nanos
Localization of NOS mRNA and Bicoid mRNA are independent of one another. Females with mutation in cappuccino, oskar, spire, staufen, tudor, valois and vasa produce embryos containing normal levels of NOS mRNA, but fail to localize this RNA properly to the posterior pole, and hence develop abdominal defects characteristic of nos mutants (Gavis, 1994). In addition, these mutants disrupt the pole plasm, the special cytoplasm at the posterior of the embryo, and the pole cells, the germ line precursors.
The Nanos 3' untranslated region, like that of the Bicoid RNA, is sufficient for RNA localization. The Bicoid RNA localization signal can be used to mislocalize Nanos, producing embryos with two sources of Nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with Nanos RNA localize only to the anterior, have greater nanos gene activity than embryos with Nanos RNA localized posteriorly. (Gavis, 1992).
Although the unlocalized NOS mRNA is stable in embryos from females mutant for any of the posterior group genes, these embryos appear to lack nos activity because they develop the abdominal defects characteristic of embryos produced by nos mutant females. Unlocalized NOSm RNA is translationally inactivated. Translational inactivation is mediated by the NOS 3'UTR and can be alleviated either by replacement of the 3'UTR with heterologous 3'UTR sequences or by posterior localization. Thus, RNA localization provides a novel mechanism for translational regulation (Gavis, 1994).
The targeting of NOS mRNA is achieved by the combined effects of multiple, partially independent sequences throughout the complex 3'UTR region (Gavis, 1996a).
A discrete translation control element within the Nanos 3' untranslated region acts independently of the localization signal to mediate translational repression of unlocalized Nanos mRNA. This region, designated as nos translational control element (TCE) is evolutionarily conserved and is predicted to form a dual stem-loop structure. It is possible that TCE-mediated repression is achieved by blocking a stimulatory effect of a poly(A)-binding protein, or that a TCE-protein complex may itself block translational initiation or elongation. One component of the Nos localization machinery, VASA protein, plays a role in overcoming TCE-mediated repression. RNAs bearing the Nos translational control element are completely inactive in embryos from vasa mutants. Thus, translation of Nos mRNA occurs by a specific derepression mechanism, requiring VAS protein (Gavis, 1996b).
Deletion of the 184-nucleotide translational control element (TCE) from the 3' UTR leads to the derepression of NOS mRNA in bulk cytoplasm and the development of lethal anterior defects. A minimal mRNA containing only the TCE rescues nos mutant embryos to adulthood. The TCE is sufficient to confer on maternal Torso mRNA all three aspects of NOS mRNA regulation: translational repression in the bulk cytoplasm, localization to the pole plasm, and translational activation at the posterior pole. These three phenomena are coupled intimately, as mutations in a pair of CUGGC pentamers within the TCE simultaneously abrogate all three regulatory events. This coupling suggests a model in which the polarized distribution of NOS protein is generated primarily by translational control and that NOS mRNA localization is a byproduct of this regulation, at least in part (Dahanukar, 1996)
In bullwinkle mutants (unlike other bicaudal mutants) at stage 10 Oskar mRNA is localized correctly to the posterior pole of the oocyte. By early embryogenesis, however, some Oskar mRNA is mislocalized to the anterior pole. Consistent with the mislocalization of Oskar mRNA, a fraction of the Vasa protein and Nanos mRNA are also mislocalized to the anterior pole of bullwinkle embryos. Mislocalization of Nanos mRNA to the anterior is dependent on functional Vasa protein. Although the mirror-image segmentation defects appear to result from the action of the posterior group genes, germ cells are not formed at the anterior pole. The bicaudal phenotype is also germ-line dependent for bullwinkle. It seems that Bullwinkle interacts with the cytoskeleton and extracellular matrix and is necessary for gene product localization and cell migration during oogenesis after stage 10a (Rittenhouse, 1995).
Mutations in the BicaudalD (BicD) gene lead to a global reorganization of the Drosophila body pattern such that the head, thoracic, and anterior abdominal segments are replaced by posterior abdominal segments and terminalia. The primary cause of this phenotype is the inhibition of two anterior factors, Bicoid and Hunchback, by mislocalized activity of the posterior determinant Nanos. The BicD gene encodes a coiled-coil protein similar to the carboxy-terminal portion of the myosin heavy chain. BicD protein is uniformly distributed throughout wild-type oocytes but is concentrated at the anterior pole of BicD mutant oocytes together with ectopic Nanos activity. Taken together, these results suggest that BicD encodes a cytoskeleton-like protein involved in transporting or anchoring the Nanos morphogen, or NOS mRNA, within the oocyte cytoplasm (Wharton, 1989).
Three mRNAs that dictate anterior, dorsoventral, and terminal specification--Bicoid, Toll, and Torso, respectively--showed increases in polyadenylate [poly(A)] tail length concomitant with translation. In contrast, posteriorly localized Nanos mRNA, although also translationally activated, is not regulated by poly(A) status. This implies the existence of at least two mechanisms of mRNA activation in flies (Salles, 1994).
Oskar protein directs the deployment of Nanos. To avoid inappropriate activation of nos, osk activity must appear only at the posterior pole of the oocyte, where the OSK mRNA becomes localized during oogenesis. Translation of OSK mRNA is, and must be, repressed prior to its localization; absence of repression allows OSK protein to accumulate throughout the oocyte, specifying posterior body patterning throughout the embryo. Translational repression is mediated by an ovarian protein, Bruno, that binds specifically to Bruno response elements (BREs), present in multiple copies in the OSK mRNA 3'UTR. Addition of BREs to a heterologous mRNA renders it sensitive to translational repression in the ovary (Kim-Ha, 1995).
Patterning of the anterior-posterior body axis during
Drosophila development depends on the restriction of
Nanos protein to the posterior of the early embryo.
Synthesis of Nanos occurs only when maternally provided
Nanos RNA is localized to the posterior pole by a large, cis-acting
signal in the Nanos 3' untranslated region (3'UTR);
translation of unlocalized Nanos RNA is repressed by a 90
nucleotide translational control element (TCE), also in
the 3'UTR. The majority
of Nanos mRNA in the embryo is not localized to the posterior
pole but is distributed throughout the cytoplasm, indicating
that translational repression is the primary mechanism for
restricting production of Nanos protein to the posterior.
Through an analysis of transgenes bearing multiple copies
of Nanos 3'UTR regulatory sequences, evidence is provided
that localization of Nanos mRNA by components of the
posteriorly localized germ plasm activates its translation by
preventing interaction of Nanos RNA with translational
repressors. This mutually exclusive relationship between
translational repression and RNA localization is mediated
by a 180 nucleotide region of the Nanos localization signal,
containing the TCE. These studies suggest that the ability
of RNA localization to direct wild-type body patterning
also requires recognition of multiple, unique elements
within the Nanos localization signal by novel factors (Bergsten, 1999).
While combinations of three individual NOS 3'UTR elements
are sufficient for wild-type localization, three copies of an
individual element cannot completely compensate for the loss
of the other two. In addition, different elements behave
uniquely when multimerized. The limited posterior
localization conferred by individual localization elements
requires osk-dependent assembly of germ plasm, suggesting that germ plasm components can recognize each element to some extent. The fact that osk, vas and tud
mutations do not produce consistent partial localization
phenotypes characteristic of NOS 3'UTR deletion mutants further suggests that Osk, Vas and Tud
recognize the localization signal as a complex, rather than by
interactions of individual proteins with individual elements.
Recognition of localization signal elements by these germ
plasm components, therefore, cannot easily explain the differential behavior of different elements upon
multimerization and the requirement for several different elements in wild-type localization.
Conservation in sequence and function of NOS localization
elements between D. melanogaster and D. virilis predicts that
these elements contain recognition sites for localization factors
common to both species. With the exception of two elements,
which contain binding sites for the Smaug
protein
proposed to mediate translational repression, there is no significant similarity among the conserved
segments of different elements, with respect to both primary
sequence and predicted secondary structure. Taken together,
these results suggest that different elements are recognized
uniquely by different cytoplasmic factors, not yet identified,
that provide an interface between NOS mRNA and the germ plasm
components. Preliminary evidence from UV-crosslinking
experiments indicates that embryo extracts contain several
proteins that interact differentially with the localization
elements. The ability
of one of the elements to act cooperatively, either with itself or with
a second element, points to an important role for these sequences
in assembling a localization complex (Bergsten, 1999).
An embryonic protein of 135 kDa, Smaug, binds NOS mRNA at a stem loop structure containing CUGGC pentamers. These stem loop structures, within the TCE are referred to as Smaug recognition elements. Analysis of point mutations in the SREs reveals a strong correlation between Smaug binding and translational repression; mutants unable to bind Smaug in vitro are not repressed translationally in vivo, whereas mutants that do bind Smaug remain repressed translationally. These results strongly suggest that Smaug acts in translational repression of unlocalized NOS mRNA. Translational repression is essential, as embryos expressing a NOS mRNA with mutated SREs develop with anterior body patterning defects and die, despite correct localization of RNA (Smibert, 1996).
Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. This study describes cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, it is demonstrated that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor (Smibert, 1999; full text of article).
Smaug acts as a translational repressor; it binds NOS mRNA at a stem loop structure found within the Nos translational control element. Signals that mediate regulation of NOS mRNA reside in its 3' UTR. In particular, the 184 nt translational control element (TCE) contains
all of the 3' UTR signals that are necessary and sufficient for NOS function.
The key components of the TCE consist of a pair of
redundant hairpins, each bearing the loop sequence CUGGC. These mediate both repression of NOS mRNA in the bulk cytoplasm as well as Oskar-dependent activation in the pole plasm. Thus, the TCE hairpins constitute the
essential cis-acting elements of a translational switch responsible for generating a polarized distribution of Nos protein in the early embryo (Dahanukar, 1999 and references).
Using a set of mutant TCE hairpins, it was asked whether binding to Smaug in vitro correlates with TCE-mediated repression of NOS mRNA in vivo. Binding was monitored in gel mobility shift experiments, and TCE activity was monitored using transgenic flies that express appropriately altered NOS mRNAs. In brief, derepression of NOS mRNA in the bulk cytoplasm, where NOS mRNA is usually held in a translationally silent repressive complex, results in a reduction in the levels of anterior Bcd and Hb proteins, which in turn results in the development of lethal head defects. Binding of Smaug to the G12U mutant TCE, which is strongly defective in vivo, is reduced by a factor of at least 50 relative to wild type; binding to the moderately defective A15G mutant TCE is reduced by a factor of at least 5; and binding to the U18C mutant TCE, which regulates nos normally, is indistinguishable from binding to the wild-type hairpin. Thus, the variation in degree of binding of these mutant hairpins to Smaug indeed correlates with the mutant's capacity to repress translation of NOS mRNA in the bulk cytoplasm of the embryo (Dahanukar, 1999).
Overproduction of Smaug represses NOS mRNA in the pole plasm. Smaug protein is distributed throughout the preblastoderm embryo, with no detectable difference between its concentration in the bulk cytoplasm and the pole plasm. Why, then, does Smaug not repress the translation of NOS mRNA in the pole plasm? One explanation is that, in fact, Smaug-dependent repression competes with Osk-dependent activation, with Osk prevailing in wild-type embryos. To investigate this idea, Smaug was overproduced by introducing up to four extra copies of a smg+ transgene, thereby generating '6× smg+' embryos. The extent of Smaug overproduction appears to be approximately proportional to the gene dose. Otherwise, wild-type 6× smg+ embryos are completely viable and exhibit no segmentation defects. Moreover, the distribution of Nos protein during early development appears normal in such embryos, suggesting that this level of Smaug does not significantly interfere with Osk-dependent activation of wild-type NOS mRNA. However, excess Smaug clearly interferes with NOS mRNA translation in two different sensitized genetic backgrounds. While it is not understood why overproduction of Smaug is without apparent consequence in the pole plasm of wild-type embryos, one simple possibility is that a 3-fold increase in Smaug concentration is insufficient to repress translation in wild-type embryos, but that higher levels of Smaug would do so (Dahanukar, 1999).
