orb
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).
fs(1) K10 mRNA transport and anterior localization is mediated by
a 44 nucleotide stem-loop structure. A similar putative stem-loop structure is found in the 3'
untranslated region of ORB mRNA, suggesting that the same factors mediate the
transport and anterior localization of both K10 and ORB mRNAs. Apart from ORB, the K10 TLS is
not found in any other localized mRNA, raising the possibility that the transport and localization of
other mRNAs, e.g., Bicoid, Oskar and Gurken, are mediated by novel sets of cis- and trans-acting
factors. Moreover, the K10 TLS overrides the activity of Oskar cis-regulatory elements
that mediate the late stage movement of the mRNA to the posterior pole (Serano, 1995).
The targeting of positional information to specific regions of the oocyte or early embryo is one of
the key processes in establishing anterior-posterior and dorsal-ventral polarity. In many
developmental systems, this is accomplished by localization of mRNAs. The germ line-specific
Drosophila orb gene plays a critical role in defining both axes of the developing oocyte, and its
mRNA is localized in a complex pattern during oogenesis. A 280-bp sequence
from the orb 3' untranslated region is capable of reproducing this complex localization pattern.
Multiple cis-acting elements appear to be required for proper
targeting of Orb mRNA. In egalitarian and Bicaudal-D mutants, localization of ORB mRNAs is disrupted (Lantz, 1994).
Microtubules are required for localization
of these mRNAs during oogenesis, as they are for Bicoid RNA.
However, the RNAs show a differential sensitivity to microtubule inhibitors. Anterior localization of
Bicaudal-D, Fs(1)K10, and ORB RNAs is completely disrupted following even mild drug
treatments. In addition, the localized RNAs respond differently
to taxol, a microtubule stabilizing agent.
Microtubules are also required for the preferential accumulation of these transcripts in the
previtellogenic oocyte, consistent with the idea that these mRNAs are transported by a
microtubule-dependent mechanism to the oocyte (Pokrywka, 1995).
The homeless gene of Drosophila is required for anterior-posterior and dorsoventral axis
formation during oogenesis. Transport and localization of
Bicoid and Oskar messages during vitellogenic stages are strongly disrupted in homeless mutants. The distribution
and/or quantity of Gurken, Orb, and Fs(1)K10 mRNAs are also affected, but to a lesser degree. In
contrast, Hu-li tai shao and Bicaudal-D transcripts are transported and localized normally in hls
mutants. 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).
The RRM-type RNA binding protein Orb plays a central
role in the establishment of polarity in the Drosophila egg
and embryo. In addition to its role in the formation and
initial differentiation of the egg chamber, orb is required
later in oogenesis for the determination of the dorsoventral
(DV) and anteroposterior (AP) axes. In DV axis formation,
Orb protein is required to localize and translate Gurken
mRNA at the dorsoanterior part of the oocyte. In AP axis
formation, Orb is required for the translation of Oskar
mRNA. In each case, Orb protein is already localized at the
appropriate sites within the oocyte before the arrival of the
mRNAs encoding axis determinants. Evidence is presented
that an autoregulatory mechanism is responsible for
directing the on site accumulation of Orb protein in the
Drosophila oocyte. This orb autoregulatory activity ensures
the accumulation of high levels of Orb protein at sites in
the oocyte that contain localized Orb message (Tan, 2001).
The results presented here suggest that autoregulation plays a critical role in
promoting the on site accumulation of Orb protein. In this
model, translationally repressed Orb mRNA synthesized in
nurse cells would be transported into and targeted to specific
sites within the oocyte. Orb protein already present at these
sites would bind to the Orb mRNA when it arrives, anchoring
the message to the cortex of the oocyte and activating its
translation. Newly synthesized Orb protein would then be
available to interact with incoming localized mRNAs
and activate their translation. Once initiated, this orb
autoregulatory activity would ensure the accumulation of high
levels of Orb protein at sites in the oocyte containing localized
Orb mRNAs (Tan, 2001).
The autoregulatory model was initially suggested by the
dominant negative activity of transgenes expressing lacZ
mRNAs that have the orb 3' UTR. As in classical
antimorphic mutations, the phenotypic effects of the lacZ Orb
3' UTR transgenes can be exacerbated by increasing the
transgene dose relative to the endogenous orb gene.
Conversely, it is possible to suppress the phenotypic effects of
the Orb 3' UTR transgenes by increasing the relative dose of
the endogenous gene. Since ß-galactosidase has no adverse
effects on orb function, the antimorphic activity of these
transgenes can be attributed to the Orb 3' UTR sequences in
the transgene mRNAs. To have an antimorphic activity, these
RNA sequences must interfere with the functioning of the
endogenous gene. The most plausible mechanism is that the
transgene 3' UTR sequences compete with mRNA from the
endogenous gene for some limiting factor that is essential for
the expression of sufficient quantities of Orb protein. Since orb
is haploinsufficient, the obvious candidate for this limiting
factor is the Orb protein itself (Tan, 2001).
One prediction of the autoregulatory model is that the
expression of ß-galactosidase from the lacZ Orb 3' UTR mRNA
should depend upon orb function. This is the case.
Orb is required for the translation of the lacZ Orb 3'UTR
mRNA, and the synthesis of ß-galactosidase from the lacZ
Orb 3' UTR message is substantially reduced in orb mutant
ovaries. In contrast, orb mutations have no effect on the
translation of lacZ mRNAs that have unrelated 3' UTR
sequences. A second prediction of the autoregulatory model is
that the lacZ Orb 3'UTR mRNA should compete with the
endogenous message for orb function. Consistent with this
prediction, increasing the relative dose of the transgene lacZ
Orb 3' UTR mRNA down regulates Orb protein expression
from the endogenous gene. Taken together with the dominant
negative activity of the lacZ Orb3'UTR transgene evident in
genetic assays these two lines of evidence provide strong
support for the autoregulatory model (Tan, 2001).
Since Orb is an RRM type RNA binding protein, the
simplest hypothesis is that Orb activates translation by
interacting with Orb mRNA rather than indirectly by
controlling the synthesis or functioning of some other protein
that binds to the 3' UTR. Two lines of evidence support the
idea that Orb associates with Orb mRNA in vivo. (1) The
defects in Orb mRNA localization are evident in orb mutant
ovaries. Both the endogenous Orb mRNA and the lacZ Orb 3'
UTR transgene mRNA are not properly localized in the
absence of wild-type orb activity. The abnormalities in
localization, particularly the loss of the anterior ring along
the nurse cell-oocyte margin, are consistent with a failure to
properly anchor Orb message to the oocyte cortex. (2)
Orb mRNA is associated with Orb protein in an
immunoprecipitable complex in vivo. This complex appears
to be specific since neither BCD nor NOS mRNAs can be detected in the
immunoprecipitates. The Orb
immunoprecipitates were assayed for 3' UTR sequences from four mRNAs:
OSK, K(10), GRK and BIC-D, that exhibit defects in localization
and translation in orb mutant ovaries, and
could potentially be targets for orb regulation in vivo. Of
these four, OSK, K(10) and BIC-D are present in Orb
immunoprecipitates, while GRK is not (Tan, 2001).
An obvious question is whether ORB binds directly to Orb mRNA or
any of the other mRNAs found in the immunoprecipitates. Orb
homologs (the CPEB proteins) in other species have been
shown to recognize a U-rich 'CPE' sequence in the 3' UTRs
of masked mRNAs. It is interesting to note that CPE-like sequences are
found in the Orb 3' UTR and also in the UTRs of OSK, K(10),
and BIC-D. In contrast, CPE-like sequences are not found in
GRK, NOS or BCD mRNAs. Also favoring a direct interaction is
the finding that a protein species that co-migrates with Orb
can be UV cross-linked to 32 P-labeled sequences from the Orb
3' UTR RNA. However, even if Orb recognizes the CPE-like
sequences in the Orb 3'UTR, the possibility
cannot be ruled out that autoregulation is nevertheless indirect, and, for example,
depends on activating the expression of some other protein
that also binds to Orb mRNA (Tan, 2001).
Assuming that Orb plays a direct role in autoregulation, two
different mechanisms could potentially account for the
autoregulatory activity. In the first, efficient translation of Orb
mRNA would depend upon localization to the oocyte cortex
and Orb protein would be required because it functions as an
anchor. In this model, the defects in the expression of ß-galactosidase
from the lacZ Orb 3' UTR mRNAs in the absence
of wild-type orb would be explained by a failure in
localization. Similarly, the antimorphic effects of the lacZ Orb
3' UTR transgenes would be explained by the displacement of
the endogenous mRNA from the cortex by the transgene
mRNA. Since lacZ mRNAs lacking the Orb 3' UTR are not
localized in the oocyte, but are translated even in orb mutants,
this postulated requirement for cortical association would have
to be a special feature of mRNAs containing the Orb 3' UTR (Tan, 2001).
For example, a translational repressor might be displaced from
the Orb 3' UTR when the message is associated with the cortex.
In the second, Orb protein would not only anchor Orb mRNA
to the cortex, but also actively promote its translation. In this
case, cortical localization would not in itself be sufficient for
the translation of either the Orb or lacZ Orb 3' UTR mRNAs.
Arguing in favor of a more active role is the fact that the Orb
homologs of Xenopus and other species, the CPEB proteins,
function in the translational regulation of masked maternal
mRNAs. These CPEB proteins are thought to bind to target
sequences in the 3' UTRs of masked maternal messages,
initially helping to ensure that the mRNAs remain
translationally silent. In response to an oocyte maturation signal, the
proteins then activate translation of the masked mRNAs by
promoting cytoplasmic polyadenylation (Tan, 2001 and references therein).
It would be reasonable to suppose that the regulatory
activities of Orb resemble the CPEB proteins of other
organisms. In this case Orb would positively autoregulate its
own expression by activating the polyadenylation of localized
Orb mRNAs. However, experiments aimed at demonstrating
this point have been inconclusive. Using an anchored-dT RT-PCR
procedure, it was found
that Orb mRNAs isolated from the two strong loss-of-function
orb mutants have much shorter poly(A) tails than wild type.
The caveat here is that the apparent reduction in poly(A) tail
length may be only an indirect consequence of the defect in
orb function. For example, orb343 is an apparent protein null and the level of Orb mRNA is quite low. The short poly(A) tails
in this mutant could be due to the fact that the message is
targeted for degradation and deadenylated by an Orb
independent mechanism. For the hypomorphic allele orbmel,
the average poly(A) length appeared to be only slightly shorter
than wild type. The presence of Orb mRNAs with extended
poly(A) tails is not surprising as substantial quantities of Orb
protein are expressed in orbmel mutant ovaries, especially in the
pre-vitellogenic stages. However, this finding leaves open the
possibility that orb activates the translation of Orb mRNAs by
some other mechanism (Tan, 2001).
