RNA is transported from nurse cells to oocyte and then imported into the oocyte nucleus. The second explanation is favored for the following reasons. First, it is thought that the oocyte nucleus is transcriptionally inactive. Second, the I factor mRNA is known to be synthesized in the nurse cells, transported into the oocyte and then imported into the oocyte nucleus. While the genetic background that was studied is not very active for I factor transcription, it is possible that the poly(A)+ RNA detected in the oocyte nucleus represents transcripts of other transposable elements or some endogenous transcripts that follow the same pattern of biogenesis and import into the oocyte nucleus (Meignin, 2008).
During oogenesis and early embryogenesis, the majority of UAP56 protein is present in the nucleus and is probably associated with DNA. These results are consistent with previous work showing that UAP56 is closely associated with salivary gland chromosomes and localized to the nuclei of Drosophila embryos and ovaries as well as acting as an enhancer of position affect variegation in Drosophila and affecting heterochromatic gene expression in yeast. These data together with the conclusion that uap56 is a spindle-group gene strongly suggest that the gene is involved in chromatin organization (Meignin, 2008).
In yeast, Sub2p associates with the TREX (TRansport-EXport) complex, which is required directly for transcription elongation, splicing and export and is recruited during transcription. In contrast, the mammalian TREX complex is recruited during splicing. It is proposed that, in Drosophila, UAP56 has an intermediate role between yeast and human cells. It is likely to bind mRNA during transcription and be involved in splicing in a similar manner to nonsense-mediated mRNA decay (NMD) (Meignin, 2008).
The new alleles of uap56 reveal a role of UAP56 in mRNA localization and translational modification in the cytoplasm, key processes in axis specification. A reduction in the intensity of tau GFP staining in the oocyte was found, although the antero-posterior gradient, which is essential for mRNA localization, was unaffected. While the possibility cannot be excluded that the reduction in efficiency of localization of injected grk mRNA is due to a lower density of MTs in the oocyte, the alternative interpretation is favored, in which UAP56 plays a more direct cytoplasmic role in promoting remodeling of factors required for grk RNA localization (Meignin, 2008).
The observations in the context of the previous work on UAP56 suggest that, as well as being a general RNA export factor in Drosophila, the protein has a specific role in the localization and post-translational modification of a sub-population of key transcripts. In this respect, UAP56 is similar to some other components of the EJC, such as Mago nashi, Y14 and eIF4AIII, which are involved in mRNA localization and post-translation control of a subset of Drosophila transcripts. Interestingly, mutants in the small repeat-associated siRNAs (rasiRNAs) pathway also show similar defects in axis specification to uap56 mutants, raising the possibility that UAP56 is a component of the rasiRNA pathway. Nevertheless, the results are surprising, given that UAP56 is thought of as a ubiquitous house keeping gene with an essential function in the export of all mRNAs. A partial loss-of-function of an equivalent essential general mRNA export factor, NXF1, does not give rise to specific developmental defects, such as the ones observed with uap56 alleles. rasiRNA mutants show an accumulation of double strand breaks in egg chambers. However, no differences were found in the accumulation of H2A staining of double strand breaks between wild type and mutant germaria, in contrast to armi, aub and spn-D mutants. It is concluded that the defects observe in axis specification in uap56 mutants are not due to double strand breaks perturbing signaling in the germ line. Therefore, it is proposed that, unlike NXF1, UAP56 is likely to remain on the RNA after export and has a role in the remodeling of RNP complexes in the cytoplasm. This idea is supported by the fact that UAP56 is detected in the cytoplasm as well as the nucleus. However, no UAP56 colocalized is observed with mRNA in the cytoplasm or recruited by injected RNA. These observation are interpreted as indicating that UAP56 may only associate with RNA transiently in the cytoplasm, while acting as a cytoplasmic RNA remodeling factor. Such a role is novel for UAP56, and it remains to be discovered whether it is a general feature of this RNA dependent helicase in a variety of model systems (Meignin, 2008).
oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, it was showm that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. Each component of the oskar mRNA complex is shown to play a distinct role in particle formation and transport (Zimyanin, 2008).
The mechanism of osk mRNA localization has been controversial, and a number of competing models have been proposed to explain its targeting to the posterior. This study observed directly how the RNA travels to the posterior by tracking the movements of osk mRNA particles at high temporal resolution in living oocytes. Surprisingly, the results are incompatible with the existing models, leading to proposal of a new mechanism for the localization of the mRNA (Zimyanin, 2008).
(1) It is clear that the mRNA is not transported in a highly directed fashion toward the posterior since the particles move in all directions with only a slight posterior bias. (2) The rapid transport of the osk mRNPs argues against a role for passive diffusion. (3) The results are inconsistent with the two-step model for osk mRNA localization, in which kinesin first transports the RNA away from the anterior and lateral cortex to the oocyte center before it is translocated to the posterior in a second step. osk mRNP particles show a similar behavior in all regions of the oocyte at stage 9, with a consistent small excess of particles moving posteriorly, and this is incompatible with the idea that particles are first transported to the center. Moreover, slow kinesin mutants have an identical effect on the speeds of particle movements in all regions of the oocyte, strongly arguing that kinesin transports the mRNA in a one-step pathway all of the way to the posterior pole (Zimyanin, 2008).
Instead, the data suggest that osk mRNA is localized by a biased random walk, in which each particle undergoes a large number of active movements in many different directions, with a small excess of movements toward the posterior. After hundreds of movements, the 14% excess of posterior movements results in a large net posterior displacement that delivers the mRNA to its destination. Given that 13% of particles are moving at any one time, the average osk mRNP will undergo a net posterior displacement during the 6-10 hr of stage 9 of 112-187 microm (6-10 × 3600 s × 0.13 × 0.04 microm/s). Since this is more than 1.5 times the length of the oocyte (80 microm), this is more than sufficient to produce a robust posterior localization of osk mRNA by the end of stage 9 (Zimyanin, 2008).