In one experiment, the effect of excess Smaug on translation of a modified, minimal NOS mRNA in wild-type pole plasm was examined. The 3' UTR of this minimal NOS mRNA (nosDeltaBX) contains essentially nothing other than the TCE and a polyadenylation signal (Dahanukar, 1996). Regulation of nosDeltaBX and wild-type NOS mRNAs is indistinguishable. In particular, translation of nosDeltaBX is dependent on the activities of pole plasm components such as Vasa and Osk, as is the case for nos+ mRNA. However, nosDeltaBX mRNA is translated relatively inefficiently. As a result, otherwise wild-type embryos (i.e., 2× smg+) in which the only source of Nos activity is translation of nosDeltaBX mRNA develop five to six abdominal segments. If instead, such embryos bear excess Smaug, they develop only two abdominal segments. Thus, excess Smaug represses the translation of nosDeltaBX mRNA in the pole plasm. In the second experiment, the effect of excess Smaug was examined on translation of nos+ mRNA that is activated as a result of ectopic Osk activity. At the anterior of Bicaudal D (BicD) embryos, sufficient Osk accumulates to activate translation of NOS mRNA, but pole plasm assembly is incomplete and no anterior pole cells form. The resulting ectopic Nos activity blocks translation of hb and bcd mRNAs, and as a consequence, the anterior of the embryo develops abdominal segments. Strikingly, overproduction of Smaug suppresses the bicaudal phenotype—wild-type head and thoracic segments are specified normally, and many of the embryos hatch. As is the case in a wild-type background, excess Smaug has no apparent effect on Nos activity generated at the posterior pole. The distribution of NOS mRNA is essentially the same in 2× smg+BicD embryos and 6× smg+BicD embryos, showing that excess Smaug has no effect on the level or distribution of NOS mRNA in this experiment. Thus, excess Smaug suppresses the Osk-dependent activation of nos+ mRNA at the anterior of BicD embryos (Dahanukar, 1999).
Oskar interacts with the RNA-binding domain of Smaug. Smaug and Osk compete in the pole plasm, the former repressing and the latter activating translation of NOS mRNA. Smaug evidently acts by binding to the TCE hairpins of NOS mRNA. The molecular mechanisms by which Osk acts are not yet clear, although it plays a central role in both pole plasm assembly and activation of NOS translation.
In particular, two lines of evidence suggest that Osk is the limiting component in the embryo for translational activation of NOS: (1) unlike other gene products required for pole plasm assembly, which are also present throughout the bulk cytoplasm, Osk is found only in the pole plasm; (2) overexpression of Osk is sufficient to activate NOS translation throughout the embryo. The mutually antagonistic activities of Osk and Smaug might be the result of a direct interaction between the two. To test this possibility, plasmids that direct the synthesis of various fragments of Osk and Smaug in yeast were constructed, and protein-protein interactions were assessed using the two-hybrid technique. Smaug interacts specifically with Osk in yeast. The region of Smaug that mediates this interaction corresponds to a 31 kDa fragment that contains the minimal RNA-binding domain. Further mutational analysis of this domain suggests that its TCE- and Osk-binding activities are not readily separable (Dahanukar, 1999). The region of Osk that mediates binding to Smaug consists of residues 290-418, a domain of the protein that may also mediate interactions with the pole plasm constituents Vas and Staufen (Dahanukar, 1999).
Taken with earlier work, these results support a simple model for the operation of a translational switch that governs expression from NOS mRNA. In the bulk cytoplasm, repression of NOS mRNA is dependent on the activity of Smaug, which binds to the essential targets in the 3' UTR. In the pole plasm, Smaug-mediated repression is antagonized by Osk, which interacts with the RNA-binding domain of Smaug. Currently, it is not know whether Osk interacts with Smaug bound to the TCE or whether Osk competes with the RNA for binding to Smaug. In either case, Smaug-dependent repression is overcome, and Nos protein accumulates in the posterior of the embryo. Osk also activates translation via other signals in the NOS 3' UTR. However, unlike the translational switch governed by Smaug, these signals are dispensable for NOS function in the embryo (Dahanukar, 1999 and references).
The Nanos translational control element (TCE) function requires formation of a bipartite secondary structure that is recognized by Smaug repressor and at least one additional factor. Translational activation requires the interaction of localization factors with sequences that overlap TCE structural motifs. The identification of separate but overlapping recognition motifs for translational repressors and localization factors provides a molecular mechanism for the switch between translational repression and activation (Crucs, 2000).
Translational repression of NOS mRNA is mediated by a bipartite cis-acting translational control element consisting of two stem-loop structures. One stem-loop, stem-loop II, is the target for binding by the previously identified Smg protein. The other, which is not required for Smg interaction but is essential for translational repression, contains a unique double-stranded recognition motif. The essential requirement for both of these stem-loops indicates that binding by one or more factors in addition to Smg is necessary for TCE-mediated translational repression. Translational activation of NOS mRNA requires recognition by localization factors of NOS 3'UTR motifs distinct from, but overlapping those that mediate translational repression. This arrangement of recognition motifs can explain the ability of a single region of the NOS 3'UTR to mediate these two mutually exclusive functions. These results provide direct molecular evidence for a model in which translational activation of nos RNA results when binding of translational repressors is prevented by binding of localization factors at distinct but overlapping sites (Crucs, 2000).
NOS TCE function requires formation in vivo of secondary structure predicted both by phylogenetic conservation and by RNA folding algorithms. Two stem-loops make unique contributions to TCE function by providing distinct recognition motifs for trans-acting translational repressors. The requirement for formation of stem-loop II can be explained entirely by its role in presenting a single-stranded binding site for Smg protein. The Smg RNA-binding domain does not interact with stem-loop III. By contrast, the specific sequence as well as structural requirements for stem-loop III activity suggest that stem-loop III may be recognized by a double-stranded RNA-binding protein. One such protein, Staufen (Stau), is required for localization of BCD RNA and an indirect in vivo assay suggests that Stau interacts with a double-stranded region of the BCD RNA localization signal. Another double-stranded RNA-binding protein, Prbp, binds to a conserved stem-loop in the 3'UTR of the mouse protamine 1 (Prm1) mRNA in vitro and is required in vivo for translational activation of Prm1 during spermatogenesis (Crucs, 2000).
Stau and Prbp belong to a large family of proteins that bind double-stranded RNA through a conserved amino acid motif, the dsRBD. Biochemical and structural studies of dsRBD family members indicate that binding is highly specific for double-stranded RNA but largely independent of RNA sequence. Since the function of stem-loop III requires alternating U:A and A:U base pairs, recognition of the helix appears to be sequence-dependent. It is possible, therefore, that TCE stem-loop III is recognized by a double-stranded RNA-binding protein with a novel sequence-specific binding domain. Alternatively, local sequence-dependent structural variation may confer specificity to an interaction with a more typical ds-RBD. The partial reduction in TCE function caused by replacement of the poorly conserved III loop with a helix-stabilizing tetraloop further suggests that TCE function is sensitive to the geometry of the stem III helix (Crucs, 2000).
The ability of TCE stem-loops II and III to repress translation when separated by 52 nucleotides argues that coaxial stacking of the stem II and III helices, potentiated by the phylogenetically conserved junctional structure, is not required for TCE function. Furthermore, the junction does create a binding site for translational repressors. However, the ability of a bulged nucleotide within the stem III helix to disrupt TCE function suggests that the position of the distal half of the stem III helix with respect to the stem II helix is indeed critical. It is possible, therefore, that translational repression requires interaction between factors bound to stem-loops II and III or between these factors and a third component. When the two stem-loops are separated in a rearranged variant, increased flexibility in the RNA molecule may continue to allow this interaction (Crucs, 2000).
Posterior localization and translational activation can be largely recapitulated by the 180 nucleotide +2 element of the NOS RNA localization signal. This element can be further subdivided into the +1 element, which is coincident with the TCE, and the +2' element. While the +2' element also contains a Smg-binding site, it confers minimal translational repression. On their own, the +1 and +2' elements each provide weak localization function. However, when combined to form the +2 element, they collaborate to produce substantial localization function. RNA localization function of the +1 element is highly sensitive to mutation. All mutations analyzed here, including compensatory mutations and the single U51A mutation that retains TCE function, eliminate localization by the +1 element. Thus, localization requires recognition of multiple sequence and possibly structural motifs distributed throughout this element. The possibility that localization requires formation of an alternate secondary structure cannot be eliminated. While all suboptimal structures predicted by RNA folding algorithms closely resemble the optimal structure and cannot explain the effect of these mutations on localization function, it is possible that binding by localization factors stabilizes an alternate structure with a low propensity to form on its own (Crucs, 2000).
A synergistic interaction between the +1 and +2' elements permits the +2 element to recapitulate much of wild-type NOS localization function. The effect of TCE/+1 element mutations on localization by the +2 element indicates that the TCE/+1 sequences are recognized in two different ways, perhaps by two different proteins or protein complexes. While each recognition event corresponds to one TCE stem-loop, it is separable from the recognition event required for the translational repression function of that stem-loop. Presumably, contacts between proteins bound to these +1 element sites and proteins bound to the +2' element or contacts between +1 and +2' element binding proteins and a common component, such as a complex of Osk, Vas, and Tud proteins, underlie the interaction (Crucs, 2000).
Biochemical experiments have shown that Osk protein interacts directly with Smg, although the functional significance of this interaction is not known. A recently proposed model, in which a ternary complex of Osk, Smg, and the TCE serves both to localize NOS RNA to the posterior and to activate NOS translation (Dahanukar, 1999), is not supported by the data presented here. Rather, these results indicate that direct interaction of localization factors with NOS 3'UTR sequences is obligate for translational activation. The recruitment of NOS mRNA, via factors bound to localization element sites, to a localization complex with Osk, Vas, and Tud proteins may facilitate a Smg:Osk interaction that inactivates Smg function. However, the close apposition or overlap of localization factor recognition sites to the motifs required for translational repressor recognition supports an alternative model in which binding of translational repressors and localization factors is mutually exclusive. In this model, the Smg:Osk interaction may serve as a fail-safe mechanism to sequester Smg and inhibit its activity. Given the structural requirements for TCE function, binding by localization factors to motifs within stem-loops II and III may prevent binding of repressors by preventing formation of TCE structure. Thus, the structure of the TCE may lie at the heart of the switch between translational repression and activation (Crucs, 2000).
Nanos protein is required in the posterior of the Drosophila embryo to promote abdominal development, but must be excluded from the anterior to permit head and thorax development. Spatial restriction of Nos is accomplished by selective translation of the 4% of NOS mRNA localized to the posterior pole and translational repression of the remaining unlocalized mRNA. Repression is mediated by a 90-nucleotide translational control element (TCE) in the NOS 3' untranslated region (UTR) and the TCE-binding protein Smaug, but the molecular mechanism is unknown. Sucrose density gradient sedimentation was used to ascertain whether unlocalized NOS mRNA is excluded from polysomes and therefore is repressed during translational initiation. Surprisingly, a significant percentage of NOS mRNA was found to be associated with polysomes, even in mutants in which all NOS mRNA is unlocalized and repressed. Using a regulated Drosophila cell-free translation system, it has been shown that ribosomes contained within these polysomes are capable of elongation in vitro, under conditions in which synthesis of Nos protein is repressed. Thus, synthesis of ectopic Nos protein is inhibited by a novel regulatory mechanism that does not involve a stable arrest of the translation cycle (Clark, 2000).