While a positive autoregulatory feedback loop would provide a
mechanism for ensuring that Orb accumulates at sites of
localized Orb mRNA, a number of important questions remain.
Some of these can be illustrated by comparing orb
autoregulation with the autoregulatory cycle of the Sex-lethal
gene (Sxl). For Sxl, autoregulation is
crucial to its function as a binary switch gene, on in females
and off in males. When the gene is on, Sxl proteins promote
their own synthesis by directing the splicing of Sxl pre-mRNAs
in the productive female pattern. When the gene is off and no
Sxl proteins are present, splicing is in the default male pattern
and the resulting mRNAs do not encode functional proteins. At
this point it seems unlikely that orb autoregulation is used as an
on/off switch; rather it is suspected that the autoregulation serves to augment the on site accumulation of Orb protein. In this view, Orb mRNAs
would be translated at a low level in the absence of Orb, while
translation would be upregulated in its presence. Consistent
with this suggestion it was found that ß-galactosidase is
expressed from the lacZ Orb 3'UTR transgene in both orb343
and orb303 ovaries even though these mutants have little or no
functional Orb. (There is, however, an important caveat that the
lacZ Orb 3' UTR mRNA does not have the long 5' UTR (which
contains several short Orfs) and consequently may not fully
reproduce the translational regulation of Orb mRNA) (Tan, 2001).
A second question relates to the initiation mechanism. In the
case of Sxl, productive splicing can only occur in the presence
of Sxl proteins. Consequently, activation of the autoregulatory
cycle depends upon a special initiation pathway that
bypasses this requirement. If Orb
mRNAs can be translated at a reduced level in the absence
of Orb protein, a special bypass mechanism would be
unnecessary. However, there remains the problem of
generating the proper spatial pattern of Orb protein
accumulation within the egg chamber. For example, if the
autoregulatory cycle is activated inappropriately in nurse cells,
it would promote the accumulation of Orb protein in these cells
instead of at the proper sites in the oocyte. One possibility
would be to link autoregulation to Orb protein localization -
for example, only Orb protein associated with the oocyte cortex
would be able to activate Orb mRNA translation. In this model,
the binding of free Orb protein to Orb mRNAs in transit in the
nurse cells or in the oocyte would either have no effect on
translation or (given the activities of CPEB proteins in other
species) might actually repress translation (Tan, 2001).
Another issue is the mechanism that limits the positive
autoregulatory feedback loop, preventing the over expression
of Orb protein. In the case of Sxl, several factors appear to be
responsible for limiting protein accumulation. One is the
comparatively low level of Sxl transcription, while another is
the instability of the Sxl protein. In addition,
in females Sxl proteins negatively regulate their own
translation by binding to target sites in the 3' UTR of the Sxl
mRNAs. For orb, there must be
mechanisms that turn off the autoregulatory cycle once
sufficient quantities of Orb protein have been synthesized at
particular sites. Since the localization of Orb mRNA changes
during oogenesis, one mechanism might be turnover (or re-localization)
of Orb mRNA. Another possibility is that high
levels of Orb protein inhibit, instead of promote, translation.
Finally, if positive autoregulation is coupled to the binding of
Orb protein to the cortex, then the cycle might be inactivated
once all Orb protein target sites are occupied. Further studies
will be required to resolve these questions (Tan, 2001).
After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Par-1 is shown to become restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. baz is required for Par-1 relocalization within the oocyte and the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization (Vaccari, 2002).
Genetic analysis of Drosophila par-1 function has revealed a requirement for the kinase during oocyte polarization; in par-1 null alleles, Orb fails to relocate to the posterior of the oocyte and eventually disappears, after which the oocyte loses its fate and becomes a nurse cell. In addition to Orb, two other oocyte determinants, BicD and Egl, also fail to relocalize within par-1 null oocytes (Vaccari, 2002).
During oocyte specification, localization of the determinants BicD, Egl, and Orb to the early oocyte relies on the asymmetric distribution of microtubules in the cyst, evident as a dense focus of MT in the oocyte. Depolymerization of the MT by colchicine abolishes the localization of BicD, Egl, and Orb and results in egg chambers with 16 nurse cells and no oocyte. It was therefore asked if the restriction of Par-1 to the oocyte during the transition from region 2a to region 2b is also MT dependent. Ovaries of flies fed with colchicine for 12 hr fail to localize Par-1 and Orb to the oocyte, indicating that Par-1 restriction to the oocyte is indeed MT dependent. This is in contrast to the localization of Par-1 to the fusome, which occurs independently of MT (Vaccari, 2002).
The presence and localization of Par-1 in the oocyte at the time of its determination and polarization complements the previously reported localization of Par-1 on the fusome prior to oocyte determination and establishes Par-1 as a unique oocyte marker, for at least two reasons. (1) Absence of any one of the oocyte determinants, BicD, Egl, or Orb, prevents the concentration of the two other determinants in this cell. In contrast, in the absence of Par-1, it is the relocalization of the determinants within the oocyte that is specifically affected. (2) BicD, Egl, and Orb are not present on the fusome, and the observed enrichment of these determining factors in the oocyte is the result of the enrichment of their RNAs in this cell during its specification. In contrast, no par-1 RNA is detected in the germline at such early stages. The idea that Par-1 is initially loaded on the fusome, where it perdures during the cyst divisions, and that it is later preferentially inherited by the oocyte, is favored. Taken together, the facts that par-1 mutants show no fusomal defects and that accumulation of Par-1 itself in the oocyte requires MT suggest that Par-1 does not affect the oocyte MT cytoskeleton from its fusomal location. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where it acts in conjunction with BicD, Egl, and Dynein to effect polarization (Vaccari, 2002).
The Dmnk (Drosophila maternal nuclear kinase, Chk2/loki) gene, encoding a nuclear protein serine/threonine kinase, is expressed predominantly in the germline cells during embryogenesis, suggesting its possible role in the establishment of germ cells. Dmnk interacts physically with Drosophila RNA binding protein Orb, which plays crucial roles in the establishment of Drosophila oocyte by regulating the distribution and translation of several maternal mRNAs. Considering similar spatiotemporal expression patterns of Dmnk and orb during oogenesis and early embryogenesis, it is suggested that Dmnk plays a role in establishment of germ cells by interacting with Orb. Although there are two forms of Dmnk proteins, Dmnk-L (long) and Dmnk-S (short) via the developmentally regulated alternative splicing, Orb can associate with both forms of Dmnk proteins when expressed in culture cells. However, immunohistochemical analysis has revealed that Dmnk-S, but not Dmnk-L, can affect the subcellular localization of Orb in a kinase activity-dependent manner, suggesting differential functions of Dmnk-S and Dmnk-L in the regulation of Orb (Iwai, 2002).
Translational regulation of maternal mRNAs in distinct temporal and spatial
patterns underlies many key decisions in developing eggs and embryos. In
Drosophila, Orb is responsible for mediating the translational activation of
mRNAs localized within the developing oocyte. Orb is a germline-specific RNA
binding protein and is one of the founding members of the CPEB family of
translational regulators. Orb associates with the Drosophila
Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein
complex that controls the localized translation of mRNAs in developing egg
chambers. One of the key orb regulatory targets is orb mRNA, and this
autoregulatory activity is critical for ensuring that Orb protein is expressed
at high levels in the oocyte. dFMR1 functions as a negative
regulator in the orb autoregulatory circuit, downregulating orb mRNA
translation (Costam, 2005).
To identify factors that could function in regulating Orb activity and/or
localization proteins that are physically associated with Orb in vivo were sought. For this purpose ovarian extracts were immunoprecipitated with Orb antibodies. Besides Orb, the immunoprecipitates contained a complex set of proteins that were absent in the control immunoprecipitates. Attention was focused on four larger proteins because they are present in nearly the same yield as Orb (as judged by
staining) and are well resolved from other polypetides. The association of these four proteins with Orb is RNase resistant; they are observed not only when an RNase inhibitor is present, but also when the immunoprecipitates are treated with RNase or when the immunoprecipitation is done in the presence of RNase (Costam, 2005).
The four proteins were identified by mass spectroscopy. The largest 150 kd species corresponds to Lingerer. Lingerer is expressed at high levels in the nervous system, imaginal discs, and gonads. It is required for viability and has been implicated in sexual behavior. The 120 kd species is CG18811-PA. Nothing is known about its function in flies; however, related proteins called Caprins have been identified in vertebrates including humans and are thought to function in cell proliferation. The species migrating slightly more slowly than Orb is Rasputin (Rin). Rin is the fly homolog of the RasGAP SH3 binding protein (G3BP),
and it is thought to regulate RNA metabolism in response to Ras signaling.
Finally, the smallest protein, which is the subject of this study, corresponds
to the Drosophila Fragile X Mental Retardation protein (dFMRP) (Costam, 2005).
Though dFMR1 is a KH domain RNA binding protein, it is associated with
Orb in an RNase resistant complex. Although this could indicate that Orb and
dFMR1 complex with each other through (direct or indirect) protein-protein
interactions, an inability to produce soluble recombinant Orb has precluded
testing for direct interactions in vitro. In addition, it should be noted that
the fact that the Orb-dFMR1 complex in ovarian extracts is RNase resistant does
not exclude the possibility that Orb and dFMR1 coassemble into mRNPs because
the two proteins recognize sequences in the same RNA species. That there must
be some type of target specificity in Orb-dFMR1 complex assembly is suggested
by both Western and confocal analysis. Though dFMR1 appears to be present in
Orb immunoprecipitations in near molar yield, Western blots indicate that only
a subfraction of the total dFMR1 in ovaries is in an immunoprecipitable complex
with Orb. One reason for this is that all of the somatic cells in the ovary
have dFMR1, whereas they do not contain Orb. However, even within the germline
not all of the dFMR1 is associated with Orb. For example, in the germarium
dFMR1 is found in stem cells and the mitotic cysts, whereas Orb is not
readily detected until the 16-cell cyst is formed. Even after the 16-cell cysts
are formed and begin to develop, a significant fraction of the germline dFMR1
is localized in nurse cells, and with the exception of perinuclear particles,
this protein does not seem to be associated with Orb. In contrast, much
of the dFMR1 protein present in the oocyte appears to be specifically
associated with Orb. Moreover, it is in the oocyte where Orb activity is known
to be required. In previtellogenic chambers, dFMR1 and Orb colocalize around
the oocyte nucleus, concentrating most heavily at the posterior pole. After the
onset of vitellogenesis, Orb and dFMR1 colocalize along most of the oocyte
cortex except at the very posterior pole where there is a relatively low level
of dFMR1 and a high level of Orb (Costam, 2005).