This model is supported by the observation that the direction of the bias correlates perfectly with the site of osk mRNA accumulation: wild-type oocytes show a posterior bias and posterior localization of the mRNA, whereas the bias is reversed in mago, TmII, and btz mutants, and osk mRNA accumulates at the anterior. The effectiveness of the biased random walk in localizing the RNA is even more clearly demonstrated by stau mutants: most of the mRNA is trapped at the anterior of stau mutant oocytes, and the mRNA that is released into the oocyte cytoplasm moves four times less frequently than in wild-type. Nevertheless, the small number of movements that occur show a normal posterior bias, which leads to a transient posterior enrichment of the mRNA that is lost at later stages because the RNA is not anchored (Zimyanin, 2008).
Similar biased bidirectional transport has been described for lipid droplets in the Drosophila embryo and for many other particles and organelles in other systems. In most cases, the bias depends on the competing activities of motors that move in opposite directions. By contrast, the results indicate that the vast majority of osk mRNA movements are directed toward microtubule plus ends and are mediated by kinesin. (1) When the microtubules are aligned around the cortex by premature cytoplasmic streaming, over 80% of fast-moving particles move in the same direction as the cytoplasmic flows, i.e., toward the plus ends. (2) More than 80% of movements are abolished by null mutations in the Khc. (3) Point mutations in kinesin reduce the speed of particle movements in all directions. Fourth, particles have never been observed that show a clear 180° reversal in the direction of their movement out of more than 3000 particle tracks analyzed, indicating that particles rarely switch between plus and minus end-directed motion (Zimyanin, 2008).
The traditional view of the oocyte microtubule cytoskeleton is that it is polarized along the anterior-posterior axis with minus ends at the anterior and plus ends at the posterior. This view is based on the localization of fusion proteins containing the motor domains of Nod and kinesin to the anterior and posterior of the oocyte, respectively, and the assumption that these act as minus and plus end markers. The microtubule organization appears much more complex, however, when visualized directly: the microtubules appear to be nucleated from both the anterior and lateral cortex and extend in all directions to form an anterior-posterior gradient. The data are consistent with this latter view because osk mRNA particles move in all directions in every region of the oocyte. More importantly, because almost all osk mRNA movements are plus end directed, each RNA track provides a snapshot of the polarity of a microtubule segment. The observation that 57% of tracks have a net posterior vector therefore indicates that the microtubules have only a weak orientation bias toward the posterior. Even if all 10%-20% of kinesin-independent osk mRNA movements are minus end directed, this would still give a posterior bias in microtubule polarity of only 62%. Thus, the data suggest a revised view of the organization of the cytoskeleton, in which the microtubules extend in all directions from the anterior and lateral cortex, with about a 20% excess of microtubules with their plus ends pointing posteriorly. One appealing aspect of this model is that it can reconcile the two opposing views of the microtubule organization. Kinβgal is an unregulated motor that constitutively moves toward the plus ends of microtubules, and it is proposed that it accumulates at the posterior by following a biased random walk similar to osk mRNA. According to this view, it is not a marker for microtubule plus ends but for regions where plus ends are most enriched (Zimyanin, 2008).
Mutants in different components of the osk mRNA localization complex produce very similar phenotypes when analyzed by in situ hybridizations to fixed samples. However, they have different effects on the dynamics of osk mRNA particles. First, hrp48 mutants abolish the formation of visible osk mRNA particles, indicating a requirement for this HnRNPA/B-like protein in the assembly of functional transport particles (Zimyanin, 2008).
Mutants in the EJC components, Mago nashi and Btz, do not affect osk mRNP particle formation but reduce the frequency of particle movement and reverse the bias, so that the particles accumulate at the oocyte anterior. This behavior is what one would expect if the movement is primarily mediated by a minus end-directed motor. Furthermore, the anterior accumulation of osk mRNA in these mutants resembles that of many other mRNAs that are transported into the oocyte by the dynein/Bic-D/Egl pathway, and which are thought to localize to the anterior by default, because this pathway remains active in the oocyte. Thus, the EJC may be required to turn off the dynein/Bic-D/Egl pathway when osk mRNA enters the oocyte so that it can then associate with the kinesin pathway (Zimyanin, 2008).
TmII mutants have the same effects on osk mRNP dynamics as EJC mutants, suggesting that Tropomyosin is required for the same step in localization. This raises the possibility that Tropomyosin plays a role in either the recruitment of the EJC to osk mRNA or the subsequent activity of the EJC in switching from the anterior to the posterior localization pathway (Zimyanin, 2008).
Stau seems to function downstream of the EJC since osk mRNA is either trapped at the anterior or moves with a normal posterior bias toward the posterior pole. The results suggest that Stau regulates several aspects of osk mRNA behavior once it enters the oocyte. First, it seems to be required for the efficient release of the mRNA from the anterior. This may reflect a role of Staufen in the coupling of the mRNA to the kinesin-dependent posterior transport pathway. osk mRNA particles that escape the anterior move with a normal bias but a reduced frequency, suggesting that Stau is also required for full kinesin activity. Finally, Stau is essential for the activation of osk mRNA translation once the mRNA has reached the posterior pole. Thus, the in vivo analysis of osk mRNA dynamics reveals that different components of the osk mRNP complex are required for at least three distinct steps in the localization pathway, namely particle formation, uncoupling from the dynein/BicD pathway, and release from the anterior and coupling to the kinesin pathway, and this may begin to explain why so many trans-acting factors are required for the localization of this mRNA (Zimyanin, 2008).
The dynamics of osk mRNA particles have several features in common with the behavior of MS2-labeled mRNAs in mammalian cells. Fusco (2003) found that RNA particles undergo stochastic movements in COS cells, in which they switch between fast microtubule-dependent movements, diffusion, and stationary phases. Furthermore, an RNA containing the β-actin localization signal showed a 5-fold higher frequency of fast movements than a random RNA. This is very similar to the behavior of osk mRNA, which undergoes fast, direct movements 4-5 times more frequently in wild-type oocytes than in EJC, TmII, and stau mutants. MS2-GFP has also been used to image CamKIIα mRNA in the dendrites of cultured neurons and labels particles that show similar bidirectional movements to osk mRNA. Since dendrites contain microtubules of mixed orientations and Stau, Barentsz, and kinesin have been implicated in dendritic mRNA localization, it will be interesting to determine whether CamKIIα mRNA localizes by a biased random walk similar to osk mRNA (Zimyanin, 2008).