Two possible models for repression are proposed. (1) Factors bound to the TCE may degrade or destabilize the nascent polypeptide chain. While this mechanism may not strictly regulate the translation cycle, it should operate cotranslationally, since the TCE works only in cis. (2) Alternatively, the TCE and its associated factors may alter the processivity of the ribosome and promote premature release of either the ribosome or nascent polypeptide, followed by degradation of the incomplete protein product. Polysome association of translationally repressed transcripts has also been observed
for the heterochronic genes lin-14 and lin-28 in C. elegans. While elongation has not yet been examined for these mRNAs, it is tempting to speculate that they may be regulated by a mechanism similar to that used for NOS. Intriguingly, the cis-regulatory element for each of these mRNAs lies within the 3'UTR and has the capacity to form a double-stranded structure (Clark, 2000).
Temporal considerations of NOS expression and function underscore the advantages of regulating translation at a step after initiation. NOS mRNA is actively translated in nurse cells before its deposition in the oocyte. Post-initiation mechanisms may be particularly effective at rapidly inactivating mRNAs, such as NOS, that are already engaged with ribosomes. Furthermore, repressing
mRNA after initiation may allow for rapid activation of silenced mRNAs. Nos protein promotes abdominal development by repressing translation of maternal Hunchback mRNA, which is activated at fertilization. Derepression of NOS mRNA that has been preloaded with ribosomes may allow Nos to be rapidly synthesized at the posterior pole upon localization, before activation of HB. Indeed, post-initiation mechanisms of translational control may prove to be a powerful mechanism for enhancing spatial and temporal fidelity of gene expression (Clark, 2000).
The bicaudal(bic) mutation was the first Drosophila
mutation identified as having a clear effect on embryonic
pattern formation. At its most extreme, the
mutation produces bicaudal embryos, that is, embryos in
which the head and thorax are missing and are replaced with
mirror-image duplications of abdominal segments.
The critical defect required for production of bicaudal
embryos has proved to be the ectopic translation of Nanos mRNA
in the anterior embryonic compartment. Expression of Nanos
in this compartment not only prevents maternal Hunchback
action in this region but also represses translation of mRNA
for the major anterior determinant Bicoid. As a result, anterior
development is lost and replaced by the posterior program.
Mutations at four loci (BicD, BicC, bullwinkle and Klp38B)
that produce bicaudal embryos all result in ectopic expression
of Nanos in the anterior.
Interestingly, all of these mutations are believed to cause
ectopic NOS mRNA translation indirectly as a result of
abnormal retention/accumulation of OSK mRNA at the anterior
of the mature oocyte/embryo. Three of these genes (BicC, BicD
and Klp38B) encode proteins with predicted roles in RNA
localization and transport. Mutations of bullwinkle have
pleiotropic effects on development, including defects in
migration of the somatically derived follicle cells surrounding
the nurse cell/oocyte complex. bullwinkle may thus cause
abnormal OSK mRNA accumulation/translation by a more
indirect route. A number of molecular
constructs that remove repressor binding sites for Smaug and
Bruno from NOS and OSK mRNAs, respectively, leading to
ectopic expression of NOS mRNA in the anterior compartment,
also yield efficient production of embryos with posterior
duplications in the anterior (Markesich, 2000 and references therein).
bicaudal was the first Drosophila mutation identified as
producing mirror-image pattern duplications along the
anteroposterior axis of the embryo. However the mutation
has been little studied due to its low penetrance and
suppressibility. The bicaudal locus has been cloned and the mutation's effects on key
elements of the posterior embryonic patterning pathway has been examined.
Mapping studies place the bicaudal mutation within a
~2 kb region, 3' to the protein coding sequence of the
Drosophila homolog of beta NAC, a subunit of Nascent
polypeptide Associated Complex (NAC). Genomic DNA
encoding beta NAC completely rescues the bicaudal
phenotype. The lethal phenotype of Enhancer of Bicaudal,
[E(Bic)], a mutation hypothesized to affect the bicaudal locus,
is also completely rescued by the beta NAC locus. It was further demonstrated that the E(Bic) mutation is caused by
a P element insertion into the transcribed region of the beta
NAC gene. NAC is among the first ribosome-associated
entities to bind the nascent polypeptide after peptide bond
formation. In contrast to other bicaudal-embryo-producing
mutations, bicaudal leads to ectopic translation of mRNA
for the posterior determinant nanos, without affecting the
localization of mRNA for its upstream regulator, oskar, in
the embryo. These findings suggest that repression of nanos
mRNA translation occurs on the ribosome and involves a
role for beta NAC (Markesich, 2000).
The combined effects of the bic mutation on the patterns of
NOS mRNA, Nanos protein and OSK mRNA localization in the
early embryo, are distinctly different from any reported
previously. For other mutations that produce bicaudal embryos,
localization of NOS mRNA and protein in the anterior is always
associated with a similar mislocalization pattern for OSK mRNA,
indicating, as in the wild-type situation, that local accumulation
of NOS mRNA and its release from translational repression is
dependent on OSK activity. In contrast, it has been found that the bic mutation
produces aberrant NOS mRNA localization and widespread high
level accumulation of Nanos protein, without alterations to the
OSK mRNA distribution pattern. Given that more than 95% of
NOS mRNA is distributed throughout the oocyte cytoplasm, these findings suggest a failure to
repress NOS mRNA translation outside the posterior pole,
resulting in widespread global production of Nanos protein (Markesich, 2000 and references therein).
How can the defects produced by the bic mutation be related
to the function of beta NAC, the protein affected by the
mutation? Most available information on the function of beta
NAC comes from in vitro biochemical studies of translational
regulation. The protein was identifed as the beta subunit of
the heterodimeric Nascent polypeptide Associated Complex
(NAC), an abundant ribosome-associated complex that can be
cross-linked to very short nascent polypeptide chains on the
ribosome. Binding of NAC to
relatively extensive regions of nascent chains (up to 17-100
amino acids from the peptidyl transfer site) suggests NAC may
have some role in processing whole protein domains as they
emerge from the ribosome. NAC
association with ribosomes has also been shown in vitro to
prevent non-specific ribosomal association with the
endoplasmic reticulum (ER).
If the ribosome is the major in vivo site of action for beta
NAC, this clearly has implications for the molecular basis of
the ectopic translation of NOS mRNA produced by the bic
mutation. The most interesting possibility is that the release
of NOS mRNA from translational repression is a direct
consequence of loss of beta NAC function at the ribosome
and thus that, in vivo, beta NAC not only binds nascent chains
but, through this interaction, may also actually regulate
further translation of growing polypeptides. Repression of
NOS mRNA translation would thus be a ribosome-related
event, involving a block in translational elongation rather than
initiation (Markesich, 2000 and references therein).
Several findings support the prediction that
NOS mRNA translational repression occurs on the ribosome.
NOS mRNA is translationally active in the early stages of
oogenesis in the nurse cells and thus much
of the NOS mRNA that is transferred to the oocyte cytoplasm
has a history of association with active ribosomes. Further,
the NOS mRNA present in newly laid eggs is
associated with polysomes.
The suggestion that the ectopic translation of NOS mRNA is
a direct and specific consequence of loss of beta NAC function
may appear to conflict with the presumably universal ability of
NAC to interact with nascent polypeptide chains. However, a
weak hypomorphic allele like bic can be expected to affect the
process most susceptible to loss of gene function, and the
robust translational repression required to prevent NOS function
throughout the embryo could represent such a highly sensitive
process. The poor penetrance and variability of the bic
mutation may reflect the fact that only a critical range of
decreased beta NAC function will produce bicaudal embryos
without major effects on vital processes. Similarly the
limitation of the effects of the mutation to anteroposterior
pattern formation could reflect the central role of translational
control in the development of this embryonic axis, as opposed
to the dorsal-ventral axis (Markesich, 2000 and references therein).
Although a direct effect of the bic mutation on NOS mRNA
translation is suggested by the biochemical function of beta
NAC, clearly it is also possible that the mutation acts to
produce aberrant NOS activity through a more indirect route. Aberrant production/localization of NOS
mRNA binding factors that are normally concentrated at the
posterior pole may be produced by the bic mutation; the
effects on NOS mRNA translation may depend primarily on
such a defect.
To date, the roles of NAC and its component proteins have
mainly been addressed through in vitro biochemical
methodologies. The demonstration of a role for beta NAC in
the translational regulation events of anteroposterior
embryonic pattern formation provides the first indication that
more complex regulatory functions are associated with the
protein in the whole organism (Markesich, 2000).
Maternally derived HB mRNA is uniformly distributed throughout the embryo; the mRNA is translationally repressed in the posterior,
giving rise to an anterior-to-posterior gradient of Hb protein. Failure of this repression results in the abnormal accumulation of Hb in the
posterior, which inhibits abdominal segmentation. Two conserved RNA-binding proteins, Pumilio (Pum) and Nanos (Nos), are specifically required to repress HB translation. Pum, which is distributed uniformly throughout the embryo, is the founding member of a large family of RNA-binding proteins. Pum binds to 32 nucleotide sites in the 3' UTR of HB (Nos
Response Elements, NREs) to regulate HB translation. Nos, which
initially is distributed as a gradient emanating from the posterior pole of the embryo, contains a conserved zinc finger that mediates nonspecific RNA binding. Nos is selectively recruited into a ternary complex on HB mRNA by NRE-bound Pum. The mechanism by
which the resulting Nos/Pum/NRE complex regulates translation is not yet understood, although deadenylation is thought to play a role (Sonoda, 2001 and references therein).
To identify targets or cofactors of the Nos/Pum/NRE ternary complex,
a yeast 'four-hybrid' experiment was performed; a Gal4
activation domain fusion library was screened for proteins that interact with the ternary complex. The bait contained the RNA-binding domain of Pum, full-length Nos, and NRE-bearing RNA. As anticipated, factors that interact
with individual components in isolation were identified. However, one factor, which proved to be a fragment of Brain tumor (Brat), interacts only with the ternary complex and not with either Nos alone, Pum alone, or a Pum/NRE binary complex. Deletion analysis revealed that recruitment of Brat is dependent on the conserved carboxy-terminal domain of Nos that mediates its interaction with Pum on HB mRNA, and not the amino-terminal domain of Nos that mediates interaction with Cup during early oogenesis. Mutational analysis further showed that a fragment of Brat consisting of little more than the NHL domain is recruited to the ternary complex. Protein-protein interaction experiments show that Nanos and Pumilio are required to recruit Brat to HB mRNA and genetic experiments show that Brat is required for repression of HB mRNA (Sonoda, 2001).
A model is presented of how Nos, Pum, and Brat act to regulate gene
expression. The model involves combinatorial interactions among cis-acting sequences in regulated mRNAs, proteins that recognize these sequences, and the
NHL domain of Brat. Recruitment of Brat occurs through protein-protein interactions
with RNA-bound Pum and Nos; formation of the resulting quaternary complex is essential for translational control of HB. Recruitment of Brat to the NRE jointly by Nos and Pum is essential for regulation of HB mRNA. Three lines of evidence show that the NHL domain plays a key role in this process: (1) the NHL domain is
sufficient to mediate interaction with the Nos/Pum/NRE complex, thereby
targeting Brat to HB mRNA; (2) single amino acid substitutions within the NHL domain attenuate interaction with the ternary complex and regulation of HB in vivo; (3) maternal expression of the wild-type NHL domain alone is sufficient to restore HB regulation in bratfs mutant embryos. This result suggests that the NHL domain contains intrinsic translation regulatory activity. However, activity of the isolated NHL domain is (necessarily) assayed
in the presence of Bratfs mutant protein, and thus, the possibility that the amino-terminal BCC domain participates somehow in HB mRNA regulation cannot be ruled out (Sonoda, 2001).
Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the
same machinery and RNA signals drive specific accumulation of maternal RNAs in
the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos;
interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor
complex that drives transcript localization in a variety of tissues (Bullock, 2001).
During Drosophila oogenesis, specification of the oocyte is associated with selective accumulation of RNA determinants supplied by the neighboring, interconnecting ovarian nurse cells. Subsequently, deposition of mRNA transcripts at selected sites within the
oocyte leads to localized translation of the proteins that establish the
prospective embryonic body axes. gurken (grk) transcripts reside first posteriorly and then anterodorsally, and sequentially establish
the anteroposterior and dorsoventral axes. bicoid (bcd) and oskar
(osk) transcripts localize to the anterior and posterior of the oocyte,
respectively, to pattern the anteroposterior body axis (Bullock, 2001).
The injection assay was used to investigate whether any maternal
transcripts that localize in the oocyte are recognized by the localization machinery of blastoderm embryos. Five such transcripts [bcd, grk, nanos (nos), osk and female sterile (1) K10 (K10)] were tested, and all accumulate in the apical cytoplasm after injection. With the exception of osk transcripts -- only a small proportion of which localize apically -- the efficiency of localization of these transcripts appears indistinguishable from that of pair-rule transcripts. Maternal transcripts also localize apically when zygotically expressed from endogenous transgenes. Preinjection with colcemid severely inhibits apical localization of the injected maternal transcripts, indicating that their localization in blastoderm embryos, like that of the pair-rule transcripts, is dependent on intact microtubules (Bullock, 2001).
The common aspect of maternal RNA localization measured in
these experiments is unlikely to be transport within the oocyte,
because the maternal transcripts tested are distinctly distributed
in late stage oocytes by means of different motors and accessory
factors. However, all the transcripts -- with the possible exception
of grk -- are synthesized in adjacent nurse cells and reach the
oocyte by transport along microtubules. To test whether this
process is analogous to apical localization in blastoderm embryos,
a bcd transcript was used containing a single nucleotide change
(4496G->U). This change prevents early oocyte-specific transport (stages
4-6) without disrupting later (stage 6 onwards) import of transcripts into the oocyte or their subsequent accumulation at the
anterior cortex. This mutation inhibits apical bcd localization in
blastoderm embryos, suggesting that transcripts localize in this injection assay through the same machinery
that transports transcripts into the early oocyte (Bullock, 2001).
These data suggest that components of the blastoderm localization machinery are also likely to function in RNA transport into the
early oocyte. Genetic screens for maternal mutations that affect
formation of the embryonic axis have identified egl and BicD as genes required for oocyte differentiation and for specific RNA
accumulation in the oocyte. However, their exact activities are
uncertain. BicD protein includes multiple heptad repeats, which may
mediate oligomerization and interactions with other proteins;
Egl includes a domain shared with 3'-5' exonucleases. During
oogenesis, these two proteins form complexes together and colocalize at the minus ends of microtubules. The integrity of the
microtubule cytoskeleton is defective in egl and BicD mutants, which has been proposed to explain subsequent defects in RNA
localization. Alternatively, Egl and BicD might act directly in
RNA transport. However, evidence that distinguishes between
these two possibilities is lacking (Bullock, 2001).
Whether Egl and BicD are present in early
embryos was examined. Both proteins are supplied maternally to the embryo.
They are noticeably enriched apical to the nuclei at blastoderm
stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the
proteins is present in the basal cytoplasm (Bullock, 2001).
Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of
protein/RNA co-transport. The complex may have an additional
role in anchoring transcripts at their destination. Alternatively,
maintenance of localized transcripts might not depend on an
independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).
Localization of Nanos (NOS) mRNA to the germ plasm at the
posterior pole of the Drosophila embryo is essential to
activate NOS translation and thereby generate abdominal
segments. NOS mRNA localization is mediated by a large cis-acting
localization signal composed of multiple, partially redundant elements within the NOS 3' untranslated region.
A protein of ~75 kDa (p75) has been identified that interacts
specifically with the NOS +2' localization signal element (nucleotides 97-185 of the NOS 3'UTR). The function of this element can be delimited to
a 41 nucleotide domain that is conserved between D.
melanogaster and D. virilis, and confers near wild-type
localization when present in three copies. Two small
mutations within this domain eliminate both +2' element
localization function and p75 binding, consistent with a role
for p75 in NOS RNA localization. In the intact localization
signal, the +2' element collaborates with adjacent
localization elements. Different +2' element
mutations not only abolish collaboration between the +2'
and adjacent +1 element (nucleotides
6-96 of the NOS 3'UTR) but also produce long-range
deleterious effects on localization signal function. These
results suggest that higher order structural interactions
within the localization signal, those that require factors such
as p75, are necessary for association of NOS mRNA with the
germ plasm (Bergsten, 2001).
Although the 2' element has very weak localization
function on its own, three tandem copies confer substantial
localization. Furthermore, the
+2' element acts synergistically with adjacent localization
elements. In particular, combination of the +2' element with
the weakly localizing +1 element produces the near wild-type
localization function of the +2 element. The +1 element is coincident with the
NOS translational control element (TCE), which mediates
translational repression of unlocalized NOS mRNA. TCE function requires the formation of two stem-loop
structures. The synergistic interaction between the +2' and +1 elements
requires +1 element motifs that overlap but are distinct from
the TCE structural motifs (Bergsten, 2001).
The central 41 nucleotides of the NOS
3'UTR +2' element are sufficient for its RNA localization and
translational regulatory activities. Remarkably, three copies of
this minimal element achieve a sufficient balance between
translational repression and RNA localization to permit wild-type
development. Small mutations distributed throughout the
41 nucleotide domain each disrupt +2' localization function
when assayed in the context of the +2'-3X trimer and in the
native context of the +2 element. These mutations define a
recognition motif for at least one protein, p75. This protein is
the first factor identified that interacts specifically with a NOS
localization signal element (Bergsten, 2001).
Binding of p75 to the +2' element is disrupted by two
mutations, A and D, that abolish +2' element localization
function. Although these mutations also disrupt translational
repression, two reasons suggest that p75 is more likely to play a role in
localization than in translational repression: (1) p75 does not
interact with the TCE; (2) two other +2' element
mutants, B and C, which have more severe effects than A or D
on translational repression, retain significant ability to bind
p75. Mutation C alters one nucleotide of the Smg binding site, whereas A, B and D lie outside the
reiterated TCE loop motif. The potential for nucleotides altered
in mutations A, B, and D to participate in formation of a Smg-binding
loop suggests that these mutations disrupt
translational repression most likely by affecting Smg binding.
Nonetheless, molecular identification and genetic analysis of
p75 will be essential to determine its role in vivo.
The ability of multiple mutations spanning the conserved
+2'ME to disrupt localization suggests that localization
depends on either the simultaneous binding of multiple
proteins to distinct sequence motifs or the binding of one or
more proteins to a complex structural motif. Recognition of the
+2' element by p75 requires the integrity of two non-contiguous
sets of nucleotides. p75 may bind as an obligate
dimer, with each molecule contacting one binding site. Folding
of the RNA may be necessary to bring the sites into close
proximity or, alternatively, may create a single binding motif
within a larger secondary structure. The fact that mutations B
and C, which lie between A and D, have some effect on p75
binding while mutations E and F, which lie outside this region,
behave as wild-type is consistent with an interaction dependent
on structural features of the RNA. The purification of p75 and
the generation of additional +2' element mutations will
facilitate the quantitative biochemical analysis required to
distinguish between these possibilities. However, the ability of
mutations to disrupt localization without affecting binding by
p75 indicates that at least one other factor is required for +2'
localization function in vivo (Bergsten, 2001).
The sensitivity of the +2' element to mutation resembles that
of the +1 element. While the secondary
structure of the TCE is well conserved, analysis of mutations
that disrupt +1 element localization function does not support
a requirement for this structural motif in localization, and RNA
folding algorithms do not predict alternative structures that
might mediate localization. Similarly,
secondary structure requirements for +2' element localization
function do not appear to be readily predicted or assayed by
mutagenesis. Multiple isoenergetic structures predicted for the
+2' element by RNA folding algorithms reveal little similarity
between structures predicted for the D. melanogaster and D.
virilis +2' sequences. If
formation of specific RNA structures is indeed required for
localization function, these alternate structures may be driven
or stabilized by the binding of localization factors such as p75
and, thus, would not be readily calculated (Bergsten, 2001).
Surprisingly, mutations distributed throughout the +2'
minimal element have a long range effect within the +2
element. Alteration of as few as two nucleotides nearly or
completely eliminates +2 element localization function and the
ability of the +1 element alone to interact with the localization
machinery. This result indicates that although the +1 element
can interact independently, albeit weakly, with the localization
machinery, this independent function is lost in the +2 element
and the intact localization signal. Rather, sequences or local
structures within the +1 element may normally participate in
formation of a higher order structure with sequences or
structures from the +2' element. +2' mutations may disrupt +2
element function by disrupting subdomains of +2 element
structure or the interaction of a +2 element-protein complex
with germ plasm components, without disrupting participation
of +1 element sequences/structures. Consistent with the
contribution of +1 and +2' element sequences to a larger
structure, the combination
of mutations in two different regions of the +1 element affects
collaboration of the +1 and +2' elements. In addition, this idea is supported by the results of altering the
spacing and relative positions of the +1 and +2' elements. The
separation of +1 and +2' element sequences could still permit
secondary or tertiary interactions to occur, whereas altering
their relative positions would not (Bergsten, 2001).
The long range effects of +2' element mutations are specific
to the localization function of the +2 element. Translational
repression by the TCE is not affected by +2' mutations,
indicating that they do not prevent formation of TCE structure
in unlocalized NOS RNA. Similarly, mutations have been identified in the +1 element that retain TCE function but
disrupt the ability of the +1 and +2' elements to interact. Based on analysis of +2' element mutations,
it is suggested that localization factors recognize or promote
formation of an alternate conformation of the NOS 3'UTR different from
that recognized by translational repressors. Since p75 binding
is independent of the germ plasm components, formation of
this structural motif, aided by factors such as p75, may be
required prior to association of NOS RNA with the germ plasm
anchor. It has been suggested that binding by
translational repressors and localization factors is mutually
exclusive, and that RNA localization activates NOS translation
by preventing binding of translational repressors. The ability to form alternative
structures can explain the mutually exclusive relationship
between translational repression and localization (Bergsten, 2001).
Translational control of gene expression plays a fundamental role in the early development of many organisms. In Drosophila, selective translation of nanos mRNA localized to the germ plasm at the posterior of the embryo, together with translational repression of nanos in the bulk cytoplasm, is essential for development of the anteroposterior body pattern. Both these components of spatial control of nanos translation initiate during oogenesis and translational repression is initially independent of Smaug, an embryonic repressor of nanos. Repression during oogenesis and embryogenesis are mediated by distinct stem loops within the nanos 3' untranslated region; the Smaug-binding stem-loop acts strictly in the embryo, whereas a second stem-loop functions in the oocyte. Thus, independent regulatory modules with temporally distinct activities contribute to spatial regulation of nanos translation. It is proposed that nanos evolved to exploit two different stage-specific translational regulatory mechanisms (Forrest, 2004).