An important question is whether the
Orb-dFMR1 association in the oocyte (and in the perinuclear particles in the
nurse cells) has regulatory consequences. Several lines of evidence argue that
it does. (1) dfmr1 interacts genetically with orb. orb
is weakly haploinsufficient and a significant fraction of the eggs laid by
females heterozygous for strong loss of function mutations such as
orb343 have D-V polarity defects indicative of a failure in
the grk-EGFR signaling pathway. This haploinsufficiency is enhanced by
transgenes, like HD19G, that express orb 3′ UTR sequences
fused to heterologous LacZ protein coding sequence. The HD19G
mRNAs compete with the endogenous orb mRNA for Orb protein binding and
further compromise the orb positive autoregulatory circuit.
D-V polarity defects of HD19G orb343/+ females can be
suppressed by reducing the dose of dfmr1 in half, whereas they can be
enhanced by adding an extra dfmr1 gene. Genetic interactions between
dfmr1 and orb are also seen in the absence of the HD19G
transgene. Moreover, in this case the D-V polarity defects seen at 18°C
for females heterozygous for orb343 or for another allele,
orbdec, can be almost completely suppressed by eliminating
dfmr1 altogether. (2) As would be expected if dfmr1 exerts
its effects on D-V polarity by modulating the orb autoregulatory
circuit, it was found that dfmr1 negatively regulates Orb protein expression.
Consistent with the suppression of D-V polarity defects, Orb accumulation in
HD19G orb343/+ females can be increased by reducing the dose
of dfmr1. Conversely, as would be expected from the finding that excess
dFMR1 enhances the frequency of D-V polarity defects, increasing the dose of
dfmr1 decreases Orb expression. (3) The effects of dfmr1 on
Orb accumulation are not restricted to circumstances in which orb
autoregulation is partially compromised. It is also seen in females that are
wild-type for orb. In this case, eliminating dfmr1 leads to the
overexpression of Orb protein. (4) orb interacts genetically with
dfmr1. Two independent orb mutations dominantly suppress the
excess germ cell phenotype seen in egg chambers from dfmr1 mutant
females. The fact that this phenotype can be suppressed by reducing the
orb gene dose would suggest that it arises, either directly or
indirectly, from the overproduction of Orb protein. (5) While
the inhibitory effects of dfmr1 on Orb
expression cannot be
unambiguously attributed to the particular subfraction of the dFMR1 protein that is complexed
specifically with Orb, the data indicate that dfmr1 activity is required
in the germline in order to regulate Orb expression (Costam, 2005).
Based on the pattern of Orb accumulation when dfmr1 activity is reduced, it would appear that
dFMR1 is initially required to repress the translation of orb mRNAs
while they are in transit from the nurse cells to the oocyte. If dFMR1 action
is direct in the nurse cells, it presumably associates with orb message
soon after it is synthesized in the nurse cell nuclei and acts to represses
translation. In this respect, it may be of interest that dFMR1-Orb
particles are observed around the edge of the nurse cell nuclei. These perinuclear particles
resemble the sponge bodies that are thought to be involved in assembling newly
synthesized mRNAs into translationally dormant mRNPs so that they can be
transported from the nurse cells to the oocyte. Although dFMR1 clearly
functions to block Orb expression in nurse cells, the fact that the amount of
Orb in nurse cells in the absence of dfmr1 activity is still much lower
than it is in the oocyte argues that there must be other factors besides dFMR1
that inhibit the translation of orb mRNA while it is in transit. As
observed in the nurse cells, Orb accumulation is upregulated in
dfmr13 oocytes. Orb expression is also upregulated in the
oocytes of HD19G orb343/+ egg chambers when dfmr1
activity is reduced, whereas it is repressed when dfmr1 activity is
increased. The effects of dfmr1 on Orb expression in the oocyte suggest
that it functions to attenuate the orb positive autoregulatory feedback
loop; however, the fact that Orb levels do not become excessive in the mutant
oocytes indicates that there must be other mechanisms to prevent Orb over
accumulation (Costam, 2005).
Unlike the nurse cells, most of the dFMR1 in the oocyte
colocalizes with Orb. Consequently, it would be reasonable to suppose that
dFMR1 modulates the orb autoregulatory circuit in the oocyte through its
association with Orb complexes that contain orb mRNA. As noted above,
while Orb-dFMR1 complexes in ovary extracts were found ot be RNase
resistant, it is suspected that complex assembly may depend upon the presence of
recognition sequences for each protein in the mRNA. In the case of orb
mRNA, previous studies indicate that Orb interacts with several sequences in
the 3′ UTR in vivo and in extracts. While the 3′ UTR does not
have sequences that match the dFMR1 consensus, there are three recognition
motifs in the 5′ UTR, at the beginning of the protein coding sequence. Thus,
a plausible idea is that these 5′ and 3′ recognition motifs serve
to recruit dFMR1 and Orb into the orb mRNA RNPs. That the dFMR1
recognition motifs may, in fact, be important for dFMR1 regulation is suggested
by the behavior of the HD19G mRNA, which contains all of the known Orb
target sequences in the orb 3′ UTR but does not have the dFMR1
motifs. The HD19G mRNA mimics the endogenous orb mRNA with
respect to its dependence on orb activity for both localization within
the oocyte and translational activation. However, HD19G mRNA responds
differently from the endogenous orb message to changes in dfmr1
activity (Costam, 2005).
Seemingly similar specificities are evident in the requirements for
orb and dfmr1 activity in regulating fs(1)K10 and
osk mRNA translation. Both of these mRNAs have Orb target sequences in
their 3′ UTRs, whereas only fs(1)K10 has potential dFMR1
recognition motifs. Whereas both fs(1)K10 and osk depend upon
orb activity for proper localization and translation, the
expression of Fs(1)K10 is upregulated in the absence of dfmr1 activity,
while there is no apparent effect on Osk. Because Orb is overexpressed in
dfmr1 mutants, one explanation for the upregulation of Fs(1)K10 is that
it is an indirect consequence of excess orb activity. However, since Osk
levels are unaltered, the idea is favored that dfmr1 functions to inhibit
orb-mediated activation of fs(1)K10 mRNA translation but does not
have a role in osk regulation. In this respect, it may be significant
that Orb specifically promotes the translation of osk mRNA localized at
the posterior pole in vitellogenic stage egg chambers. The posterior pole
differs from elsewhere along the cortex in that there seems to be much less
dFMR1 associated with Orb. Presumably other factors, such as Bruno and another
KH domain RNA protein, Bicaudal-C, would function in repressing osk
translation and counteracting orb activation. Much like dFMR1, these
proteins coimmunoprecipitate with Orb and are necessary for proper repression
of osk translation (Costam, 2005).
An intriguing question is whether the connection between Orb and dFMR1 in fly ovaries is relevant to the neurological phenotypes induced by inactivation of the fragile X gene in humans and mice. It is interesting in this regard that recent studies have implicated both CPEBs and FMRPs as key players not only in CNS development but also in learning and memory. Moreover, much as is observed in the fly germline, CPEBs and FMRPs appear to function antagonistically in regulating the localized translation of specific target mRNAs. If the activity and/or expression of mammalian CPEBs is negatively regulated by FMRPs, as is the case for Orb in flies, then it seems possible that the loss of FMRP activity may lead to excess CPEB activity in the nervous system and perturb the proper spatial or temporal regulation of translation (Costam, 2005).
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. (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).
The data indicate that yps is unlikely to regulate the expression or localization of Orb protein itself. (1) The distribution and levels of Orb produced by hypomorphic orb alleles are not altered in a ypsJM2 background. In addition, genetic analysis of yps, orb double mutants, shows that ypsJM2 specifically rescues defects in OSK mRNA localization and translation, but not orb-associated defects in dorsoventral chorion patterning or grk mRNA localization. Taken together, these results indicate a specific effect of yps on orb's function in localizing and/or translating OSK mRNA (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).
Doubly mutant ypsJM2orbmel/ypsJM2orbF303 ovaries display a novel phenotype, not observed in ypsJM2 or orbmel/orbF303 females: a small proportion (5%) of mid- and late-stage egg chambers are bipolar. Strong allelic combinations of orb also generate a high proportion of egg chambers with the oocyte mispositioned, but these egg chambers arrest oogenesis before budding from the germarium, or shortly thereafter. The low frequency of late-stage bipolar egg chambers observed in ypsJM2orbmel/ypsJM2orbF303 females may result from partial rescue of egg chambers that would normally have arrested at very early stages in orbmel /orbF303 ovaries, with a phenotype similar to orbF303/orbF303 egg chambers. Alternatively, this may be a novel phenotype resulting from the additive loss of both yps and orb. In either case, this phenotype suggests an earlier, as yet uncharacterized function of yps. In support of this idea, yps is expressed in the germarium (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. Thistarget 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).
During Drosophila oogenesis, the posterior determinant, Oskar, is tightly localized at the posterior pole of the oocyte. The exclusive accumulation of Oskar at this site is ensured by localization-dependent translation of oskar mRNA: translation of oskar mRNA is repressed during transport and activated upon localization at the posterior cortex. Previous studies have suggested that oskar translation is poly(A)-independent. This study shows that a long poly(A) tail is required for efficient oskar translation, both in vivo and in vitro, but is not sufficient to overcome Bruno response element-mediated repression. Moreover, accumulation of Oskar activity requires the Drosophila homolog of Cytoplasmic Polyadenylation Element Binding protein (CPEB), Orb. Since posterior localization of oskar mRNA is an essential prerequisite for its translation, it was critical to identify an allele of orb that does localize oskar mRNA to the posterior pole of the oocyte. Flies bearing the weak mutation orbmel localize oskar transcripts with a shortened poly(A) that fails to enhance oskar translation, resulting in reduced Oskar levels and posterior patterning defects. It is concluded that Orb-mediated cytoplasmic polyadenylation stimulates oskar translation to achieve the high levels of Oskar protein necessary for posterior patterning and germline differentiation (Castagnetti, 2003).