During Drosophila oogenesis, Oskar mRNA is transported to the posterior pole of the oocyte, where it is locally translated and induces germ-plasm assembly. Oskar protein recruits all of the components necessary for the establishment of posterior embryonic structures and of the germline. Tight localization of Oskar is essential, as its ectopic expression causes severe patterning defects. This study shows that the Drosophila homolog of mammalian Lasp1 protein, an actin-binding protein previously implicated in cell migration in vertebrate cell culture, contributes to the accumulation of Oskar protein at the posterior pole of the embryo. The reduced number of primordial germ cells in embryos derived from lasp mutant females can be rescued only with a form of Lasp that is capable of interacting with Oskar, revealing the physiological importance of the Lasp-Oskar interaction (Suyama, 2009).
This study shows that the Drosophila homolog of mammalian Lasp1
interacts via its SH3 domain with Oskar, binds directly to F-actin in vitro,
and colocalizes with F-actin in vivo. Western analysis of lasp mutant
oocytes shows a slight reduction in Oskar levels that is not readily
visualized by in situ hybridization. Posterior oskar mRNA maintenance
in the ovary, which depends on Oskar protein, also appears to be relatively
normal. However, in the embryos laid by lasp mutant mothers
(lasp mutant embryos), oskar protein and mRNA at the
posterior pole are strongly reduced. The overlap of Oskar, Lasp and actin
distributions at the posterior pole during oogenesis and early embryogenesis, as well as the actin-and Oskar-binding properties of Lasp, suggest that Lasp connects Oskar to actin. This is further supported by the genetic interaction between oskar and lasp,
and the dependence of ectopic Oskar activity on Lasp. It is therefore proposed
that Lasp links Oskar to the actin cytoskeleton and helps to restrict Oskar
activity to the posterior pole after its localized translation. Even though the
possiblility cannot be excluded that Lasp also protects Oskar from degradation, the reduced amounts of oskar mRNA and Oskar protein observed in lasp
mutants are probably due to the interdependence of oskar mRNA
localization and Oskar protein. Less well-anchored Oskar cannot keep its mRNA
restricted to the posterior pole, which in turn leads to less mRNA available
for localized translation (Suyama, 2009).
Lasp concentrates in actin-rich subcellular regions, including focal
adhesions and lamellipodia in migrating cells, and motile growth cones of cultured neurons. This study has shown that the NEB repeats of Drosophila Lasp bind
actin in vitro and that Lasp colocalizes with actin in vivo. lasp
mutant egg-chambers display no obvious actin defects, consistent with the
observation that rabbit Lasp1 has no detectable effect on actin polymerization
and that cells depleted of Lasp1 can form focal adhesions. Lasp1 has
also been shown to be a substrate of protein kinase A (PKA). After
PKA stimulation of parietal cells in isolated gastric glands, Lasp1
redistributes from cortical regions to the actin-rich intracellular
canalicular region. In Drosophila, egg chambers lacking PKA catalytic
subunit activity contain multinucleate nurse cells and abnormal ring canals.
In addition, pka mutant oocytes fail to respond to a signal from
posterior follicle cells and consequently fail to reorganize the microtubule
cytoskeleton. Oocytes lacking Lasp function show neither of these
phenotypes. By contrast, excess PKA activity, as in Pka-RI mutant
oocytes, causes the accumulation of high levels of ectopic Oskar protein and
embryos displaying the bicaudal phenotype. This ectopic accumulation could be due to delocalized oskar mRNA translation, or to the stabilization of Oskar protein produced ectopically that would normally be targeted for degradation. Given the reduced Oskar levels observed in lasp mutant embryos and the dependence of
ectopic Oskar activity on Lasp, it would be interesting to
determine whether PKA phosphorylation of Lasp contributes either to the
accumulation of Oskar or to its anchoring in the embryo (Suyama, 2009).
Oskar anchoring becomes crucial at stage 10B of oogenesis, when cytoplasmic
streaming starts, a vigorous process that ensures the mixing and even
distribution of cytoplasmic mRNAs and proteins in the egg.
Treatment of embryos with the actin-severing drugs Cytochalasin D and
Latrunculin A disrupts the localization of oskar mRNA and protein,
but not of bicoid mRNA, which is similar to what was observed in lasp
mutant embryos and consistent with a role of Lasp in actin-dependent Oskar
anchoring. Several mechanisms contribute to oskar RNA and protein
anchoring. Bifocal (Bif) and Homer (Hom) redundantly promote Oskar anchoring
at the oocyte posterior pole via an actin-dependent and an unknown,
actin-independent process, respectively.
Remarkably, the Oskar detachment defects of Latrunculin A-treated hom
egg chambers are stronger than in hom/bif double mutants, suggesting
there are additional actin-dependent attachment mechanisms. However, no genetic interactions have been detected between lasp and
hom/bif, as neither hom/lasp nor bif/lasp double
mutants showed an Oskar localization defect in ovaries (Suyama, 2009).
The ubiquitous expression of Oskar in the oocyte leads to its enrichment
all around the cortex, indicating that Oskar protein can be recruited all
around the F-actin-rich oocyte cortex. Since
Lasp is uniformly distributed along the actin-rich cortex, it could
participate in Oskar anchoring at ectopic cortical locations. Consistent with
this, the deleterious effects of ectopic anterior (osk-bcd) or
cortical (osk-K10) Oskar on embryonic patterning are suppressed in
lasp mutant embryos. In further support of tbe anchoring model, only
Lasp with a functional SH3 domain (i.e. one capable of interacting with Oskar
in vitro and in the two-hybrid assay), is able to rescue the grandchildless
phenotype and the reduction in pole cell number observed in osk-, lasp-/lasp- embryos (Suyama, 2009).
The interaction of Oskar with Lasp depends on its SH3 domain, because a
single W>-A point mutation on the binding surface of the SH3 domain
abolishes its binding to Oskar. SH3 domains
usually interact with short, proline-rich sequences with the consensus RxxPxxP
(class I binding site) or PxxPxR (class II binding site). The region
of Oskar that is sufficient to interact with the SH3 domain of Lasp (aa
290-369) does not encode a perfect class I or class II binding
site, nor does it contain any of the less frequently occurring SH3-binding
motifs. Three PxxP motifs are present within the SH3-binding region of Oskar, but all of
them lack the neighboring basic amino acid. Nevertheless, Oskar is highly specific for the SH3 domain of Lasp, as it does not interact with six other SH3 domains tested, and the interaction capability is conserved in Oskar of D. virilis (Suyama, 2009).