Selective translation of posteriorly localized nos mRNA achieves
the restricted distribution of Nos protein required to regulate hb
and cycB mRNAs in the posterior of the embryo without affecting
hb or bcd translation in the anterior. Although some
maternal mRNAs, including osk and gurken are translated in
the oocyte, others, such as hb and bcd,
are translationally repressed during oogenesis and activated only after
fertilization. For nos, translational activation during oogenesis would permit accumulation of Nos protein in the posterior of the embryo early enough to
block hb translation. However, the translational status of
nos in late stage oocytes has remained enigmatic, owing to the
impermeability of these tissues to immunostaining. Use of a gfp-nos
fusion gene as a reporter for nos translation has allowed this issue to be addressed; it has been established that translation of localized nos
mRNA indeed begins during oogenesis (Forrest, 2004).
Achieving the restricted Nos protein distribution in the embryo requires
that translational activity of nos in the oocyte be spatially
regulated. However, the only known repressor of nos translation, Smg,
is absent from the ovary. This dilemma has been solved by showing that a distinct,
Smg-independent mechanism mediates translational repression of unlocalized
nos mRNA in late oocytes.
Translational repression of unlocalized nos is mediated by a 90 nucleotide translational control element (TCE), the function of which requires formation of two stem-loops (II and III). Failure to repress nos in late
oocytes, as exemplified by the behavior of the nos-tub:TCEIIIA
transgene (mutated in stem-loop III of the RNA), results in unrestricted production of Nos protein that perdures to
embryogenesis. The resulting embryos die, lacking anterior structures. Thus, by
showing that the program for spatially restricted synthesis of Nos operates
during oogenesis, these results reveal how temporal demands are reconciled with
spatial constraints on nos translation needed for embryonic
patterning (Forrest, 2004).
The elucidation of temporally distinct functions of the two TCE stem-loops
explains the enigmatic structural complexity of this regulatory element. Both stem-loops (II and III) retain function, despite their
separation by a 52-nucleotide spacer, suggesting that they operate
independently. Indeed, phylogenetic analysis of the nos
3'UTR reveals that TCE stem-loops II and III are not juxtaposed in all
Drosophilid species, indicating that
the distance between stem II and III is not under tight evolutionary
constraint (Forrest, 2004).
After fertilization, Smg binds to TCE stem-loop II to mediate repression in
the preblastoderm embryo. It is not known if the ovarian repressor remains bound to
stem-loop III in the embryo, but its function is superceded by Smg. A minor
requirement for stem-loop III in the embryo suggested by in vitro
translation experiments may reflect the need to maintain the ovarian
repression mechanism until Smg reaches sufficient levels in the embryo.
Accordingly, the requirement for stem-loop III would decrease over time after
fertilization. A more significant contribution by stem-loop III might have
been missed, however, if the stem-loop III-dependent repressor is unstable in
the embryonic extract (Forrest, 2004).
The smg mutant phenotype indicates that nos is not the
only target of Smg in the embryo. Although the ovarian repressor has not yet been
identified, a candidate ovarian stem-loop III
binding factor has been isolated recently that appears to
regulate multiple maternal mRNAs. Thus, it seems likely that nos has
evolved to co-opt existing stage-specific regulatory proteins for its
advantage. The nos TCE can repress
translation in subsets of cells in both the central and peripheral nervous
systems. Although the repressors are not known in these cases
either, it is possible that the ability to interact with yet additional
proteins will underlie the multifunctionality of the TCE (Forrest, 2004).
Other RNAs may use a similar strategy of recognition by stage-specific
factors to maintain translational regulation across developmental transitions.
In the Drosophila oocyte, translational repression of unlocalized
osk mRNA occurs through the interaction of Bruno (Bru) with specific
sequence motifs in the osk 3'UTR. Since Bru
is not present in the embryo, where the majority of osk mRNA remains unlocalized, an
embryonic repressor may be required to maintain the repression initiated by
Bru. Intriguingly, the existence of binding sites for multiple, distinct
microRNAs within individual 3'UTRs suggests a
similar paradigm for controlling translation through multiple developmental
stages or in different tissues (Forrest, 2004).
The translational quiescence of unlocalized nos in late oocytes
contrasts sharply with its translational activity in the nurse cells.
Deposition of both actively translated nos mRNA and the previously
synthesized Nos protein into the oocyte during nurse cell dumping presents a
challenge for restricting Nos to the posterior of the oocyte. Although it
cannot be determined whether nos is repressed in oocytes prior to stage
10, the results indicate that the majority of nos RNA, which enters
the oocyte during dumping, must switch from a translationally active state in
the nurse cells to an inactive state in the oocyte. This switch could be
mediated by interaction of nos with an ovarian repressor restricted
to the oocyte. Alternatively, a repressor may bind to nos RNA in the nurse cells, but become activated during or after passage into the oocyte (Forrest, 2004).
Translationally repressed nos RNA is
associated with polysomes, indicating that repression is imposed at a late
step in the translation cycle. However, recent evidence that Smg interacts with Cup
to prevent recruitment of eIF-4G by eIF-4E suggests that translation is
blocked at the initiation step. The identification of a Smg-independent mechanism for translational repression during oogenesis may explain these divergent results.
Indeed, a post-initiation mechanism may be ideally suited to rapidly repress
polysomal nos RNA entering the oocyte from the nurse cells (Forrest, 2004).
In addition to translationally active nos RNA, substantial amounts
of Nos protein enter the oocyte during nurse cell dumping. Perdurance of this
protein up to embryogenesis would probably disrupt anterior development. Nos protein entering the oocyte from stage 10 nurse cells is
cleared from the oocyte by stage 13. Nos protein made in the nurse cells may
therefore be specifically targeted for degradation. Alternatively, Nos might
have a short half-life regardless of its site of synthesis. Despite
considerable effort, no ubquitinated forms of Nos protein have been detected,
although the transient nature of ubquitinated intermediates may preclude
detection. Similarly, a genetic interaction has not been detected between
mutations in numerous components of the ubiquitin degradation pathway and
nos transgenes. Thus, how Nos is degraded remains an unanswered
question. Regardless of mechanism, however, continuous translation of
wild-type nos RNA at the posterior pole or of unlocalized
nos-tub3'UTR RNA throughout the oocyte would result in
accumulation of Nos protein. Thus post-translational control of Nos protein
stability as well as post-transcriptional regulation of nos RNA
contribute to the correct spatial distribution of Nos in the early embryo (Forrest, 2004).
The development of a functional germline is essential for species propagation. The nanos (nos) gene plays an evolutionarily conserved role in germline development and is also essential for abdominal patterning in Drosophila. A small fraction of nos mRNA is localized to the germ plasm at the posterior pole of the Drosophila embryo, where it becomes incorporated into the germ cells. Germ plasm associated nos mRNA is translated to produce a gradient of Nos protein that patterns the abdomen, whereas the remaining unlocalized RNA is translationally repressed to allow anterior development. Using transgenes that compromise nos mRNA localization and translational regulation, it was shown that wild-type body patterning can ensue without nos mRNA localization provided that nos translation is properly modulated. In contrast, localization of nos to the germ plasm, but not translational regulation, is essential for nos function in the developing germ cells. It is proposed that an imperative for nos localization in producing a functional germline has preserved an inefficient localization mechanism (Gavis, 2008).
In wild-type embryos, nos mRNA localization serves two functions: to provide a local source for production of a posterior-to-anterior Nos protein gradient that directs abdominal development and to enrich nos mRNA in newly formed pole cells. This study shows that nos mRNA localization is dispensable for anterior-posterior patterning but plays an indispensable role in germ cell development (Gavis, 2008).
Nos directs abdominal development by inhibiting translation of maternal hb mRNA in the posterior of the embryo, where Hunchback represses transcription of abdominal. When expressed ectopically in the anterior of the embryo, Nos can inhibit translation of bcd mRNA as well, thereby preventing expression of anterior segmentation genes activated by Bcd. Thus, proper development of the anterior-posterior body axis can ensue only if Nos levels are sufficiently high in the posterior to prevent hb translation but sufficiently low in the anterior to allow bcd translation. Normally, the necessary differential in Nos levels is achieved by the Nos protein gradient. However, the same outcome can be achieved by a uniform level of Nos protein. These results also indicate that repression of hb and bcd translation occurs at different thresholds for Nos. A partially compromised translation control element (TCE) allows wild-type anterior-posterior patterning by unlocalized nos mRNA, by modulating the level of Nos such that it is above the threshold required to repress hb in the posterior but below the threshold required to inhibit bcd translation in the anterior. This differential sensitivity of hb and bcd to Nos protein most likely results from differences in the number and quality of the Nanos Response Elements (NREs), the motifs that mediate repression by Nos, in their 3′UTRs (Gavis, 2008).
In contrast to axial patterning, germ cell development absolutely requires localized nos mRNA. Although Nos protein produced at the posterior prior to pole cell formation may be incorporated into pole cells, the relatively short half-life of Nos protein suggests a requirement for its continued synthesis in pole cells as embryogenesis proceeds. That requirement is met by the incorporation of nos mRNA into pole cells, where it can provide a source for Nos protein production until a later time when zygotic transcription of nos is activated. Like Drosophila nos, the Xenopus nos homolog Xcat-2, C. elegans nanos-2 (nos-2) and zebrafish nanos-1 (nos-1) RNAs are localized to germ plasm during oogenesis and ultimately become incorporated into the embryonic germ cells. Thus, although its functional importance has not yet been demonstrated in these other organisms, germ plasm localization appears to be a conserved mechanism for ensuring the passage of maternally synthesized nos mRNA to the germline (Gavis, 2008).
The importance of nos localization for fertility, and consequently species propagation, may help to explain two complexities of nos localization: redundancy within the nos mRNA localization signal and persistence of an inefficient localization mechanism. Redundancy among nos localization signal elements would guard against perturbations that compromise the nos mRNA localization signal or its cognate localization factors and consequently decrease fertility. The mechanism by which nos is localized is inefficient, with only ~4% of nos mRNA in the embryo associated with the germ plasm. Intriguingly, targeting of nos mRNAs to the germ plasm, and ultimately the germ cells in C. elegans and zebrafish, is inefficient as well. Moreover, these organisms use similar mechanisms to accommodate the inefficiency of nos localization. Similarly to Drosophila, C. elegans nos-1 and zebrafish nos-2 become progressively restricted to the germ cells as embryogenesis proceeds through their degradation in the soma and protection by the germ plasm. Moreover, in C. elegans and probably also in zebrafish, translational repression of the unlocalized nos RNA pool restricts synthesis of Nos protein to the germ plasm. Although Nos does not have known patterning functions in C. elegans or zebrafish, the ability of Nos to suppress somatic cell fate in Drosophila suggests that ubiquitous expression of Nos could be toxic in these organisms as well. While the etiology of inefficient germ plasm localization of nos is unclear, the necessity of nos localization for germline function may drive the preservation of this mechanism and the superimposition of controls that accommodate somatic development to it (Gavis, 2008).
Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. This study identifies a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. The ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis (Kalifa, 2006).
Thus far, a single protein, Smaug (Smg), has been shown to interact with the TCE. Smg, which is present only after fertilization, binds to unpaired nucleotides in stem-loop II (the Smg recognition element; SRE), and is required for repression of nos in the early embryo. In contrast, stem-loop III function in the ovary depends on the double-stranded sequence and structure of the helical stem. The essential, Smg-independent function of stem-loop III during oogenesis suggests that this motif is the binding site for an ovarian repressor of nos translation. A factor that recognizes stem-loop III has yet to be identified, however (Kalifa, 2006).