Cytoplasmic polyadenylation of mRNA requires CPEB to recruit the enzyme
poly(A) polymerase (PAP) on the regulated mRNA. Until recently, only one
family of PAP, containing both a catalytic domain and an RRM-like domain, was
known. A novel family of PAP has now been identified (Wang, 2002) that differs from
the canonical PAP for the absence of the RRM-like domain. The prototype of
this family is represented by C. elegans GLD-2 whose binding to the
RNA is mediated by GLD-3, a KH domain containing protein of the BicC family.
Interestingly, Drosophila BicC interacts physically
with Orb in co-immunoprecipitation experiments. Since the phenotype of
BicC mutants implicates BicC protein as a negative regulator of
osk translation (Saffman,
1998), tests were performed to see whether Orb interacts with the translational
repressor Bru. Indeed, a physical interaction between Bru and
Orb has been detected, as revealed by
the co-immunoprecipitation of Bru with Orb. By contrast, no direct
interaction has been detected between Bru and BicC (Castagnetti, 2003).
The relevance of these interactions in vivo is further confirmed by the
genetic interactions between the BicC locus and the orb and
aret loci -- the latter encoding Bru protein. Females
heterozygous for BicC show a number of AP patterning defects, ranging
from head defects to bicaudal embryos. The
BicC phenotype is suppressed when the mutation is combined with an
orb allele or the strong aret allele,
aretQB72. 85% of embryos produced by
Bic-CYC33/+ females fail to hatch and of those 60% are
bicaudal. The null allele orbF343 efficiently suppresses
the Bic-C phenotype and only 13% of the embryos produced by
BicCYC33/+; orbF343/+ fail to hatch,
none of which show a bicaudal phenotype. The strength of the
phenotype and the extent of the suppression depends on the orb
allele. Embryonic viability is also improved, up to 72%, in embryos produced
by BicCYC33/ aretQB72 females (Castagnetti, 2003).
Thus, combining measurement of osk poly(A) tail length in vivo with
quantification of the translation activity of the corresponding mRNAs in vivo
and in vitro, polyadenylation has been shown to corrolate with the
translational status of the mRNA. A mutation in Orb, the Drosophila
CPEB, leads to shortening of the osk poly(A) tail and to a reduction
in Osk accumulation. The fact that, at least in vitro, a long poly(A) tail
neither overcomes BRE-mediated repression nor is necessary for repression of
osk reporter transcripts, suggests that cytoplasmic polyadenylation
is not the decisive event in translational activation of osk at the
posterior pole. Rather, it appears that the presence of a 200 A long tail on
osk mRNA promotes its efficient translation, allowing accumulation of
Osk to levels sufficient for both abdomen and germline formation to
proceed (Castagnetti, 2003).
The prevailing model, which is based on studies of translational control in
the Xenopus oocyte, suggests that the polyadenylation status of a
transcript correlates with its translational status: a short poly(A) tail
corresponding to a silenced mRNA and poly(A) tail elongation triggering
translational activation. In Xenopus, upon progesterone treatment a
wave of cytoplasmic polyadenylation activates translation of deadenylated and
silenced maternally derived mRNAs. In
Drosophila, translation of bicoid mRNA, which encodes the
anterior determinant of the embryo, is repressed until egg activation, when
poly(A) tail elongation triggers translation initiation.
Although the correlation between adenylation and translation still holds for
several transcripts, a growing body of evidence suggests that the two events
may be coincidental but not directly connected. Interestingly, deadenylation
and translational repression of Drosophila hunchback (hb)
and mouse tPA mRNAs can occur independently of each other. The transcripts
are deadenylated concomitant with translational repression, yet repression can
occur in the absence of ongoing deadenylation. In arrested primary mouse
oocytes, polyadenylation of the tPA mRNA is necessary to counteract the
default deadenylation that affects most other oocyte mRNAs, thus preventing
its degradation (Castagnetti, 2003).
Observations made in this study suggest that silencing and awakening of osk mRNA
translation can occur in the absence of changes in poly(A) tail length and, in
fact, osk mRNA bears a long poly(A) tail at all stages of oogenesis,
including when it is unlocalized and translationally silent. However, it is
still formally possible that at intermediate stages of oogenesis osk
mRNA undergoes a deadenylation that goes undetected in measurements on
bulk RNA, and that elongation of the poly(A) tail causes displacement of the
repressor complex, leading to translational derepression. This hypothesis is
supported by the fact that the repressor protein Bru shares a 50% sequence
identity with the Xenopus deadenylation promoting factor EDEN-BP.
However, no obvious pattern of deadenylation has been detected in vitro when
Bruno is added to the embryonic extract, nor could a shortening be detected of
the poly(A) tail of translationally silenced osk transcript recovered
from ovarian extract. Nevertheless, the results show that
BRE-mediated repression is effective independent of the length of the
poly(A) tail on osk transcripts, and that a silenced mRNP can be
assembled on a naked osk transcript, whether or not it bears a
poly(A) tail. These results suggest that polyadenylation is not the sole
determining event leading to translational derepression of osk mRNA
at the posterior pole, but that the maintenance of a long poly(A) tail, by
cytoplasmic polyadenylation, accounts for the enhancement of osk
translation and is required for efficient osk translation, to ensure
sufficient accumulation of Osk at the posterior pole of the
Drosophila oocyte to promote abdominal patterning and germline
differentiation (Castagnetti, 2003).
Furthermore, the physical interaction detected between Orb and Bru, and Orb
and BicC suggests the existence of a multi-protein complex containing both
positive and negative regulators of osk translation. In this
scenario, translational silencing and polyadenylation are linked through Bru
protein, offering a possible explanation as to how CPEB might be recruited to
mRNAs in Drosophila, where no canonical CPE has so far been
identified. Transcripts properly repressed by Bru, upon localization, could be
adenylated by the recruitment of Orb by Bru itself. Loss of Bru repression
would, therefore, result in loss of Orb binding with consequent deadenylation
and translational silencing. In this model, modulation of the poly(A) tail
would be part of the mechanism that regulates translation, ensuring a second
level of control over ectopic expression while localizing all the components
necessary for efficient translation. Remarkably, mutations in the BRE sites do
not result in ectopic osk translation,
suggesting the existence of a second layer of translational control. Moreover,
during embryonic development when osk translation is no longer
required, both Orb and Bru proteins are depleted in the embryo and
osk mRNA undergoes complete deadenylation (Castagnetti, 2003).
Bicaudal C (Bic-C) encodes an RNA-binding protein required maternally for patterning the Drosophila embryo. A set of mRNAs have been identified that associate with Bic-C in ovarian ribonucleoprotein complexes. These mRNAs are enriched for mRNAs that function in oogenesis and in cytoskeletal regulation, and include Bic-C RNA itself. Bic-C binds specific segments of the Bic-C 5' untranslated region and negatively regulates its own expression by binding directly to NOT3/5, a component of the CCR4 core deadenylase complex, thereby promoting deadenylation. Bic-C overexpression induces premature cytoplasmic-streaming, a posterior-group phenotype, defects in Oskar and Kinesin heavy chain:βGal localization as well as dorsal-appendage defects. These phenotypes are largely reciprocal to those of Bic-C mutants, and they affect cellular processes that Bic-C-associated mRNAs are known, or predicted, to regulate. It is concluded that Bic-C regulates expression of specific germline mRNAs by controlling their poly(A)-tail length (Chicoine, 2007).
Precise coordination of translational control and mRNA localization regulates the temporal and spatial expression of proteins that define the dorsal/ventral and anterior/posterior axes of the Drosophila embryo . These axes are established during oogenesis through the activities of the TGF-α homolog Gurken (Grk) and subsequent posterior accumulation of Oskar (Osk). During oogenesis, osk and grk mRNAs are localized in particular regions of the oocyte cytoplasm, and their localization is dynamic, highly regulated, and essential for their developmental functions. Translation from both of these mRNAs is also under complex regulation (Chicoine, 2007 and references therein).
In wild-type oogenesis, rapid circular streaming of the oocyte cytoplasm begins in late stage 10 and continues until stage 12, when the nurse cells transfer their cytoplasm into the oocyte. Cytoplasmic streaming has been linked to osk localization, because its disruption prevents anterior to posterior translocation of injected osk mRNA in stage-10b to -11 oocytes. Furthermore, Kinesin-1 mutants blocked specifically in cytoplasmic streaming display an abnormal persistence of osk in the center of stage-10 oocytes. Oocytes produced by females homozygous for a hypomorphic orb allele (orbmel) initiate cytoplasmic streaming prematurely. Because orb encodes an RNA-binding protein related to Xenopus cytoplasmic polyadenylation element binding protein (CPEB), this suggests that the timing of this process is regulated through one or more mRNA intermediates (Chicoine, 2007).
Stability and translational activity of maternally transcribed mRNAs are frequently regulated by cytoplasmic proteins that affect their polyadenylation state. In Xenopus oocytes, mos and cyclin B1 mRNAs undergo cytoplasmic elongation of their poly(A) tails at meiotic maturation, and this induces their translation. Cytoplasmic polyadenylation requires CPEB; CPSF (Cleavage and Polyadenylation Specificity Factor) and Symplekin, two factors also involved in nuclear polyadenylation; and Gld2, a cytoplasmic poly(A) polymerase. Cytoplasmic poly(A)-tail elongation depends on phosphorylation of CPEB at meiotic maturation. Before maturation, the polyadenylation complex also contains PARN, a deadenylase whose activity appears to counterbalance Gld2-dependent poly(A)-tail elongation. CPEB phosphorylation leads to a remodeling of the mRNP, which has been proposed to result in the release of PARN from the complex, thus leading to polyadenylation and translational activation (Chicoine, 2007 and references therein).
Regulation of poly(A)-tail length also contributes to regulation of Drosophila mRNAs involved in axis patterning. Orb has been implicated in cytoplasmic polyadenylation of osk mRNA and accumulation of Osk protein at the posterior pole of the oocyte. There is no Drosophila PARN ortholog, and the CCR4-NOT complex, which contains the deadenylase CCR4, POP2, and four NOT proteins, is the major deadenylase in Drosophila (Temme, 2004). The CCR4-NOT deadenylation complex can be recruited to specific mRNA targets in Drosophila embryos, and in yeast, by RNA-binding proteins such as Smaug, Nanos, and PUF-family members, resulting in activated deadenylatio. The mutant phenotypes of twin, the gene encoding CCR4, and measurements of cyclin A and B mRNA poly(A) tails in twin mutants, suggest that regulated deadenylation also occurs in Drosophila oogenesis, but an activator of the CCR4 deadenylase complex in ovaries has not yet been identified (Chicoine, 2007).