Interestingly, the Lasp SH3 domain shares a high degree of similarity with
several proteins involved in clathrin-mediated endocytosis: the actin-binding
proteins Cortactin and Abp1, as well as Syndapin and Amphiphysin, both of
which bind Dynamin and contain an F-actin- or an Arp2/3-interacting domain.
It has been reported that both Lasp and Dynamin II localize to the apical
membrane of parietal cells, and that they interact in vitro. Thus,
it is possible that Lasp is not only an actin binding-protein, but is also
involved in vesicle trafficking. Endocytic trafficking and actin-based
mechanisms also contribute to Oskar anchoring, and it is thus possible that
Lasp, together with other functionally related proteins, represents a link
between these processes. Furthermore, one of the two Oskar isoforms
(Long-Oskar) plays a crucial role in the anchoring of both Oskar isoforms and
oskar mRNA at the oocyte cortex at stage 10 of oogenesis.
Oocytes lacking Oskar have a dramatically reduced endocytic compartment and
lack the thick actin bundles that are normally observed at the posterior pole.
Thus Oskar anchoring in the oocyte appears to be a dynamic process, with
Oskar-stimulated recycling endocytosis and filamentous actin outgrowths
playing an important role in the maintenance of Oskar at the cell cortex. It is
thus conceivable that Oskar, Lasp, and other possibly redundant actin-binding
proteins act in feedback loops with components of the endocytic pathway to
maintain Oskar at the posterior pole (Suyama, 2009).
Sm and Sm-like proteins are RNA-binding factors found in all three domains of life. Eukaryotic Sm proteins play essential roles in pre-mRNA splicing, forming the cores of spliceosomal small nuclear ribonucleoproteins (snRNPs). Recently, Sm proteins have been implicated in the specification of germ cells. However, a mechanistic understanding of their involvement in germline specification is lacking and a germline-specific RNA target has not been identified. This study demonstrates that Drosophila SmB and SmD3 are specific components of the oskar messenger ribonucleoprotein (mRNP), proper localization of which is required for establishing germline fate and embryonic patterning. Importantly, oskar mRNA is delocalized in females harboring a hypomorphic mutation in SmD3, and embryos from mutant mothers are defective in germline specification. It is concluded that Sm proteins function to establish the germline in Drosophila, at least in part by mediating oskar mRNA localization (Gonsalvez, 2010).
Previous studies in C. elegans, Xenopus and mice showed that Sm proteins localize to cytoplasmic granules that are functionally similar to the Drosophila pole plasm. Using VFP-tagged constructs, this study demonstrates that SmB and SmD3 localize to the Drosophila pole plasm. Does the localization of the tagged constructs reflect the true localization of the endogenous proteins? Several lines of evidence suggest that it does. VFP-SmB rescues the lethality of a transposon insertion in SmB and produces viable adults in which the tagged protein is properly localized. Expression of VFP-SmD3 or GFP-SmD3 rescues the oskar mRNA-delocalization phenotype of protein trap allele SmD3pt mutant, and both constructs localize to the pole plasm. Additionally, the transgenes are not overexpressed in comparison to their endogenous counterparts. Thus, the posterior enrichment of VFP-SmB and VFP-SmD3 does not appear to be the result of a tagging artifact (Gonsalvez, 2010).
The results suggest that germ granules in Drosophila contain a novel type of Sm complex. SmB and SmD3, but not the other Sm proteins, have been shown to co-purify with human telomerase RNPs. Furthermore, the U7 snRNP, which is required for nuclear processing of histone mRNA, specifically lacks SmD1 and SmD2. Thus, there is precedence for the formation of non-canonical Sm complexes that contain subsets of the seven core Sm proteins (Gonsalvez, 2010).
The splicing function of Sm proteins is required for cell viability. Therefore, examination of Sm protein function in germline specification requires a viable mutant allele that is not compromised for splicing. Such an allele is not available for SmB. However, SmD3pt is homozygous viable and defective in oskar mRNA localization. In early egg chambers, oskar mRNA occupies the entire volume of the ooplasm. By stage 8, the message can be detected at the anterior margins of the oocyte and also within a transient central focus. By stage 9, oskar mRNA localizes to the posterior pole of the oocyte where it remains anchored for the remainder of oogenesis. In SmD3pt mutants, the localization of oskar mRNA is identical to that in wild type until stage 8. A significant fraction of stage 9 and 10 egg chambers, however, show varying patterns of oskar mRNA delocalization. The penetrance of the mutant phenotype is temperature sensitive but is not significantly different between stage 9 and 10 egg chambers. Consistent with oskar mRNA delocalization, Stau, a core component of the oskar mRNP, was also delocalized in SmD3pt egg chambers (Gonsalvez, 2010).
Why is oskar mRNA delocalized in mutant oocytes? Smd3pt is hypomethylated in comparison to the wild-type protein. The results indicate, however, that loss of methylation is unlikely to be the cause of oskar mRNA delocalization. It is also possible that the GFP insertion within Smd3pt somehow compromises the activity of the protein. Using anti-GFP antibodies, it was found that Smd3pt is able to associate with oskar mRNA in vivo. The temperature-sensitive nature of the SmD3pt allele was exploited to ask whether oskar mRNA delocalization correlates with loss of Smd3pt binding. No significant differences were found in the association of SmD3pt with oskar mRNA at permissive (21°C) and non-permissive (27°C) temperature. It is concluded that when oskar mRNA is delocalized, SmD3pt remains associated with the message (Gonsalvez, 2010).