By using a biochemical approach to isolate TCE binding
proteins, a previously uncharacterized Drosophila protein, Glorund, has been identified. Members of the hnRNP family participate in all aspects of nuclear and cytoplasmic RNA metabolism, including mRNA processing, nuclear export, localization, translation, and stability. Although all mRNAs are associated with hnRNPs and many hnRNPs recognize numerous mRNAs, recent studies have identified roles for several hnRNPs in localization and/or translational control of specific mRNAs. hnRNPs K and E1, which repress translation of 15-lipoxygenase (LOX) mRNA during erythrocyte differentiation, and hnRNP I, which participates in localization of Vg1 and VegT mRNAs in Xenopus oocytes, recognize specific primary sequence motifs in the 3'UTRs of their target mRNAs that are required for regulation. In Drosophila oocytes, two hnRNP A/B family members, Squid and Hrp48/ Hrb27C, play roles in localization and translational control of gurken (grk) and osk mRNAs. The sequence motifs in the grk and osk mRNAs that are targeted by these hnRNPs are not well defined, however. Glo is most closely related to mammalian hnRNPs F and H, whose RNA binding domains contain RNA recognition motifs (RRM) that deviate from RRMs found in the more common hnRNP A/B class. Glo is the first hnRNP F/H family member to be implicated in translational repression. Glo interacts specifically with a double-stranded motif in TCE stem-loop III, and the ability of the TCE to bind Glo correlates with its translational regulatory function. Furthermore, through the analysis of a glo null mutation, evidence is provided that Glo is required for translational repression of unlocalized nos mRNA in late oocytes. Thus, Glo acts prior to Smg to establish the repressed state of nos during oogenesis through its interaction with TCE stem-loop III (Kalifa, 2006).
Several lines of evidence establish Glo as a repressor of nos translation. (1) Binding of Glo to the double-stranded UA-rich motif of TCE stem-loop III in vitro correlates with the ability of this element to repress translation in vivo. (2) Analysis of a GFP-Nos reporter in glo mutant ovaries shows increased accumulation of GFP-Nos in late oocytes. (3) Loss of glo results in derepression of unlocalized, translationally silent nos RNA in late oocytes. The translational activity of nos RNA during oogenesis is both spatially and temporally dynamic. nos is translated first in the nurse cells, but becomes repressed in the oocyte after nurse cell dumping. The rapid inactivation of nos translation upon entry into the oocyte has been proposed to occur by a mechanism that blocks translation downstream of the initiation step. The identification of Glo will now facilitate investigation of this mechanism. Since Glo is present in both the nurse cells and oocyte, the ability of Glo to interact with nos or repress its translation must be largely restricted to the oocyte. Evidence that Nos protein produced in nurse cells is targeted for degradation in the oocyte suggests that there are significant physiologic differences between the nurse cells and oocyte that can affect protein behavior. Thus, Glo could be negatively regulated by a nurse cell-specific cofactor or modification event, or positively regulated by an oocyte-specific factor or modification (Kalifa, 2006).
Following fertilization, repression of nos translation is
mediated primarily by the interaction of Smg with stemloop II. Because misexpression of Smg in the female germline severely disrupts oogenesis, Smg cannot fulfill the role of an ovarian repressor of nos translation. At the same time, although Glo is normally present in the early embryo, derepression of nos RNA in smg mutants indicates that Glo cannot substitute for Smg during embryogenesis. Thus, Glo and Smg fulfill temporally distinct roles in nos regulation. The minor requirement observed for stem-loop III in embryonic repression may, however, reflect a role for Glo at the beginning of embryogenesis while Smg is accumulating. The ability of Glo and Smg to bind to the TCE simultaneously would therefore ensure that repression is maintained across the transition from oogenesis to embryogenesis (Kalifa, 2006).
The translational activity of posteriorly localized nos RNA requires that repression by Glo and Smg is alleviated at the posterior of the oocyte and embryo, respectively. However, Glo, like Smg, is uniformly distributed. Thus, both proteins must be prevented from functioning at the posterior pole. Genetic evidence that binding of localization factors and translational repressors to nos RNA is mutually exclusive suggests that localization factors at the posterior may compete with Glo and Smg for binding to the nos 3'UTR. Alternatively, factors at the posterior pole may inactivate the repressors locally by posttranslational modification (Kalifa, 2006).
The effect of eliminating glo is less severe than anticipated from analysis of the effect of TCE stem-loop III mutations that eliminate Glo binding (Crucs, 2000). For example, approximately 25% of glo mutant embryos develop and hatch as larvae, whereas all embryos produced by females carrying the nos-tub:TCEIIIA transgene die with anterior defects. Thus, it is possible that a second ovarian factor, which recognizes TCE stem-loop III similarly to Glo, can partially compensate for loss of glo function. Given that the nos-tub:TCEIIIA transgene lacks all nos 3'UTR sequences outside of the TCE (Crucs, 2000), an alternative explanation is favored that additional factors binding to other regions of the nos 3'UTR can contribute to repression during oogenesis. Attempts to identify such regulatory sequences and factors are currently in progress (Kalifa, 2006).
Glo is the closest Drosophila homolog to mammalian hnRNPs F and H. A second Drosophila protein, Fusilli (Fus), contains RNA binding domains that are more distantly related to hnRNPs F and H and even more distantly related to Glo, but show greatest similarity to a human protein of unknown function. Thus, hnRNPs F and H appear to be represented in Drosophila by a single protein. Members of the F/H family have been implicated as general splicing factors through their interaction with the nuclear cap binding proteins and as regulators of alternative splicing in mammalian neuronal cells. An hnRNP F/H-related protein, guanine-rich sequence factor 1 (GRSF-1), stimulates translation of influenza virus-encoded mRNAs by binding to sequences in their 5'UTRs. Glo is the first hnRNP F/H family member to be identified as a translational repressor, however. The ability to recognize the double-stranded UA motif in TCE stem-loop III sets Glo apart from its mammalian splicing counterparts, which bind preferentially to poly(rG) sequences. In addition, Glo differs from the mammalian proteins by the insertion of a glycine- and asparagine- rich domain between the second and third q-RRMs. These differences may reflect the unique acquisition by Glo of functions such as translational repression. It will therefore be of interest to determine whether the mammalian proteins participate in translational control in addition to splicing and, likewise, whether Glo also functions as a splicing factor (Kalifa, 2006).
Like Hrp48/Hrb27C, which was first identified as a splicing regulator and subsequently shown to regulate both localization and translation of grk and osk mRNAs, Glo may serve multiple functions. The pleiotropy of the glo mutant phenotype, including its zygotic lethality, suggests that glo acts at different developmental stages to regulate RNAs in addition to nos. Expression of Glo in the central nervous system (CNS) at late stages of embryogenesis is particularly intriguing in light of increasing evidence for translational control in neuronal development and synaptic function. Furthermore, the TCE can mediate translational repression in subsets of cells in the CNS and this repression is Smg independent. Glo is therefore a good candidate to mediate repression of RNAs with TCElike motifs in the CNS (Kalifa, 2006).
Shortening of the poly(A) tail (deadenylation) is the first and often rate-limiting step in the degradation pathway of most eukaryotic mRNAs and is also used as a means of translational repression, in particular in early embryonic development. The nanos mRNA is translationally repressed by the protein Smaug in Drosophila embryos. The RNA has a short poly(A) tail at steady state and decays gradually during the first 2-3 h of development. Smaug has also been implicated in mRNA deadenylation. To study the mechanism of sequence-dependent deadenylation, a cell-free system was developed from Drosophila embryos that displays rapid deadenylation of nanos mRNA. The Smaug response elements contained in the nanos 3'-untranslated region are necessary and sufficient to induce deadenylation; thus, Smaug is likely to be involved. Unexpectedly, deadenylation requires the presence of an ATP regenerating system. The activity can be pelleted by ultracentrifugation, and both the Smaug protein and the CCR4.NOT complex, a known deadenylase, are enriched in the active fraction. The same extracts show pronounced translational repression mediated by the Smaug response elements. RNAs lacking a poly(A) tail are poorly translated in the extract; therefore, SRE-dependent deadenylation contributes to translational repression. However, repression is strong even with RNAs either bearing a poly(A) tract that cannot be removed or lacking poly(A) altogether; thus, an additional aspect of translational repression functions independently of deadenylation (Jeske, 2006; full text of article).
Anteroposterior patterning of the Drosophila embryo depends on a
gradient of Nanos protein arising from the posterior pole. This gradient
results from both nanos mRNA translational repression in the bulk of
the embryo and translational activation of nanos mRNA localized at
the posterior pole. Two mechanisms of nanos translational repression
have been described, at the initiation step and after this step. This study
identifies a novel level of nanos translational control. The Smaug protein bound to the nanos 3' UTR recruits the
deadenylation complex CCR4-NOT (see Twin), leading to rapid deadenylation and subsequent
decay of nanos mRNA. Inhibition of deadenylation causes stabilization
of nanos mRNA, ectopic synthesis of Nanos protein and head defects.
Therefore, deadenylation is essential for both translational repression and
decay of nanos mRNA. A mechanism is proposed for translational
activation at the posterior pole. Translation of nanos mRNA at the
posterior pole depends on oskar function. Oskar prevents
the rapid deadenylation of nanos mRNA by precluding its binding to
Smaug, thus leading to its stabilization and translation. This study provides
insights into molecular mechanisms of regulated deadenylation by specific
proteins and demonstrates its importance in development (Zaessinger, 2006).
Post-transcriptional mechanisms of gene regulation play a prominent role
during early development. Because the oocyte and developing embryo go through
a phase in which no transcription takes place, gene expression relies on a
pool of maternal mRNAs accumulated during oogenesis and is regulated at the
level of translation or mRNA stability. It has been shown in several
biological systems that poly(A) tail shortening contributes to translational
silencing, whereas translational activation requires poly(A) tail extension.
Poly(A) tail shortening, or deadenylation, is also the first step in mRNA
decay. Subsequent steps occur only after the poly(A) tail has been shortened
beyond a critical limit. Rapid deadenylation of unstable RNAs is caused by
destabilizing elements, for example AU-rich elements (AREs) found in the
3' UTRs of several mRNAs. A number of proteins have been identified that
bind to destabilizing RNA sequences and accelerate deadenylation as well as
subsequent steps of decay (Zaessinger, 2006).
In yeast, deadenylation is mostly catalyzed by the multi-subunit CCR4-NOT
complex, and this complex is also involved in deadenylation in Drosophila (Temme, 2004) and in mammalian cells. A second conserved deadenylase, the heterodimeric PAN2-PAN3 complex, appears to act before the CCR4-NOT complex. A third enzyme, the poly(A)-specific ribonuclease (PARN) is
present in most eukaryotes but has not been found in yeast and
Drosophila (Zaessinger, 2006).
Translational regulation of maternal mRNAs in Drosophila is
essential to the formation of the anteroposterior body axis of the embryo.
During embryogenesis, a gradient of the Nanos (Nos) protein arises from the
posterior pole and organizes abdominal segmentation. This
gradient results from translational regulation of maternal nos mRNA.
The majority of nos transcripts is uniformly distributed throughout
the bulk cytoplasm and is translationally repressed and subsequently degraded during the first 2-3 hours of embryonic development. A
small proportion of nos transcripts is localized in the pole plasm,
the cytoplasm at the posterior pole that contains the germline determinants.
This RNA escapes repression and degradation, and its translation product forms a concentration gradient from the posterior pole.
Both translation activation at the posterior pole and repression elsewhere in
the embryo are essential for abdominal development, and head and thorax
segmentation, respectively (Zaessinger, 2006 and references therein).
Translation of nos mRNA is repressed in the embryo by Smaug (Smg),
which binds two Smaug response elements (SREs) in the proximal part of the
nos 3' UTR. The SREs are also essential for the decay of nos
mRNA. Repression of nos translation appears to be a multistep process,
involving at least one level of regulation at the initiation step and
another after nos mRNA has been engaged on polysomes.