Bic-C is required maternally for specifying anterior position during early Drosophila development and for oogenesis (Mohler, 1986; Schüpbach, 1991; Mahone, 1995). Females heterozygous for Bic-C mutations produce embryos of several different phenotypic classes, including bicaudal embryos that consist only of a mirror-image duplication of 2-4 posterior segments. Homozygous Bic-C females are sterile because the centripetal follicle cells fail to migrate over the anterior surface of the oocyte at stage 10. Most egg chambers degenerate shortly after this event. Bic-C protein contains five KH (hnRNP K homology) domains and a SAM (Sterile Alpha Motif) domain. KH and SAM domains are RNA-binding motifs. The KH and SAM domains can also bind to domains of the same type, and SAM domains can bind SH2 domains. Bic-C binds RNA homopolymers in vitro, and a substitution mutation in its third KH domain (G296R) results in substantially decreased RNA-binding activity in vitro and a strong mutant phenotype in vivo. No specific target RNA for Bic-C has heretofore been identified, although Osk accumulation is premature in homozygous Bic-C oocytes (Chicoine, 2007).
This study reports that Bic-C associates with Bic-C mRNA in an ovarian mRNP complex and in gel-shift experiments, and that it can repress its own expression in vivo. Bic-C was overexpressed in germline cells, and premature cytoplasmic streaming, abrogation of posterior osk localization, and dorsal-appendage defects, were observed. The latter phenotype was suppressed, and embryonic viability was increased, by mutations in twin. Furthermore, hiiragi [(hrg) which encodes poly(A) polymerase] and orb mutations are potent dominant enhancers of the Bic-C-overexpression phenotypes. Accordingly, a direct association is found between Bic-C and the NOT3/5 subunit of the CCR4-NOT deadenylation complex, and Bic-C is required for poly(A)-tail shortening of endogenous Bic-C mRNA during early stages of oogenesis. These data show that Bic-C negatively regulates target mRNAs, including Bic-C, by recruiting the CCR4-NOT deadenylation complex, thus identifying an ovarian activator of this complex. Moreover, the results provide direct evidence in support of the hypothesis that Bic-C and Orb act antagonistically to regulate poly(A)-tail lengths of specific mRNA targets essential for embryonic patterning (Chicoine, 2007).
Several lines of evidence indicate that Bic-C negatively regulates its own expression by binding to an element within its 5'UTR and recruiting the CCR4 deadenylase complex through a direct association with NOT3/5. Several other RNA-binding proteins, such as Nova-1, FMR1P, HuD, PABP, and Orb, bind specifically to their own mRNAs, and, in most cases, these interactions are autoregulatory. Posttranscriptional mechanisms of autoregulation may provide a means of 'fine tuning' levels of regulatory RNA-binding proteins with respect to their target mRNAs, creating the proper equilibrium between silenced and active targets (Chicoine, 2007).
Bic-C-mediated autoregulation is likely essential for development, since overexpression of Bic-C in the female germline induces premature cytoplasmic streaming, which, in turn, produces defects in pole-plasm assembly, posterior patterning, and dorsal-appendage formation. These phenotypes are largely reciprocal to those observed when Bic-C function is reduced, and they are suppressed by reduction of endogenous Bic-C activity. Although the nos::vp16 promoter used to drive UASP-containing transgenes does not recapitulate the normal transcriptional regulation of Bic-C, the level of Bic-C expression it supports is approximately equal to that of wild-type. No attempt was made to overexpress Bic-C from its own promoter, because it was anticipated that doing so in a noninducible manner would result in dominant female sterility resulting from the overexpression phenotypes that were observed. Germline expression of the UASP-Bic-C transgene restored fertility to Bic-CYC33-homozygous females, albeit at a low frequency, possibly due to a lack of fine-tuned regulation, since overexpression phenotypes were observed. This demonstrates that the Bic-C protein produced from UASP-Bic-C is functional. Furthermore, a decrease was observed in the frequency and severity of dorsal-appendage defects induced by Bic-C overexpression through a concomitant reduction of endogenous Bic-C dosage. It is thus likely that the phenotypes observed upon Bic-C overexpression result from an increase in the wild-type function of Bic-C (Chicoine, 2007).
Bic-C-overexpression phenotypes suggest that its targets include mRNAs involved in regulating the onset of rapid cytoplasmic streaming. Consistent with this, overexpression of Bic-CG296R, a form with reduced RNA-binding activity (Saffman, 1998), did not affect cytoplasmic streaming. While the possiblility cannot be excluded that the G296R mutation abrogates other unknown functions of Bic-C, it is noteworthy that several mRNAs that coimmunoprecipated with Bic-C (par-1, Cp190, Cip4, Klp10A, RhoGAP18B, and CG17293) have proven or predicted roles in regulating the actin or tubulin cytoskeleton. It will be important to determine in future experiments whether Bic-C influences cytoplasmic streaming through a regulatory effect on one or more of these potential target mRNAs (Chicoine, 2007).
The results identify Bic-C as an activator of the CCR4 deadenylase complex. Recent data indicate that this complex can be targeted to mRNAs through interactions between different RNA-binding proteins and several of its subunits. PUF proteins interact with the POP2/CAF1 subunit of the complex, as is also likely for Smaug, whereas Nanos binds the NOT4 subunit to recruit the complex to CyclinB 3'UTR. Bic-C directly associates with the NOT3/5 subunit. It is speculated that the ability of different RNA-binding proteins to target different components of the CCR4 complex provides additional regulatory independence and diversity. An uncommon characteristic of activated deadenylation by Bic-C is that binding to the 5'UTR of the regulated mRNA is required, whereas recruitment of the deadenylation complex by other regulatory proteins occurs through 3'UTRs. Circularization of mRNAs through an association between poly(A)-binding protein and eukaryotic initiation factor 4G, which is part of the 5' cap-binding structure, places the 5' and 3'UTRs in close juxtaposition and enables them to function coordinately. Therefore, 3'UTR-binding proteins influence translation initiation. Conversely, this study demonstrates that Bic-C interacts with elements in the 5'UTR and influences poly(A)-tail length. Consistent with this, direct targeting of the yeast CCR4 deadenylation complex to a reporter mRNA results in its rapid decay, regardless of whether the targeting site is in the 3' or 5'UTR of the reporter (Chicoine, 2007 and references therein).
orb mutants produce a premature cytoplasmic-streaming phenotype similar to that of Bic-C overexpression, reduction of orb activity suppresses Bic-C-mutant phenotypes, and this study observed a remarkable enhancement of the Bic-C-overexpression phenotype in a heterozygous orb-mutant background. Because Bic-C overexpression disrupts posterior recruitment of pole-plasm components prior to any detectable effects on Orb levels or distribution, it is concluded that Bic-C and Orb directly regulate the expression of a common set of target mRNAs, rather than Bic-C operating solely through an effect on orb mRNA itself. Consistent with this, Bic-C and Orb proteins have been found in coimmunoprecipitation experiments to be in common mRNP complexes in ovaries. Orb has a role in cytoplasmic polyadenylation of osk mRNA, and genetic interactions suggest that Orb achieves this function together with poly(A) polymerase. Taken together, these data support the model that regulation of the poly(A)-tail length of specific mRNAs results from concomitant polyadenylation and deadenylation regulated by specific RNA-binding proteins. Consistent with this, in Xenopus oocytes, PARN deadenylase is present in the cytoplasmic polyadenylation complex and counteracts polyadenylation prior to meiotic maturation. Both deadenylation and polyadenylation depend on CPEB, the Orb homolog that interacts with both PARN deadenylase and Gld2 poly(A) polymerase. A role in translational repression has not yet been described for Orb, but the observations that Bic-C is involved both in direct activation of deadenylation by CCR4, and also in poly(A)-tail length elongation in later oogenesis, suggests that it is central to poly(A)-tail length regulation and potentially responsible for a switch in the balance between deadenylation and polyadenylation. This switch appears to take place at mid-oogenesis, before stage 9, for Bic-C mRNA, but it could be timed differently for other mRNA targets. The transition from promoting deadenylation to promoting polyadenylation could depend on specific regulatory proteins bound to each mRNA and/or on posttranslational modifications to Bic-C itself (Chicoine, 2007).
Cytoplasmic polyadenylation has an essential role in activating maternal mRNA translation during early development. In vertebrates, the reaction requires CPEB, an RNA-binding protein and the poly(A) polymerase GLD-2. GLD-2-type poly(A) polymerases form a family clearly distinguishable from canonical poly(A) polymerases (PAPs). In Drosophila, canonical PAP (see Hiiragi) is involved in cytoplasmic polyadenylation with Orb, the Drosophila CPEB, during mid-oogenesis. This study shows that the female germline GLD-2 is encoded by wispy. Wispy acts as a poly(A) polymerase in a tethering assay and in vivo for cytoplasmic polyadenylation of specific mRNA targets during late oogenesis and early embryogenesis. wispy function is required at the final stage of oogenesis for metaphase of meiosis I arrest and for progression beyond this stage. By contrast, canonical PAP acts with Orb for the earliest steps of oogenesis. Both Wispy and PAP interact with Orb genetically and physically in an ovarian complex. It is concluded that two distinct poly(A) polymerases have a role in cytoplasmic polyadenylation in the female germline, each of them being specifically required for different steps of oogenesis (Benoit, 2008).
In many species, the oocyte and early embryo develop in the absence of transcription. Therefore, the first steps of development depend on maternal mRNAs and on their regulation at the level of translation, stability and localization. Regulation of mRNA poly(A) tail length is a common mechanism of translational control. Deadenylation or poly(A) tail shortening results in mRNA decay or translational repression. Conversely, poly(A) tail elongation by cytoplasmic polyadenylation results in translational activation. How the poly(A) tail length of a particular mRNA and, consequently, its level of translation are determined has been a matter of investigation for many years. It is becoming clear that poly(A) tail length results from a balance between concomitant deadenylation and polyadenylation (Benoit, 2008).
The molecular mechanisms of cytoplasmic polyadenylation have been investigated in Xenopus oocytes. The specific RNA-binding protein in the reaction is CPEB (Cytoplasmic polyadenylation element binding protein), which binds the CPE in the 3'-UTR of regulated mRNAs. Two other factors, CPSF (Cleavage and polyadenylation specificity factor) and Symplekin, are required in addition to a poly(A) polymerase. Before meiotic maturation, the polyadenylation complex also contains PARN, a deadenylase whose activity counteracts poly(A) tail elongation. At meiotic maturation, CPEB phosphorylation results in the release of PARN from the complex, thus leading to polyadenylation and translational activation (Benoit, 2008).