How do SmD3 and SmB function in oskar mRNA localization? In considering this question, it is worth examining the function of Sm proteins in snRNP assembly. An early event in assembly of spliceosomal snRNPs involves the association of an Sm core with the snRNA. By binding the snRNA, the Sm proteins are thought to aid in the three-dimensional folding of the RNA such that additional components can be specifically added to the maturing snRNP. By analogy, SmD3 and SmB might bind oskar mRNA early in the life cycle of the message, thereby enabling proper folding of the RNA and subsequent recruitment of additional essential components. When SmD3pt is brought in trans over a strong loss-of-function SmD3 mutant, the penetrance of oskar mRNA delocalization is greatly increased. By contrast, removing one copy of SmB does not exacerbate the SmD3pt mutant phenotype. Thus, SmB and SmD3 do not appear to function redundantly in oskar mRNA localization (Gonsalvez, 2010).
Unlike oskar mRNA, bicoid and gurken mRNAs are properly localized in SmD3pt egg chambers. Additionally, no defect was observed in the repositioning of the oocyte nucleus to the dorsal-anterior corner of SmD3pt egg chambers. These processes are microtubule dependent and require a properly polarized oocyte, suggesting that overall polarity is maintained in SmD3pt egg chambers (Gonsalvez, 2010).
In contrast to bicoid and gurken mRNAs, the plus-end-directed microtubule motor, Khc, was not enriched at the posterior pole in SmD3pt mutants that contained delocalized oskar mRNA. One interpretation suggests that microtubule plus ends are not anchored at the posterior in SmD3pt mutants. According to this view, the defect in oskar mRNA localization would be a secondary consequence of prior defects in the localization of microtubule plus ends. This hypothesis posits that the primary function of SmD3 is in the regulation of microtubule polarity. This possibility cannot be entirely ruled out. However, the intertwined nature of oocyte plus end polarity and the Oskar pathway causes a slightly different hypothesis to be favored. Oskar protein participates in a positive-feedback mechanism that reinforces posterior recruitment of microtubule plus ends. According to this view, an egg chamber that fails to express Oskar protein would fail to recruit sufficient microtubule plus ends to the posterior pole. Thus, Khc would appear delocalized in these egg chambers. This is believed to be the case in SmD3pt mutants. Consistent with the notion of an Oskar-Khc connection, SmD3pt egg chambers that contain a central focus of oskar mRNA and protein recruit Khc to the same focus (Gonsalvez, 2010).
Is the function of Khc compromised in SmD3pt egg chambers? The fact that Khc is not localized to the posterior pole of mutant egg chambers suggest that at least this aspect of its function is compromised. However, global functions of Khc in the female germline appear to be relatively unaffected in SmD3pt mutants. Loss of Khc results in delocalization of bicoid mRNA, gurken mRNA and protein, mispositioning of the oocyte nucleus, and mislocalization of oskar mRNA around the entire cortex of the oocytes. None of these phenotypes was observed in SmD3pt mutants (Gonsalvez, 2010).
The Sm family of proteins is of ancient evolutionary origin. The Escherichia coli Sm ortholog, Hfq, functions to modulate the translation and stability of several RNAs, including mRNAs and tRNAs. Based on these ancestral functions, the involvement of eukaryotic Sm proteins in splicing is generally thought to be a derived function. The finding that spliceosomal Sm proteins are also associated with oskar mRNA suggests that some of these ancestral functions in mRNA regulation have been retained. In the context of oskar, SmD3 is required for regulating the localization of the message. As a consequence of this function, hypomorphic mutants in SmD3 do not form germ cells and display defects in developmental patterning. This study represents the first demonstration of a eukaryotic Sm protein regulating the cytoplasmic fate of an mRNA (Gonsalvez, 2010).
mRNA localization coupled with translational control is a widespread and conserved strategy that allows the localized production of proteins within eukaryotic cells. In Drosophila, oskar (osk) mRNA localization and translation at the posterior pole of the oocyte are essential for proper patterning of the embryo. Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs. In cultured mammalian cells, Ge-1 protein is required for P body formation. Combining genetic, biochemical and immunohistochemical approaches, this study shows that, in vivo, Drosophila Ge-1 (dGe-1) is an essential gene encoding a P body component that promotes formation of these structures in the germline. dGe-1 partially colocalizes with osk mRNA and is required for osk RNP integrity. This analysis reveals that although under normal conditions dGe-1 function is not essential for osk mRNA localization, it becomes critical when other components of the localization machinery, such as staufen, Drosophila decapping protein 1 and barentsz are limiting. These findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo (Fan, 2011).
P bodies have been described in many eukaryotes and consist of aggregates of translationally inactive RNPs. The number and size of these dynamic structures depends on the availability of mRNAs not associated with the translational machinery. Proteins of the mRNA degradation machinery, such as the decapping protein Dcp1 and Dhh1, and translational repressors, such as RAP55 and 4E-T, are enriched in P bodies. Although P bodies are conserved structures, their disruption seems to affect neither mRNA decay nor translational repression. It has therefore been proposed that the role of P bodies might be to compartmentalize mRNA decay and translation repression, possibly enhancing the efficiency of these processes (Fan, 2011 and references therein).
In yeast, the Yjef-N dimerization domain and the prion-like Glutamine/Asparagine (Q/N)-rich domain of two P body components, Edc3 and Lsm4, respectively, are required for P body assembly, suggesting that P body formation might be a self-assembly process. However, in higher eukaryotic cells the Yjef-N domain of Edc3 plays only a minor role in P body assembly and the Q/N domain of yeast Lsm4 is not found in its eukaryotic homologues, suggesting that Lsm4 either performs its function by a different mechanism or does not promote P body formation in these organisms. Interestingly, a conserved protein with no homologue in yeast, Ge-1, contains at its C-terminus a Q/N domain that promotes oligomerization of the protein in vitro, and at its N-terminus a WD40 repeat, a domain often involved in protein-protein interactions. Ge-1 can associate with Dcp1 in vitro to enhance decapping activity and Arabidopsis Ge-1 is involved in postembryonic development, by regulating the decapping process. In different eukaryotic cells, Ge-1 knockdown causes P bodies to disappear. While it is clear that Ge-1 plays a critical role in P body formation in cells, its in vivo functions in metazoans remain largely unexplored (Fan, 2011 and references therein).