Repression at the initiation step is thought to involve an interaction between Smg and the protein Cup. The latter associates with the cap-binding initiation factor eIF4E, displacing the initiation factor eIF4G.
Translation of nos mRNA at the posterior pole depends on Oskar (Osk)
protein, although its mechanism of action has remained unknown (Zaessinger, 2006).
Bulk nos mRNA has a short poly(A) tail, and it was thought that
nos translational control was independent of poly(A) tail length
regulation. More recently, Smg and its yeast homologue Vts1 were shown
to be involved in the degradation of mRNAs. Smg
induces degradation and deadenylation of Hsp83 mRNA during early
embryogenesis. This appears to result from recruitment by Smg of the CCR4-NOT
deadenylation complex on Hsp83 mRNA, although the Smg-binding sites
in this mRNA have not been identified. However, Hsp83 mRNA
deadenylation was reported not to repress its translation. This study shows that nos mRNA is subject to regulation by active
deadenylation by the CCR4-NOT deadenylation complex. This deadenylation
depends on Smg and on the SREs in the 3' UTR of nos mRNA. The model is confirmed of the CCR4-NOT complex recruitment by Smg, in this case,
onto nos mRNA, using genetic interactions between mutants affecting
smg and the CCR4 deadenylase (properly named Twin and not to be confused with Twins), and showing the presence in a same protein complex of endogenous Smg and CAF1 (properly termed Pop2 and not to be confused with Caf1), a protein of the CCR4-NOT complex. Active deadenylation of nos mRNA contributes to its translational repression in the bulk embryo and is essential for the
anteroposterior patterning of the embryo. Moreover, Osk activates
translation of nos by preventing the specific binding of Smg protein
to nos mRNA, thereby precluding active deadenylation and
destabilization of nos mRNA (Zaessinger, 2006).
This paper shows that poly(A) tail length regulation is central to
nos translational control. Poly(A) tail length regulation is a major
mechanism of translational control, particularly during early development.
nos translational control has been reported to be independent
of poly(A) tail length. This conclusion came from the absence of nos
poly(A) tail elongation between ovaries and early embryos, and the
lack of nos poly(A) tail shortening between wild-type and
osk mutant embryos in which nos mRNA is not translated at
the posterior pole. However, later studies suggested that this lack of poly(A) tail change was not unexpected, as nos mRNA translation starts in ovaries, and the pool of translationally active nos mRNA in
embryos is very small (4%) and remains undetected among the amount of
translationally repressed nos in whole embryos. It has now been found that
nos mRNA deadenylation by the CCR4-NOT complex, recruited to the
3' UTR by Smg, is required for nos translational repression in
the bulk embryo. In addition, these data also suggest that nos
translation at the posterior pole depends on the prevention of this
deadenylation. nos mRNA is regulated at several levels, including
localization, degradation, translational repression and translational
activation. Localization at the posterior pole depends on two mechanisms: an
actin-dependent anchoring at late stages of oogenesis, after nurse cells
dumping and localized stabilization. Localization and translational
control are coupled in that the localized RNA escapes both translational
repression and degradation. A mechanism is proposed for this coupling.
Translational repression and RNA degradation both involve Smg-dependent
deadenylation. Deletion of the SREs in a nos transgene, as well as
mutations in smg or in twin, which encodes the major
catalytic subunit of the deadenylating CCR4-NOT complex, abrogate poly(A) tail
shortening. Lack of deadenylation prevents the timely degradation of the RNA
and also relieves translational repression. Deadenylation could repress
nos mRNA translation by two mechanisms. Interaction of the
cytoplasmic poly(A) binding protein (PABP) with mRNA poly(A) tails is
important for the activation of translation initiation.
Therefore, poly(A) shortening of nos mRNA would lead to PABP
dissociation and inhibition of translation. In addition, deadenylation leads
eventually to nos mRNA decay, which should also contribute to
translational repression. Consistent with the Smg-dependent deadenylation of
nos mRNA, describe in embryos, a recent study documented
SRE-dependent deadenylation of chimeric transcripts containing the 3'
UTR of nos mRNA in cell-free extracts from Drosophila
embryos (Jeske, 2006). In this system, deadenylation of the chimeric RNAs also strongly contributes to translational repression, along with at least another deadenylation-independent mechanism (Zaessinger, 2006).
In this analysis, twin and smg mutants, although both
impaired in nos mRNA poly(A) tail shortening, did not show the same
defects. twin mutants fail to show nos poly(A) tail
shortening during embryogenesis, whereas in smg mutant embryos or
when poly(A) tails are measured from nos(ΔTCE) transgene, a
poly(A) tail elongation is visible. This suggests that nos mRNA is
also regulated by cytoplasmic polyadenylation which balances the deadenylation reaction, and that Smg binding to the RNA reduces the polyadenylation reaction. Consistent with a dynamic regulation of poly(A) tail length of maternal mRNAs resulting from a tight balance between regulated deadenylation and polyadenylation, it was found that in mutants for the GLD2 poly(A) polymerase, which is involved in cytoplasmic polyadenylation, nos mRNAs are precociously degraded in 0-1 hour embryos (Zaessinger, 2006).
Ectopic expression of osk in the bulk cytoplasm of the embryo is sufficient to impair nos mRNA binding to Smg and its deadenylation and destabilization. Therefore, it is proposed that, in wild-type embryos, Osk at the posterior pole inhibits Smg binding to the anchored nos mRNA, preventing deadenylation, decay and translational repression. This results in localized nos stabilization and translation. Osk might achieve this by a direct binding to Smg; it was shown to interact with Smg in vitro, through a region overlapping the RNA-binding domain in Smg. Alternatively, Osk could prevent Smg function independently of its binding to Smg, through its recruitment by another protein in nos-containing mRNPs. Consistent with a potential presence of Smg and Osk in the same protein complex, it was possible to co-immunoprecipitate Osk with Smg in embryos overexpressing Osk (Zaessinger, 2006).
Two mechanisms of nos translational repression have already been
described. A first mode of translation inhibition appears to act during
elongation, as suggested by polysome analysis and by
the involvement of the Bicaudal protein, which corresponds to a subunit of the nascent polypeptide associated complex. The
second mode of repression involves Smg and is thought to affect initiation. It requires the association of Smg with the protein Cup, which also binds eIF4E. The association of Cup with eIF4E competes with the eIF4E/eIF4G interaction, which is essential for translation initiation. This study
identifies deadenylation by the CCR4-NOT complex as a novel level of
nos translational repression, also involving Smg. Smg protein
synthesis is probably induced by egg activation during egg-laying. Smg is
absent in ovaries and accumulates during the first hours of embryogenesis,
with a peak at 1-3 hours. Its amount is low during the first hour and possibly nonexistent during the first 30 minutes. This correlates with the presence at that time of high levels of nos mRNA in the bulk embryo that are not destabilized. nos translational repression is active, however, as this pool of mRNA is untranslated. Thus a Smg-independent mode of repression
must be efficient during the first hour of development. This might correspond
to repression at the elongation step and/or involve the Glorund protein, a
Drosophila hnRNP F/H homologue newly identified as a nos
translational repressor in the oocyte (Kalifa, 2006). Glorund has a role in repression of unlocalized nos mRNA in late oocytes and has been suggested to also act at the beginning of embryogenesis while Smg is
accumulating to ensure the maintenance of translational repression at the
oogenesis to embryogenesis transition (Kalifa, 2006). Analysis of glorund mutants revealed that the embryonic phenotypes
are less severe than expected and led to the proposal that at least an
additional level of nos translational repression is active in oocytes
(Kalifa, 2006). Overexpression of Osk in the germline with nos-Gal4
results in long poly(A) tails of nos mRNA, even in 0-1 hour embryos
in which Smg protein is poorly expressed. This suggests that the short poly(A)
tail of nos mRNA in 0-1 hour wild-type embryos could in part result
from active deadenylation during oogenesis, which would depend on a regulatory
protein different from Smg. Deadenylation could therefore be involved in
nos regulation during oogenesis, and would also be prevented by Osk
in the pole plasm, as in embryos (Zaessinger, 2006).
Genetic evidences indicate that all three levels of translational
repression are additive. Although the importance of the Smg/Cup/eIF4E mode of
nos translational repression for the anteroposterior patterning of
the embryo has not been addressed, the other two levels of repression are
essential, as ectopic Nos protein leads to disruption of the embryo
anteroposterior axis in twin or bicaudal
mutants. This demonstrates that none of the three levels of
repression is sufficient by itself and suggests that all three regulations are
required to achieve complete translational repression of nos. As Osk
acts by preventing the binding of Smg to the nos 3' UTR, it is
likely to inhibit both Smg-dependent mechanisms of translational
repression (Zaessinger, 2006).
The presence of Smg in discrete cytoplasmic foci and its partial
colocalization in these foci with components of the CCR4-NOT deadenylation
complex, and with components of P bodies, suggest that Smg-dependent
deadenylation and translational control of nos occur in P bodies. P
body dynamics and function have not been addressed in a complete organism
during development. Consistent with the apparent complexity of P body
function, including mRNA decay and translational repression,
embryos different subsets of Smg-containing structures were identified in embryos: these subsets either do or do not contain the CCR4-NOT deadenylation complex and the Xrn1 5'-3' exonuclease. This suggests the existence of different types of P bodies that may have distinct functions (Zaessinger, 2006).
The CCR4-NOT complex is involved in default
deadenylation of bulk mRNAs in somatic cells
(Temme, 2004). This study finds that the same deadenylation complex has a role in active, sequence-specific deadenylation of a particular mRNA. Activation of
deadenylation by CCR4-NOT results from the recruitment of the deadenylation
complex by a regulatory RNA-binding protein to its specific mRNA target (this
study) (Semotok, 2005). Several RNA-binding proteins are expected to interact with the CCR4-NOT complex to regulate the deadenylation of different pools of mRNAs in different tissues. CCR4 controls poly(A) tail lengths of Cyclin A and B mRNAs during oogenesis (Morris, 2005); the regulatory protein has not been identified, but it cannot be Smg, which is not expressed in ovaries. A similar mode of active deadenylation involving the recruitment of the deadenylation complex by
ARE-binding proteins has been proposed in mammalian cells. A study in yeast has identified the PUF (Pumilio/FBF) family of RNA-binding proteins as activators of CCR4-NOT-mediated deadenylation through a direct interaction between PUF and POP2 (the CAF1 homologue). Although default deadenylation by CCR4 is not essential for viability (Temme, 2004), active deadenylation by CCR4 of specific mRNAs is essential for certain developmental processes, in particular during early development (Zaessinger, 2006).
Anterior-posterior axis patterning of the Drosophila embryo requires Nanos activity selectively in the posterior. This spatial asymmetry of Nanos is generated by the localization of nanos mRNA to the posterior pole of the embryo, where it is subsequently translated. Posterior localization of nanos is mediated by a complex cis-acting localization signal in its 3' untranslated region comprising several partially redundant localization elements. This localization signal redundancy has hampered the identification of trans-acting factors that act specifically to effect posterior localization of nanos. This study used a biochemical approach to identify Rumpelstiltskin, a Drosophila heterogeneous nuclear ribonucleoprotein (hnRNP) M homolog, which binds directly to an individual nanos localization element. Rumpelstiltskin associates with nanos mRNA in vitro and in vivo, and binding by Rumpelstiltskin correlates with localization element function in vivo. Through analysis of a rumpelstiltskin null mutation by genetic strategies that circumvent redundancy, it was demonstrated that Rumpelstiltskin regulates anterior-posterior axis patterning by functioning as a direct-acting nanos mRNA localization factor (Jain, 2008).