CPSF and Symplekin are also required for nuclear polyadenylation, a cotranscriptional reaction that leads to the synthesis of a poly(A) tail at the 3' end of all mRNAs. A canonical poly(A) polymerase (PAP) is responsible for poly(A) tail synthesis during nuclear polyadenylation. Particular isoforms of PAP were first thought to be required for cytoplasmic polyadenylation. Moreover, TPAP (Papolb - Mouse Genome Informatics), a testis-specific PAP in mouse, is cytoplasmic in spermatogenic cells and has been shown, using a Tpap knockout, to be required for cytoplasmic polyadenylation of specific mRNAs and for spermiogenesis. More recently, a new family of atypical poly(A) polymerases, the GLD-2 family, has been characterized, with a first member identified in C. elegans. GLD-2-type proteins exist in all eukaryotes, where they have different functions (Benoit, 2008).
In C. elegans, GLD-2 is required for entry into meiosis from the mitotic cycle in the gonad, and for meiosis I progression. C. elegans GLD-2 has a poly(A) polymerase activity in vitro and in vivo. In Xenopus oocytes, GLD-2 is found in the cytoplasmic polyadenylation complex, within which it directly interacts with CPEB and CPSF, and it has a poly(A) polymerase activity in vitro in the presence of the other factors of the complex. GLD-2 is in complexes with mRNAs, such as cycB1 and mos, that are regulated by cytoplasmic polyadenylation. It is thus very likely that GLD-2 plays a role in cytoplasmic polyadenylation during Xenopus meiotic maturation. However, although cytoplasmic polyadenylation of mos and cycB1 mRNAs is required for meiotic maturation, the functional role of Xenopus GLD-2 in meiotic maturation has not been addressed. Unexpectedly, although mouse GLD-2 (Papd4 - Mouse Genome Informatics) is found in oocytes at metaphases I and II, a recent study shows that oocyte maturation in GLD-2 knockout mice is not altered, demonstrating that if mouse GLD-2 acts as a poly(A) polymerase at this stage, another protein acts redundantly (Benoit, 2008 and references therein).
Cytoplasmic poly(A) tail elongation is also crucial in early embryos to activate the translation of mRNAs, including that of bicoid (bcd), which encodes the anterior morphogen. Polyadenylation and translation occur upon egg activation, a process that also induces the resumption of meiosis from the metaphase I arrest in mature oocytes, and which is triggered by egg laying, the passage of the egg through the oviduct. A link has been established between cytoplasmic polyadenylation and meiotic progression at egg activation because mutants defective for meiotic progression are also defective for poly(A) tail elongation (Benoit, 2008).
This study analyzed the function of Drosophila GLD-2 in the female germline. This protein is encoded by wispy (wisp), a gene previously identified genetically, and it therefore referred to as Wisp. Wisp has a poly(A) polymerase activity in vitro and in vivo, and it is required for poly(A) tail elongation of maternal mRNAs during late oogenesis and early embryogenesis. Wisp is required for meiotic progression in mature oocytes. A key target of Wisp during this process is cortex (cort) mRNA, which encodes a meiosis-specific activator of the anaphase-promoting complex (APC). This demonstrates the role of polyadenylation and translational activation in meiotic progression. In addition, the respective roles of conventional PAP and of Wisp in oogenesis were investigate, and PAP and Orb were shown to be involved earlier than Wisp and The. These results establish the requirement of two poly(A) polymerases for cytoplasmic polyadenylation at different steps of oogenesis (Benoit, 2008).
Two genes, CG5732 and CG15737, encoding GLD-2 homologs are present in the Drosophila genome. The corresponding proteins share the characteristics of GLD-2 family members in other species. They have a catalytic DNA polymerase β-like nucleotidyltransferase domain containing three conserved aspartic acid residues that is included in a larger conserved central domain, a PAP/25A-associated domain, and they lack an RNA-binding domain. The region that is N-terminal to the central domain is variable in size and non-conserved in the Drosophila GLD-2. Several CG5732 cDNAs described in FlyBase are from adult testis, indicating that CG5732 is expressed in this tissue. RT-PCR verified that CG5732 is not expressed in ovaries. This study focused on CG15737, which was expressed in ovaries (Benoit, 2008).
This study characterized Wisp, one of the two GLD-2-type poly(A) polymerases in Drosophila. Wisp has a function in the female germline. Wisp is a bona fide poly(A) polymerase: it has poly(A) polymerase activity in a tethering assay that depends on a conserved residue in the catalytic domain. Wisp is required for poly(A) tail lengthening of a pool of mRNAs in late stages of oogenesis. GLD-2 poly(A) polymerases do not have an RNA-binding domain; instead, they interact with RNA through their association with RNA-binding proteins. In Xenopus oocytes, GLD-2 interacts with CPEB in a complex that is active in cytoplasmic polyadenylation. Wisp interacts directly with Orb. Consistent with a role for Wisp and Orb together in an ovarian cytoplasmic polyadenylation complex, wisp mutants are dominant enhancers of a weak orb allele. In C. elegans, GLD-2 has been reported to interact with the KH-domain RNA-binding protein GLD-3, which has homology with Drosophila BicC. Although this study found Wisp and BicC together in an ovarian RNP complex, their association is mediated by RNA, suggesting that the proteins do not interact directly. It has recently been reported that BicC functions in deadenylation: BicC recruits the CCR4-NOT deadenylase complex to mRNAs. However, a role was found for BicC in poly(A) tail elongation during oogenesis (Benoit, 2008 and references therein).
In addition to its function in oogenesis, Wisp-dependent cytoplasmic polyadenylation is required for the translation of essential determinants of the anteroposterior patterning of the embryo. bcd mRNA poly(A) tail elongation was known to be required for the deployment of the Bcd gradient from the anterior pole of the embryo. This study now shows that Osk and Nos accumulation at the posterior pole also depends on Wisp. This highlights the general role of poly(A) tail length regulation in Drosophila early development (Benoit, 2008).
In Drosophila, meiosis starts in the germarium, where several cells per germline cyst enter meiotic prophase. Meiosis is then restricted to a single oocyte that remains in prophase I during most of oogenesis. Progression to metaphase I (oocyte maturation) occurs in stage 13, with maintenance of metaphase I arrest in mature stage 14 oocytes. Arrested oocytes are then activated by egg laying, which induces the resumption of meiosis (Benoit, 2008).
The earliest phenotypes in wisp-null mutant are defects in metaphase I arrest and in the progression beyond this stage. This suggests that Wisp-dependent cytoplasmic polyadenylation and translational activation are essential for meiosis during and after metaphase I (but not for oocyte maturation). Consistent with this, massive translation appears to be dispensable for the completion of meiosis, but translational activation of specific mRNAs, at least of cort, is required. cort was identified as a Wisp target: cort poly(A) tail elongation and Cort accumulation in mature oocytes require Wisp. Moreover, defects in Cort accumulation in wisp mutant oocytes result in impaired CycA destruction, an event thought to be critical for meiotic progression. Wisp regulates many mRNAs at oocyte maturation, several of which might be involved at various steps of meiosis. Identification of these specific targets will be necessary to fully unravel the role of Wisp during meiosis (Benoit, 2008).
Cytoplasmic polyadenylation has been linked to meiotic progression at egg activation given that some maternal mRNAs undergo poly(A) tail elongation at egg activation. Moreover, bcd polyadenylation is affected in mutants that are defective in meiosis, such as cort mutants. It has been proposed that the link between cytoplasmic polyadenylation and egg activation results from the inactivation of canonical PAP activity by phosphorylation via the MPF (Mitotic promoting factor: Cdc2/CycB). CycB degradation by APC-Cort would both induce meiotic progression and release PAP inactivation, leading to polyadenylation (Benoit, 2008).
This model can be adapted with results presented in this study and in the recent literature. Two waves of cytoplasmic polyadenylation occur successively, one during oocyte maturation and one at egg activation. They both depend on Wisp poly(A) polymerase. The first wave is Orb-dependent and the pathway that triggers its activation is unknown. This polyadenylation induces the synthesis of Cort (and probably other proteins), which in turn is required for the second wave of cytoplasmic polyadenylation at egg activation. Cort could act in this process through the destruction of cyclins or of other proteins more specifically involved in the regulation of the polyadenylation machinery (Benoit, 2008).
A striking result in this paper is the requirement of two poly(A) polymerases for cytoplasmic polyadenylation during oogenesis. Since the discovery of GLD-2 poly(A) polymerases, it has been assumed that these proteins were responsible for cytoplasmic polyadenylation. The current data reveal a higher level of complexity to this regulation. The phenotypes of wisp mutants indicate a function of Wisp late in oogenesis. Entry into meiosis and restriction of meiosis to one oocyte, as well as DNA condensation in the karyosome, are unaffected in wisp mutants. By contrast, orb-null mutants arrest oogenesis in the germarium, with defects in the synchronous mitoses of cystoblasts and in the restriction of meiosis to one oocyte (Huynh, 2000). This study found that orb phenotypes corresponding to early defects in oogenesis, including oocyte determination and dorsoventral patterning, are dominantly enhanced by hrg mutants, strongly suggesting that canonical PAP and Orb act together in cytoplasmic polyadenylation during the first steps of oogenesis. Because Orb forms complexes with both PAP and Wisp, the same pools of mRNAs can be regulated by the two different complexes, at different steps of oogenesis. The inclusion of one or other poly(A) polymerase could allow for different types of regulation. In addition, it is possible that the presence of both poly(A) polymerases together in the complex could be required for some step of oogenesis (Benoit, 2008).
In Xenopus, GLD-2 catalyzes polyadenylation during oocyte maturation (Barnard, 2004; Rouhana, 2005), but the enzymes involved after fertilization have not been identified. Moreover, polyadenylation at earlier stages of oogenesis remains unexplored (Benoit, 2008).
CPEB function has been addressed genetically in mouse and the defect in the female germline of Cpeb-knockout mice was found to be during prophase I. By contrast, GLD-2 expression in the oocytes appears to start at metaphase I. Moreover, no female germline defective phenotype was observed in GLD-2 knockout mice. This demonstrates some level of redundancy in poly(A) polymerase function in mouse female meiosis, and indicates that the involvement of different types of poly(A) polymerase for translational activation in oogenesis and meiotic progression is common to other species (Benoit, 2008).
Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. This study found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. These data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control (Mukai, 2015).