The requirement for dGe-1 for P body formation during oogenesis raises the possibility that the mild effect of dGe-1 on osk mRNA localization might be a consequence of P body disruption. It has been shown that upon nutritional restriction, sponge bodies, which are P body-like structures in the Drosophila germline that contain many P body components, are enlarged, and interestingly, osk RNPs are detected within them (Snee, 2009). Therefore, a direct interaction between osk RNPs and P body-like structures or P bodies themselves may occur, but how this interaction would influence osk mRNA localization remains unclear. In the future, it will be interesting to determine the dynamic relationship between P bodies and osk RNPs, for instance by live-imaging of shared and distinct components and tracking their movement relative to these two RNP structures (Fan, 2011).
Alternatively, dGe-1 might act directly in an aspect of osk mRNA regulation that is not absolutely essential, such as optimization of the localization process. In support of this hypothesis, localization and biochemical analyses suggest that, although in the absence of dGe-1 osk RNP assembly may be mildly impaired, under normal laboratory conditions small osk RNPs can form and are sufficiently functional for mRNA localization and posterior patterning to proceed. However, a strong increase in osk localization and posterior patterning defects is observed in the dGe-1 mutant when one wt copy of dDcp1, stau or btz, each of which encodes a component of osk RNPs, is removed. Similarly, depletion of dGe-1, which disrupts P body assembly in Drosophila S2 cells, only mildly affects miRNA-mediated mRNA decay, and the knockdown of dDcp1 or Me31B, both of which are involved in mRNA decapping, has no obvious effect on this process. It is therefore possible that dGe-1 functions to locally concentrate the proteins essential for osk mRNA localization, rendering osk RNP transport more efficient or robust, in particular when some components are limiting. This study suggests a novel function of dGe-1 in optimization of the process of osk mRNA localization (Fan, 2011).
Finally, the demonstration of the involvement of dGe-1 in osk mRNA localization in the Drosophila oocyte raises the possibility that P body components and P bodies themselves may contribute to RNA localization, a highly conserved phenomenon, in other eukaryotic cell types and organisms. In addition, the defects in Grk protein expression in dGe-1 mutant ovaries point to a more general role of dGe-1 and presumably of P bodies in mRNA regulation (Fan, 2011).
Asymmetric mRNA localization is an effective mechanism for establishing cellular and developmental polarity. Posterior localization of oskar in the Drosophila oocyte targets the synthesis of Oskar to the posterior, where Oskar initiates the assembly of the germ plasm. In addition to harboring germline determinants, the germ plasm is required for localization and translation of the abdominal determinant nanos. Consequently, failure of oskar localization during oogenesis results in embryos lacking germ cells and abdominal segments. oskar accumulates at the oocyte posterior during mid-oogenesis through a well-studied process involving kinesin-mediated transport. Through live imaging of oskar mRNA, this study has uncovered a second, mechanistically distinct phase of oskar localization that occurs during late oogenesis and results in amplification of the germ plasm. Analysis of two newly identified oskar localization factors, the RNA-binding proteins Rumpelstiltskin and Lost, that are required specifically for this late phase of oskar localization shows that germ plasm amplification ensures robust abdomen and germ cell formation during embryogenesis. In addition, these results indicate the importance of mechanisms for adapting mRNAs to utilize multiple localization pathways as necessitated by the dramatic changes in ovarian physiology that occur during oogenesis (Sinsimer, 2011).
Posterior localization of osk underlies the formation of germ plasm at the posterior of the oocyte and, ultimately, the development of the germ line and abdomen of the animal. Whereas osk localization during mid-oogenesis has been well studied, this study has identified a second, previously uncharacterized phase of osk localization that occurs late in oogenesis, beginning with nurse cell dumping. The concomitant accumulation of Vas at the posterior during this period indicates that this phase of osk localization leads to germ plasm amplification. This late phase of osk localization specifically requires Rump, which binds to osk, and Lost, which interacts with Rump. In lost- rump- embryos, the reduction of germ plasm at the posterior leads to fewer germ cells and abdominal segmentation defects. Thus, the localization of osk during mid-oogenesis is not sufficient to ensure wild-type germline and abdominal development. Rather, the second phase of osk localization and germ plasm accumulation is crucial for embryogenesis. These results are consistent with results from a previous study showing that continued production of Osk during late stages of oogenesis is essential for abdomen formation and they reveal the mechanism behind this accumulation (Sinsimer, 2011).
The failure of osk to accumulate at the posterior of lost- rump- mutants at late stages of oogenesis could result from the failure of osk to reach to the oocyte posterior altogether or the failure to become entrapped and anchored there. Nurse cell dumping and ooplasmic streaming occur normally in lost- rump- mutants, suggesting that the defect does not lie in the ability of osk to reach the posterior cortex. The lack of localized osk in embryos from mutants such as staufen, which disrupt osk localization during mid-oogenesis, or osk mis-sense mutants, which lack functional Osk protein and fail to maintain osk at the posterior after stage 9, suggests that the late phase of osk localization depends on the prior localization and translation of Osk and recruitment of germ plasm components during mid-oogenesis. Consistent with this evidence, Osk is required for posterior accumulation of synthetic osk mRNA following injection into stage 11 oocytes. Thus, Rump and Lost probably mediate the interaction of osk with pre-existing Osk or other germ plasm proteins, and/or the actin cytoskeleton. Eliminating both Rump and Lost produces a more severe RNA localization defect than eliminating either one but does not completely abolish localization. Partially redundant contributions by RNA-binding factors like Rump has been previously predicted for the osk and nos RNA localization signals. The results suggest that there is functional overlap among higher order interactions of RNA-binding proteins and adaptors like Lost that make that RNP assembly and function robust to perturbation (Sinsimer, 2011).
Not only is the late phase of osk localization affected in lost- rump- mutants, but osk that had accumulated at the posterior of the oocyte during mid-oogenesis is de-localized. Although difficult to quantify, a transient decrease was also noticed in osk at the posterior in wild-type oocytes immediately following the onset of nurse cell dumping. One possible explanation is that the Osk-dependent anchoring mechanism established during mid-oogenesis cannot withstand the force of ooplasmic streaming and is, therefore, unable to maintain osk at the posterior cortex. Retention of both previously anchored and newly arriving osk at the cortex would, therefore, require the transition to a more robust anchoring system, through the activity of Rump and Lost. Once established, this anchor would support the continued accumulation of germ plasm through the remainder of oogenesis (Sinsimer, 2011).
Because nos localization depends on the germ plasm, the nos localization defect observed in lost- rump- oocytes and embryos could be secondary to the osk localization defect. However, several pieces of evidence suggest that lost and rump might also regulate nos localization in parallel. Rump and Lost were both isolated by co-purification with the nos +2? localization element. Rump binds to a specific sequence motif in the nos +2? localization element, and mutation of these sequences or elimination of Rump compromises +2? element localization function independently of osk localization. Finally, nos localization is frequently more diffuse in lost mutants whereas osk is largely unaffected. As an attractive hypothesis, nos might be transported to or anchored at the posterior together with osk, as part of the same RNP. Incorporation of nos and osk into this RNP and/or the ability to interact with Osk or other anchoring factors would then depend on the interaction of Lost and Rump with each mRNA. Alternatively, Lost and Rump might contribute to the assembly of an independent nos RNP that is competent to associate with the germ plasm at the posterior pole. Development of methods to image osk and nos simultaneously during late oogenesis will be crucial to distinguish between these possibilities (Sinsimer, 2011).
A late-acting localization pathway is responsible for the majority of anteriorly localized bcd in the embryo. The late phase of bcd localization is genetically distinct from bcd localization during mid-oogenesis but does not depend on Rump or Lost, indicating that these proteins are specific for posterior localization at late stages. Taken together, these results indicate that mRNA localization pathways functioning during late stages of oogenesis amplify localized mRNA distributions generated during mid-oogenesis to endow the embryo with the requisite concentrations of determinant mRNAs and germ plasm components needed for body axis and germline development. This work suggests that localization factors such as Lost and Rump adapt mRNAs for utilization of multiple, mechanistically distinct localization pathways necessitated by dramatic changes in ovarian physiology and the oocyte cytoskeleton during oogenesis (Sinsimer, 2011).
McDermott, S. M., Meignin, C., Rappsilber, J. and Davis, I. (2012). Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification. Biol Open 1: 488-497. PubMed ID: 23213441
In the Drosophila oocyte, mRNA transport and localised translation play a fundamental role in axis determination and germline formation of the future embryo. gurken mRNA encodes a secreted TGF-alpha signal that specifies dorsal structures, and is localised to the dorso-anterior corner of the oocyte via a cis-acting 64 nucleotide gurken localisation signal. Using GRNA chromatography, this study characterised the biochemical composition of the ribonucleoprotein complexes that form around the gurken mRNA localisation signal in the oocyte. A number of the factors already known to be involved in gurken localisation and translational regulation, such as Squid and Imp, were identified, in addition to a number of factors with known links to mRNA localisation, such as Me31B and Exu. Previously uncharacterised Drosophila proteins were identified, including the fly homologue of mammalian SYNCRIP/hnRNPQ, a component of RNA transport granules in the dendrites of mammalian hippocampal neurons. It was shown that Drosophila Syncrip binds specifically to gurken and oskar, but not bicoid transcripts. The loss-of-function and overexpression phenotypes of syncrip in Drosophila egg chambers show that the protein is required for correct grk and osk mRNA localisation and translational regulation. It is concluded that Drosophila Syncrip is a new factor required for localisation and translational regulation of oskar and gurken mRNA in the oocyte. It is proposed that Syncrip/SYNCRIP is part of a conserved complex associated with localised transcripts and required for their correct translational regulation in flies and mammals (McDermott, 2012).
This study has identified Drosophila Syp as a novel conserved component of localised RNP granules. Syp associates specifically with grk and osk and is required for their localisation and translational regulation in the Drosophila germline. Although SYNCRIP has been studied in mammalian cells using biochemical approaches, this study is the first to address the function of Syp in vivo, and particularly in generating cellular asymmetry in the germline. Despite a number of genetic screens that have been carried out to identify the genes required for axis specification in flies, Syp was not previously identified as being required for axis determination. This work together with a number of other biochemical based studies illustrates that there are many other essential factors that are still to be identified as having a role in axis specification (McDermott, 2012).
Evidence is presented in this study that Syp is required for axis specification and germline formation by affecting the localisation and translation of grk and osk mRNAs. The phenotypes of loss of function mutations in the gene, and overexpression of the protein support an interesting role for Syp in regulating grk and osk mRNAs. It is noted that the loss of function phenotypes are of low penetrance and are such that further studies are required to uncover the precise mechanism of Syp function. However, Syp is not the only component of grk and osk mRNPs that has a partially penetrant loss of function phenotype. Indeed, others such as IGF-II mRNA-binding protein (Imp) give a stronger phenotype only when in combination with other mutations. Unexpectedly, overexpression of Syp gives the same phenotype as its loss of function, but at a higher penetrance. These results are potentially interesting, but difficult to interpret with certainty. The interpretation is favoured that a certain stoichiometry is necessary within the RNP complexes in which Syp is found. Overexpression of Syp may cause the displacement of certain translational repressors, allowing grk mRNA to be prematurely translated in the nurse cells. In contrast, with loss of function of Syp, grk mRNA localisation and translational regulation may be disrupted in different ways because loss of Syp could lead to decreased stability of the RNP complex. This could in turn lead to the loss of certain components necessary for localization and translational regulation. The eggshell phenotypes observed in the syp germline clones also include some defects that are not typical of a disruption of grk mRNA localisation and translation. These results are interpreted as indicating that Syp has additional target mRNAs in the germline whose localisation and/or translation are affected in the syp mutant. On the basis of the morphological defects observed in a number of eggs these targets may include mRNAs involved in follicle cell migration or actin organisation during oogenesis, such as bullwinkle (bwk) or chickadee (chic). Mislocalisation and/or altered translation of these mRNAs could result in the shorter eggs and the bwk-like dorsal appendage defects that are observed (McDermott, 2012).