To examine histone modifications in differentiating germ cells, wild-type ovaries were stained using monoclonal antibodies specific for histone modifications. The H3K36me3 histone modification, associated with active genes, accumulated in differentiating cystoblasts. H3K36me3 signals were increased in the differentiating cystoblasts that expressed the bam reporter gene (bam-GFP). By contrast, the H3K27me3 modification associated with gene repression accumulated in early germ cells, and its signals decreased as the cells differentiated. These results suggest that the H3K36me3 levels were upregulated in differentiating cystoblasts. Next, H3K36me3 levels were examined in the ovaries of the third instar larvae and bam86 mutant adult females, both of which contain undifferentiated germ cells. Although H3K27me3 signals were detected in these undifferentiated germ cells, strong H3K36me3 signals were not detected. Taken together, these data supported the idea that H3K36me3-mediated epigenetic regulation may be involved in germ cell differentiation.
(Mukai, 2015).
Set2 methyltransferase is responsible for the H3K36me3 modification. Immunostaining revealed that, in the germarium region, Set2 was expressed in most of the germline cells, and that nuclear Set2 levels increased in differentiating cystoblasts. To determine whether Set2 participates in H3K36me3 accumulation and differentiation, Set2 expression was inhibited by using an UAS-Set2.IR line. Set2 levels in germ cells were reduced by the expression of Set2 RNAi. Specifically, while Set2 signals in differentiating cystoblasts were detected in 100% of control (nanos-Gal4/+) germaria, the Set2 signals in the cystoblasts were significantly reduced in 57% of the germaria, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver. Next, H3K36me3 levels were investigated in the ovaries expressing Set2 RNAi. As expected, H3K36me3 levels were reduced as a consequence of Set2 RNAi treatment. In control ovaries, H3K36me3 signals in differentiating cystoblasts were detected in 97% of germaria. By contrast, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver, H3K36me3 signals in cystoblasts were severely reduced in 41% of the germaria. Moreover, germ cell differentiation was impaired because of the expression of Set2 RNAi. In 96% of the control germaria, cysts with branched fusomes were observed. However, fragmented fusomes were detected in 34% of the germaria expressing Set2 RNAi. These results indicate that Set2 was required for both H3K36me3 accumulation and cyst formation. Mosaic analysis was performed by using a Set2 null allele Set21. Strong H3K36me3 signals were observed in 80% of the control germline clones. By contrast, H3K36me3 levels were considerably reduced in 74% of the Set2- cystoblasts. Furthermore, a differentiation defect was observed that was similar to that induced by Set2 RNAi treatment in 84% of Set2- mutant cysts. These results suggest that Set2 is intrinsically required both for H3K36me3 accumulation in cystoblasts and for differentiation (Mukai, 2015).
To investigate the potential regulatory link between Set2 and Bam, their genetic interaction was analyzed. Reduction in Set2 activity by introduction of a single copy of Set21 dominantly increased the number of germaria with the differentiation defect in bam86/+ flies. Fragmented fusomes were observed in 26% of germaria from the Set21/+; bam86/+ females , as compared to 5% in bam86/+ and 3% in Set21/+ females. These results indicated that Set2 cooperates with bam to promote cyst formation. To determine whether bam expression requires Set2 activity, Bam expression in Set2- germline clones by immunostaining. Indeed, Set2 activity in germ cells was dispensable for bam expression. Conversely, nuclear Set2 expression in the germ cells was significantly reduced by bam mutation, suggesting that bam is involved in the regulation of Set2 in these cells. This result is consistent with the observation that H3K36me3 levels were reduced by bam mutation. Moreover, reducing of bam activity by introducing of a single copy of bam86 dominantly increased the number of germaria with weaker H3K36me3 signals in Set21/+ flies. Decreased H3K36me3 signals in the cystoblasts were observed in 29% of germaria from the Set21/+; bam86/+ females, as compared to 3% in Set21/+ and 2% in bam86/+ females. These data prompted an exploration of the mechanism of regulation of Set2 activity by bam (Mukai, 2015).
To address whether bambam is sufficient for H3K36me3 accumulation, H3K36me3 levels were examined in the ovaries carrying the hs-bam transgene, which is used to ectopically express bam+ by heat shock treatment (Ohlstein and McKearin, 1997). No GSCs with a strong H3K36me3 signal were observed in germaria from wild-type females 1 hour post-heat shock (PHS; n = 42). However, H3K36me3 levels in the GSCs were significantly increased in 51% of the germaria from hs-bam females 1 hour PHS (n = 65), indicating that ectopic bam expression is sufficient for H3K36me3 accumulation. Because Set2 is responsible for H3K36me3, it is speculated that bam may regulate Set2 activity to control H3K36me3 accumulation and GSC differentiation. To determine whether Set2 activity is required for these bam-mediated processes, the effect was studied of a reduction in Set2 activity on the GSC differentiation induced by bam. When bam+ was ectopically expressed by heat shock, GSC differentiation was induced as previously reported. In 71% of ovaries from hs-bam flies dissected 24 hours PHS, it was found that differentiating cysts, instead of GSCs, occupied the tip of germaria. By contrast, when both bam and Set2 RNAi were ectopically expressed, GSC loss was significantly suppressed. These data suggest that Set2 activity is regulated by Bam, and that Set2 acts downstream of bam and promotes differentiation (Mukai, 2015).
Nuclear Set2 levels were increased in differentiating cystoblasts. Furthermore, nuclear Set2 levels in germ cells were reduced by bam mutation. It is speculated that bam may regulate Set2 nuclear localization. Therefore, whether bam expression is sufficient for Set2 nuclear accumulation was examined. The subcellular localization of Set2 was examined in hs-bam flies cultured at 30°C. First, H3K36me3 levels were examined in the GSCs. H3K36me3 levels in GSCs were increased in 36% of the germaria from the hs-bam females, as compared to 6% in wild-type females. This result suggests that the ectopic expression of bam is sufficient for H3K36me3 accumulation. Next, Set2 subcellular localization was examined in GSCs of hs-bam females cultured at 30°C. Nuclear Set2 levels in GSCs were increased in 54% of the germaria from the hs-bam females, as compared to 12% in wild-type females. These results suggest that bam promotes the nuclear accumulation of Set2 (Mukai, 2015).
To understand the mechanism by which Set2 regulates germ cell differentiation, the genetic interaction between Set2 and the differentiation genes A2BP1 and orb, both of which are required for cyst differentiation, were examined. Reduction of Set2 activity by introduction of a single dose of Set21 dominantly increased the number of germaria exhibiting a differentiation defect in orbdec/+ flies. In 24% of germaria from the Set21/+; orbdec/+ females, fragmented fusomes were observed, as compared with 4% in orbdec/+ and 7% in Set21/+ females. By contrast, the reduction of Set2 activity did not significantly affect cyst formation in A2BP1KG06463/+ ovaries). These results implied that Set2 function is required to specifically regulate orb expression and promote cyst formation. To confirm this, orb expression was examined in Set2- cyst clones. Deletion of Set2 led to the delayed activation of orb. Although 74% of the control cyst clones located at the boundary of germarium regions 1 and 2a initiated orb expression, only 31% of Set2- cyst clones expressed orb. Most (61%) of the Set2- cyst clones in germarium region 2b recovered orb expression. These observations suggest that Set2 was required for the proper activation of orb in differentiating cysts. Next, the H3K36me3 state of the orb locus was investigated in the ovaries. ChIP assays demonstrated that the H3K36me3 enrichment in the 3'-UTR region of orb was significantly higher than in the 5'-UTR region. It has been reported that the H3K36me3 modification exhibits a 3'-bias, such that H3K36me3 is preferentially enriched at the 3' regions of actively transcribed genes. These results support the idea that orb expression in differentiating cysts is controlled in part by H3K36me3-mediated epigenetic regulation (Mukai, 2015).
Next, the H3K36me3 status was investigated in the orb gene in bam86 mutant ovaries. ChIP assays showed that bam mutation reduced the amount of H3K36me3 in the 3'-UTR region of the orb gene. The H3K36me3 modification is linked to transcriptional elongation. Therefore, the results suggested that bam activates orb expression through the epigenetic control. Additionally, H3K4me3 and RNA polymerase II levels in the 5'-UTR region of the orb gene were also reduced by bam mutation, implying a role for bam in transcriptional initiation. To investigate this possibility, further investigation will be needed in order to identify the enzymes responsible for H3K4me3 and exploring the interactions between bam and those enzymes (Mukai, 2015).
These results have shown that H3K36me3 levels are regulated by bam. As a cytoplasmic protein, Bam may indirectly regulate Set2 nuclear localization. Set2 exerts its functions through the interactions with cofactors. Understanding the mechanism by which Bam regulates Set2 will require the identification of the cofactors that mediate the nuclear transport of Set2. These data suggest a link between Bam and epigenetic transcriptional control. Bam may counteract epigenetic silencing in GSCs through H3K36me3-mediated epigenetic regulation. This study showed that orb expression is activated by epigenetic regulation. Because orb encodes a cytoplasmic polyadenylation element-binding protein, Orb may control translation in differentiating cysts in a polyadenylation-associated manner. Bam antagonizes the Nanos/Pumilio complex, which suppresses the translation of target mRNAs that encode differentiation factors . However, the ientity of the target mRNAs and the mechanisms for transcriptional activation have not yet been elucidated. Because Set2 is required for bam-induced GSC differentiation, studies focused on identifying the genes marked by H3K36me3 and on their epigenetic regulation will aid in the identification of the differentiation genes. Because Set2 is linked to transcriptional elongation, differentiation genes in GSCs might be poised for expression, but may be kept awaiting bam expression for full activation. It is anticipated that these results will facilitate a better understanding of the epigenetic mechanisms that regulate gametogenesis (Mukai, 2015).
The Orb CPEB protein regulates translation of localized mRNAs in Drosophila ovaries. While there are multiple hypo- and hyperphosphorylated Orb isoforms in wild type ovaries, most are missing in orbF303, which has an amino acid substitution in a buried region of the second RRM domain. Using a proteomics approach this study identified a candidate Orb kinase, Casein Kinase 2 (CK2). In addition to being associated with Orb in vivo, ck2 is required for orb functioning in gurken signaling and in the autoregulation of orb mRNA localization and translation. Supporting a role for ck2 in Orb phosphorylation, it was found that the phosphorylation pattern is altered when ck2 activity is partially compromised. Finally, it was shown that the Orb hypophosphorylated isoforms are in slowly sedimenting complexes that contain the translational repressor Bruno, while the hyperphosphorylated isoforms assemble into large complexes that co-sediment with polysomes and contain the Wisp poly(A) polymerase (Wong, 2011).
While only two Orb phosphoisoforms are resolved on SDS-PAGE gels, a combination of phosphatase treatment, analysis of phosphoisoforms in different mutants, and fractionation on Phos-Tag gels indicate that Orb must be phosphorylated at multiple sites on Tyr, Ser and/or Thr residues. At least seven distinct isoforms are resolved on Phos-Tag gels, four faster migrating species and three slower migrating species. Since the degree of retardation in this gel system depends largely upon the number of phosphate residues, the set of more rapidly migrating 'hypophosphorylated' isoforms are expected to have between zero and three phosphate residue, while the set of more slowly 'hyperphosphorylated' isoforms are expected to have four or more phosphate residues. Mobility in the Phos-Tag gel is also influenced by the location of the phosphorylated amino acid, and several bands appear to be doublets. Thus, there may be isoforms that have the same number of phosphorylated residues, but differ in which amino acids are modified. Further studies will be required to determine the number and location of the phosphorylated residues associated with each of the different isoforms (Wong, 2011).
Several lines of evidence argue that phosphorylation is critical for orb activity. One comes from the dramatic effects of the orbF303 mutation: the 'hyperphosphorylated' isoforms are absent and the two more slowly migrating 'hypophosphorylated' isoforms are largely missing as well. While this would link phosphorylation to Orb function, it is not immediately clear why the Tyr742 mutation has such drastic effects. The simplest model is that Tyr742 must be phosphorylated to generate the other phosphoisoforms. However, Tyr742 is predicted to be on the buried side of the second RRM α-helix and should have essentially no solvent accessibility. Thus, unless it is modified during translation, a scenario in which phosphorylation of this Tyr is obligatory for subsequent phosphorylation elsewhere seems unlikely. A more likely possibility is that the second OrbF303 RRM domain does not fold properly and this weakens or eliminates RNA binding. In the absence of RNA-binding, it is possible that oogenesis might arrest at a point prior to when most phosphoisoforms are generated. Arguing against this is the fact that the fast and slow phosphoisoforms are found in mutants in other genes that cause even earlier oogenesis arrest. Another possibility is that phosphorylation of the sites in the Orb protein that generate the collection of more slowly migrating isoforms requires prior binding to target RNAs. This idea is suggested by the structural changes that are induced when proteins that have two RRM domains interact with RNA. For example, when Sxl binds to its target RNAs, the two RRM domains rearrange so that they clamp around the RNA, while the linker region separating the two domains is converted from an unordered structure into a distorted but spatially fixed helix. In addition to stabilizing RNA:protein interactions, this rearrangement alters the ability of Sxl to physically interact with other splicing co-factors. If the Orb RRM domains and linker region also undergo similar conformational changes upon RNA binding, this could provide a mechanism for coupling binding to phosphorylation. Since the linker region separating the two RRM domains would be an obvious target for binding dependent conformational changes that could potentially modulate phosphorylation, it is intriguing that a tryptic peptide (which contains two potential CK2 sites) from this linker region is phosphorylated in vivo. Unfortunately, it was not possible to test this mechanism properly folded wild type (or mutant) Orb protein that had RNA-binding activity could not be generated (Wong, 2011).
Another line of evidence arguing that Orb activity is linked to its phosphorylation status is the difference in the spectrum of proteins associated with the hypo- and hyperphosphorylated isoforms. While Me31B and PABP are in complexes with both isoforms, Bruno appears to interact primarily with the 'hypophosphorylated' isoforms. This interaction fits with the striking difference in the distribution of Bruno and Orb proteins on sucrose gradients and with the limited co-localization of the two proteins to sponge bodies near the anterior of the oocyte. In contrast to Bruno, the poly(A) polymerase Wisp, which is needed to activate translation, interacts preferentially with the 'hyperphosphorylated' isoforms. Although a precursor-product relationship remains to be established for orb target mRNAs, this specificity would be consistent with a model in which hypophosphorylated isoforms are in complexes with mRNAs that are translationally repressed. Translational activation would then depend upon phosphorylation of the hypophosphorylated isoforms and reorganization of the Orb complex. Bruno and/or other repressive factors would be displaced from the complex, while the Wisp poly(A) polymerase would be recruited and could potentially begin extending the poly(A) tails. Supporting a model of this type, preliminary studies indicate that like ck2, mutations in wisp dominantly enhance the HD19G orbF343/+ DV polarity defects, while mutations in the Bruno gene arrest have the opposite effect (Wong, 2011).
The conclusion that the two isoforms are incorporated into complexes that differ substantially in their composition, and likely also their function, is supported by sucrose gradient fractionation of ovary extracts. 'Hypophosphorylated' isoforms are found mostly near the top of the gradient in comparatively small complexes (<80S). This region of the gradient is also greatly enriched in the translational repressors Bruno and Me31B, while these repressors are largely absent from the more rapidly sedimenting fractions that contain the polysomes. In contrast, 'hyperphosphorylated' isoforms are found in 80S complexes and polysomes. As would be expected if the hyperphosphorylated Orb in these big complexes is directly associated with ribosomes, rather than with some other type of very large RNP, a large collection of ribosomal proteins and translation initiation/elongation factors are found in Orb immunoprecipitates. The kinase most closely tied to CPEB phosphorylation in vertebrates is Aurora, which has been shown to phosphorylate the Ser174 residue in the N-terminal half of the Xenopus CPEB. However, Aurora's role in Orb phosphorylation is uncertain as this residue is not conserved in Orb and no genetic interactions were detected between aurora mutations and orb. Though these results don't exclude a role for Aurora, they suggest that other kinases may phosphorylate Orb. Using a proteomics approach two candidate Orb kinases were identified, SRPK2 and CK2, and this study has focused on CK2 (Wong, 2011).
Like orb, ck2 is needed during oogenesis for the formation of the DV polarity axis of the egg and embryo. Chorion defects characteristic of disruptions in the grk signaling pathway are observed in eggs laid by females heterozygous for the dominant negative ck2αTik allele or homozygous for the very weak loss-of-function ck2βand allele. Moreover, the genetic interactions between ck2 and orb in DV polarity would argue that these defects arise, at least in part, because ck2 is required for orb function in this particular signaling pathway. The most compelling of these interactions is between orb and the strong loss-of-function ck2βmbuδA26-2L allele. Unlike ck2αTik, eggs laid by ck2βmbuδA26-2L/+ females have no apparent DV polarity defects; however, when this mutation is introduced into a background partially compromised for orb activity, a very strong interaction is observed and almost three quarters of the eggs laid by trans-heterozygous females have chorion defects (Wong, 2011).
In addition to the genetic interactions in DV polarity, it was found that ck2 has a direct impact on orb autoregulation. Orb is required for localizing and activating the on-site translation of orb mRNA in the developing oocyte. Strikingly, both of these autoregulatory activities are disrupted in females that are only partially compromised for ck2. orb mRNA is not properly localized in vitellogenic stage egg chambers. In addition, the accumulation of Orb protein is reduced compared to wild type. Since the females harboring these ck2 mutant combinations are viable and morphologically normal, it would appear that like DV polarity, these orb regulatory activities are especially sensitive to reductions in ck2 activity (Wong, 2011).
The effects of ck2 on orb function correlate with changes in the phosphoisoform profile. In ck2 backgrounds that have modest effects on polarity and/or orb activity, there is a small shift towards the hypophosphorylated isoforms. In backgrounds that have stronger effects on orb activity and/or show synergistic genetic interactions with orb, the changes in phosphoisoform profile are more pronounced. This is, perhaps, most evident in females heterozygous for the amorphic ck2βmbuδA26-2L allele. In addition to the hypo- and hyperphosphorylated isoforms visible on regular SDS-PAGE gels, these females have an Orb species that has a similar mobility to dephosphorylated Orb after λ phosphatase-treatment (Wong, 2011).
While these findings indicate that ck2 is required both for orb activity and to generate the normal array of Orb phosphoisoforms, the fact that this kinase has been implicated in many cellular processes raises the possibility that the effects on orb are an indirect consequence of pleiotropic defects in oogenesis induced by ck2 mutations. Unfortunately, this possibility can not be excluded; however, arguing against it is the fact that all of the experiments were done under conditions in which ck2 activity is only partially compromised. Though this might not eliminate pleiotropic effects, it should certainly minimize them and at the same time reveal cellular processes that are especially sensitive to reductions in ck2 activity and thus most likely to be intimately connected to ck2 function. DV polarity, orb activity and Orb phosphorylation would fit into this category. As for how ck2 impacts orb activity and the Orb phosophoisoform profile, the simplest explanation is that it is directly responsible for phosphorylating Orb. Consistent with this idea, there are twelve potential CK2 sites, of which nine are conserved even in distantly related Drosophila species. Of the conserved sites, two are in the linker region separating the two Orb RRM domains and, as mentioned above, could be potential candidates for RNA binding dependent phosphorylation. The seven remaining sites are in short conserved sequence blocks in the otherwise poorly conserved N-terminal half of the Orb protein. Interestingly, six of these are in a closely spaced cluster. The physical association between CK2 and Orb would also support a direct mechanism. On the other hand, it is also possible that ck2 acts indirectly through intermediate kinases. In this case, the activity of this kinase cascade would have to be especially sensitive to changes in ck2 levels. However, even in this indirect scenario, the effects of ck2 mutations on orb activity and the phosphoisoform profile provide further evidence linking the regulatory functions of the Orb protein to its phosphorylation status (Wong, 2011).
Whether the effects of ck2 on orb are direct or indirect, there are indications that other kinases must phosphorylate Orb. For one, there are likely to be phosphorylated tyrosine residues since the Orb phosphoisoforms are not completely collapsed by the Ser/Thr specific Protein Phosphatase 1, but are collapsed by λ phosphatase. Consistent with this possibility, preliminary experiments indicate that Orb is recognized by phosphotyrosine antibodies. Secondly, other Ser/Thr kinases might be needed to activate Orb. Studies have demonstrate that srpk2 mutations disrupt oogenesis and have DV polarity phenotypes. Though the reported phenotypes seem different from those of well-characterized orb mutants, this study found that a P-element-induced mutation in srpk2 dominantly enhanced the orb DV polarity defects. Finally, since known orb target mRNAs exhibit different patterns of localization and translation, they are likely to be associated with a unique set of regulatory proteins and depend upon different signaling cascades for translational activation. Thus, a more likely scenario is that CK2 is just one of several potential Orb kinases, and that translation of different orb target mRNAs might require the deployment of specific constellations of modifying enzymes (Wong, 2011)
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