The physical association of Syp with these mRNAs further supports a function for Syp in the regulation of grk and osk mRNAs, although it is unclear whether this is through a direct or indirect interaction. Syp does not appear to colocalise with localised grk or osk in the oocyte, and so Syp may function before the mRNAs reach their final destination in the oocyte in order to influence localisation and translation. Syp was also identified biochemically with a number of the factors already known to be required for grk and osk mRNA localisation and translational regulation. These include Sqd, Imp, PTB, PABP, Me31B and BicC. The homologues of these factors were also identified in biochemical studies of SYNCRIP interactors in mammalian cells. Therefore, it is proposed that the current results have uncovered a conserved module of RNA binding proteins that are required for both mRNA transport and translational regulation. The Syp associated complex that this study has uncovered binds to grk via a relatively small stem loop sequence, the GLS. While Syp also associates with osk mRNA, the minimal region necessary and sufficient for osk mRNA localisation has not yet been defined. Therefore, it is unknown whether a small region is required in this case, and if so whether it is in any way similar to the GLS, structurally or in primary sequence (McDermott, 2012).
Taking the results of this study in the context of other data and the previous publications on SYNCRIP and its associated proteins in mammalian hippocampal neurons, it is proposed that Syp may be present at neuronal synapses in a complex with at least some of the same proteins that are required for grk mRNA localisation. This idea is supported by the fact that at least some of these factors, namely Sqd, Imp, PTB, PABP and Me31B are also present in the Drosophila nervous system. Our expression studies show that Syp is absent from embryos but is highly expressed in the larval nervous system. Given the role of Syp in mRNA localization and translational regulation in the oocyte, and its presence in larval brains, it is proposed that Syp could have a similar function in the nervous system. In comparison with the oocyte, much less is known about localised transcripts and their translational regulation in the nervous system, and it remains to be determined to what extent this proposal is valid and in which neuronal tissues Syp is required in larvae. Nevertheless, this work is the first demonstration that Syp functions in mRNA localisation and translational control in the oocyte and coupled with the work on mammalian SYNCRIP showing association with RNP granules in the dendrites of hippocampal neurons it is attractive to propose that Syp protein also has a conserved function in regulation of neuronal mRNAs (McDermott, 2012).
As a way to spatially control the expression of genes within cells, RNA localization is being recognized as an important process by which proteins are restricted to specific subcellular domains, which occurs in more diverse types of tissue than previously considered. Although many localized RNAs have been identified, information on cis-acting elements of localization is still limited. As transcripts of oskar (osk) are known to localize to the posterior pole of oocytes, this study computationally analyzed a conserved sequence among eight Drosophila species and tested its role as a localization element. Dimerization of osk mRNA did not occur when the motif was deleted, but this did not affect assembly of osk mRNA-containing ribonucleoprotein (RNP) complexes. Without the motif, however, large RNP complex particles accumulated in nurse cells, and only a small fraction of these RNP complexes was transported into oocytes and properly localized to the posterior pole. Therefore, this motif may be required for the early transport of osk mRNA into oocytes. Also, as dimerization of osk mRNA does not seem to be a prerequisite for the assembly of RNP complexes, a dimerization-independent mechanism may also serve to localize osk mRNA to the posterior pole (Kim, 2014).
Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).
Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).
Quantitative analysis of germ plasm-localized mRNAs has revealed several intriguing features about the localization process and the coordinate regulation of their integration into the pole cells. nos, pgc and cycB mRNAs are transported within the oocyte as single mRNAs and are co-packaged into granules specifically at the posterior cortex concomitant with localization. Thus, localization serves not only to concentrate these transcripts at the posterior but also generates large, multi-copy polar granules to coordinate the efficient incorporation of these transcripts into the pole cells (Little, 2015).
Polar granules are heterogeneous with respect to both the amount of a particular mRNA and the combination of different mRNAs. Although nos mRNA content of polar granules varies over a large range, there is a tendency towards higher values fitting a log-normal distribution. Log-normal distributions are often associated with exponential growth processes, whereby the rate at which an object grows is proportional to the size of the object. Thus, a log-normal distribution suggests that large granules grow at faster rates compared with small ones as assembly is accelerated through positive feedback. In addition, this study found that for granules containing both nos and pgc or nos and cycB, the quantities of the two different mRNAs are correlated, and there is a greater fraction of granules completely lacking one species of mRNA entirely than granules containing just a few copies of that mRNA. Together, these data suggest that for each type of mRNA, cooperative interactions generate homotypic RNPs that then 1) accelerate the recruitment of additional mRNAs of the same type, and 2) facilitate granule assembly by promoting interactions with similarly sized homotypic clusters of other mRNAs. These results also predict the existence of dedicated molecular pathways, one to form homotypic clusters, and another to assemble homotypic clusters of many different transcripts into higher-order granules. These higher-order granules may form by fusion of smaller homotypic granules and indeed fusion of granules labelled with GFP-Vas is observed by live imaging. Alternatively, clusters of different mRNAs may grow alongside each other on a predefined granule scaffold. The localization of Caenorhabditis elegans germ granules -- P granules -- occurs through a phase transition in which soluble RNP components condense at the posterior of the embryo. It is interesting to consider whether formation of homotypic clusters occurs by a condensation of single transcript RNPs mediated by RNA-binding proteins (Little, 2015).
In contrast to other posteriorly localized RNAs, which travel as single molecules, osk forms oligomeric complexes beginning in the nurse cells. Previous studies indicated that reporters containing the osk 3'UTR can hitchhike on wild-type osk mRNA by 3'UTR-mediated dimerization. Hitchhiking is not required for osk transport, but co-packaging may be important for translational repression of osk before localization. Consistent with this, multi-copy RNPs are competent for localization by both kinesin-dependent transport during mid-oogenesis and diffusion/entrapment during late stages of oogenesis (Little, 2015).
Previous ISH-immuno-electron microscopy analysis of stage 10 oocytes showed co-localization of osk with Stau, but not with Osk protein in polar granules and the results indicate that osk/Stau RNPs are continuously segregated from the germ plasm granules. This physical separation has functional consequences. Whereas co-packaging of nos, pgc and cycB coordinates their transport to posterior nuclei and consequent segregation to the pole cells, osk is specifically excluded. Although it is not known why targeting of Osk to polar granules seems to alter their function, it is clearly detrimental to germline development. It will be interesting to determine whether osk/ Stau granules contain other mRNAs whose functions are required specifically during oogenesis or early embryogenesis but must be excluded from pole cells (Little, 2015).
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