Using a biochemical approach, Rump, the Drosophila hnRNP M homolog, was identified as a bona fide nos mRNA localization factor. Rump corresponds to the p75 binding activity that was previously shown to recognize the nos +2' localization element (Bergsten, 2001). Recombinant and endogenous Rump recognize two CGUU motifs in the +2' element. A point mutation within either of these motifs is sufficient to disrupt both interaction with Rump and mRNA localization function. The demonstration that sequences required for Rump binding are also required for posterior localization provides strong evidence that Rump plays a direct role in mediating nos mRNA localization through its interaction with the nos 3'UTR (Jain, 2008).
Rump was previously identified in Drosophila S2 cells through its cross-reactivity with an antibody to the orthologous C. tetans protein, Hrp59 (Kiesler, 2005). In the current experiments, Drosophila ovarian, embryonic and S2-cell Rump protein migrate with an apparent mobility of ~70 kDa, consistent with its calculated molecular weight of 67 kDa. Mammalian hnRNP M proteins have been implicated in splicing through their association with pre-mRNA at an early step in spliceosome assembly. Studies in Chironomus have shown that Hrp59 binds to pre-mRNA co-transcriptionally and remains associated with the RNA until it reaches the nuclear envelope, and recent analysis of the Drosophila S2 cell protein shows that it regulates alternative splicing of its own mRNA (Jain, 2008).
Rump has multiple functions at various stages of the Drosophila life cycle. In addition to defects in abdominal segmentation, many embryos produced by rump mutant females arrest development prematurely and adult rump mutant males are largely sterile. Neither the maternal-effect developmental arrest nor the zygotic sterility phenotypes is characteristic of nos mutants, indicating that Rump regulates both nos-dependent and nos-independent processes. This functional pleiotropy is consistent with the isolation of numerous Rump target mRNAs from S2 cells, the steady state levels of which are decreased by RNAi knockdown of rump (Kiesler, 2005). Since nos is not expressed in S2 cells, its behavior in this assay cannot be determined. However, no alteration in the abundance or splicing of nos mRNA is detected in rump mutant ovaries or embryos. Instead, the data demonstrate a specific and previously unidentified role for Rump as an mRNA localization factor (Jain, 2008).
The different regulatory functions of Rump might be distinguished by distinct recognition motifs in Rump target mRNAs. Rump protein isolated from S2 cells by immunoprecipitation with anti-Hrp59 antibody binds preferentially to a purine-rich motif with similarity to exonic splicing enhancers. This motif, with GGAGG at its core, is enriched among Rump target mRNAs in S2 cells identified by whole-genome analysis (Kiesler, 2005). The GGAGG core sequence does not exist within the nos +2' element, nor within the entire nos 3'UTR. The ability to recognize different motifs might be conferred by different RRMs within Rump. Similarity between RRM3 and the human splicing regulator ASF (also known as SF2 and SFRS1 - Human Gene Nomenclature Database) RRM suggests that RRM3 might be specifically responsible for interaction with splicing targets (Kiesler, 2005), whereas RRMs 1 and/or 2 might recognize a different set of Rump targets. In addition, binding of Rump to nos might be cooperative, as mutation of one or both CGUU motifs is equally deleterious. Consistent with this idea, Rump self-interaction was detected in a yeast two-hybrid screen, suggesting that Rump might function as a dimer (Jain, 2008).
Multifunctionality appears to be a common feature of proteins that regulate mRNA localization and localized translation. For example, Drosophila Hrp48 (also known as Hrb27C - FlyBase), an hnRNP A/B family member that participates in localization and translational regulation of osk and gurken mRNAs, also functions as a regulator of alternative splicing and mammalian homologs of Xenopus Vg1RBP60, an hnRNP I protein involved in Vg1 mRNA localization, play various roles in nuclear RNA biogenesis. Similarly to Rump, activities of two other nos regulators, Glorund (Glo) and Smaug (Smg), may be distinguished through multiple, distinct binding specificities. Glo, an hnRNP F/H homolog, represses translation of unlocalized nos during oogenesis through its interaction with an AU-rich double-stranded region within the nos 3'UTR, whereas its mammalian counterparts regulate alternative splicing through their interaction with a G-rich motif. Smg represses translation of unlocalized nos in the embryo through its interaction with Smaug Recognition Elements (SREs) in the nos 3'UTR, but its function in degradation of maternal mRNAs is SRE-independent (Jain, 2008).
Elimination of rump function has a more variable effect on localization of nos-tub:nos+2 RNA than does mutation of Rump binding sites. Thus, another ovarian protein with a similar recognition motif might be able to compensate for the loss of Rump. Alternatively, mutation of the Rump binding site(s) might have a more global effect on the structure of the localization element, disrupting the function of other localization factors. The CGUU motifs recognized by Rump flank one of the two SREs recognized by Smg within the nos 3'UTR and both the (A)G:C and (D)G:C mutations disrupt the function of the SRE, consistent with the ability of these mutations to cause pleiotropic effects. By contrast, mutation of the SRE had only a small effect on p75 binding to the +2' element, suggesting that Rump binding is less sensitive to local perturbations. Finally, several linker-scanning mutations in the +2' element disrupt the localization function of this element but do not disrupt Rump binding in vitro, suggesting that at least one additional, as yet unknown, factor mediates localization via the +2' element (Jain, 2008).
During early and mid-oogenesis, Rump accumulates in the nurse cell nuclei where nos is synthesized, suggesting that it might first associate with nos mRNA in the nucleus. In this regard, Rump may resemble Xenopus Vg1RBP60/hnRNP I, which associates with Vg1 and VegT mRNAs in the nucleus and travels with the RNAs to the cytoplasm where the RNP is remodeled. Owing to the impenetrability of the late-stage oocyte to antibodies, it has not been possible to determine the distribution of Rump in late oocytes when nos becomes posteriorly localized. However, Rump appears cytoplasmic in newly fertilized embryos and is not enriched at the posterior of the embryo, suggesting that it does not maintain a stable association with nos once anchored at the germ plasm. Biochemical evidence for a continued association of Rump with nos during embryogenesis, together with the uniform distribution of Rump protein, indicate that in the early embryo Rump is most likely to be associated with unlocalized, translationally repressed nos mRNA. The close apposition of Rump and Smg binding sites raises the possibility that binding by these factors is mutually stabilizing (Jain, 2008).
Taken together, data presented in this study suggest a model whereby Rump associates with nos mRNA during oogenesis, possibly in the nucleus, where it participates in the generation or stabilization of localization-competent nos RNP complexes. A small fraction of nos RNPs are entrapped by the germ plasm and this association results in reorganization of the RNP and release of Rump. The majority of nos mRNA remains unlocalized and, consequently, bound to Rump. Whether Rump plays a role in coordinating translational control with nos mRNA localization remains to be determined (Jain, 2008).
Piwi-associated RNAs (piRNAs), a specific class of 24- to 30-nucleotide-long RNAs produced by the Piwi-type of Argonaute proteins, have a specific germline function in repressing transposable elements. This repression is thought to involve heterochromatin formation and transcriptional and post-transcriptional silencing. The piRNA pathway has other essential functions in germline stem cell maintenance and in maintaining germline DNA integrity. This study uncovered an unexpected function of the piRNA pathway in the decay of maternal messenger RNAs and in translational repression in the early embryo. A subset of maternal mRNAs is degraded in the embryo at the maternal-to-zygotic transition. In Drosophila, maternal mRNA degradation depends on the RNA-binding protein Smaug and the deadenylase CCR4, as well as the zygotic expression of a microRNA cluster. Using mRNA encoding the embryonic posterior morphogen Nanos (Nos) as a paradigm to study maternal mRNA decay, it was found that CCR4-mediated deadenylation of nos depends on components of the piRNA pathway including piRNAs complementary to a specific region in the nos 3' untranslated region. Reduced deadenylation when piRNA-induced regulation is impaired correlates with nos mRNA stabilization and translational derepression in the embryo, resulting in head development defects. Aubergine, one of the Argonaute proteins in the piRNA pathway, is present in a complex with Smaug, CCR4, nos mRNA and piRNAs that target the nos 3' untranslated region, in the bulk of the embryo. It is proposed that piRNAs and their associated proteins act together with Smaug to recruit the CCR4 deadenylation complex to specific mRNAs, thus promoting their decay. Because the piRNAs involved in this regulation are produced from transposable elements, this identifies a direct developmental function for transposable elements in the regulation of gene expression (Rouget, 2010).
In Drosophila embryos, Nos is expressed as a gradient that emanates from the posterior pole and organizes abdominal segmentation. The majority of nos mRNA is distributed throughout the bulk cytoplasm, translationally repressed and subsequently degraded during the first 2-3 h of development. This repression is essential for head and thorax segmentation. A small amount of nos transcripts, localized at the posterior pole of the embryo, escapes degradation and is actively translated, giving rise to the Nos protein gradient. nos mRNA decay in the bulk cytoplasm depends on the CCR4-NOT deadenylation complex and its recruitment onto nos by Smaug (Smg). This contributes to translational repression in the bulk of the embryo and is required for embryonic antero-posterior patterning (Zaessinger, 2006; Rouget, 2010 and references therein).
Smg has been suggested to be not the only activator of nos mRNA decay during early embryogenesis. Zygotically expressed miRNAs have been reported to activate maternal mRNA deadenylation in zebrafish embryos and decay in Drosophila embryos. This study investigated the potential involvement of other classes of small RNAs in mRNA deadenylation and decay before zygotic expression. Because piRNAs are expressed maternally in the germ line and are present in early embryos, the possible role of the piRNA pathway in maternal mRNA deadenylation was analyzed. Piwi, Aubergine (Aub) and Ago3 are specific Argonaute proteins, Armitage (Armi) and Spindle-E (Spn-E) are RNA helicases, and Squash (Squ) is a nuclease involved in piRNA biogenesis and function. Poly(A) test assays were performed to measure nos mRNA poly(A) tail length in embryos spanning 1-h intervals during the first 4 h of embryogenesis. In contrast to the progressive shortening of nos mRNA poly(A) tails observed in wild-type embryos correlating with mRNA decay during this period, nos poly(A) tail shortening was affected in embryos from females mutant for the piRNA pathway (herein referred to as mutant embryos). This defect in deadenylation correlated with higher amounts of nos mRNA in mutant embryos, as quantified by reverse transcription-quantitative PCR (RT-qPCR). In situ hybridization revealed stabilized nos mRNA in the bulk cytoplasm of mutant embryos where it is normally degraded in the wild type. Consistent with previous data showing that nos mRNA deadenylation is required for translational repression (Zaessinger, 2006), defective deadenylation in mutant embryos resulted in the presence of ectopic Nos protein throughout the embryo. The presence of Nos in the anterior region results in the repression of bicoid and hunchback mRNA translation and in affected head skeleton. It was found that the piwi1 mutant embryos that were able to produce a cuticle had head defects (Rouget, 2010).
The piRNA pathway has a role during early oogenesis in preventing DNA damage, possibly through the repression of transposable element transposition. DNA double-strand breaks arising in mutants of the piRNA pathway correlate with affected embryonic axis specification, and this developmental defect is suppressed by mutations in the Chk2 DNA-damage signal transduction pathway. This study found that defects in nos mRNA deadenylation and decay observed in aub or armi mutants were not suppressed by Chk2 (mnkP6) mutations, indicating that these defects did not result from activation of the Chk2 pathway earlier during oogenesis. Moreover, affected deadenylation of nos mRNA in piRNA pathway mutants did not depend on oskar (Rouget, 2010).
A potential direct role of the piRNA pathway in the regulation of nos mRNA deadenylation and decay in the embryo was addressed. Aub and Piwi accumulate in the pole plasm and in pole cells of the embryo. However, lower levels of Aub and Piwi are found throughout the entire embryo. Ago3 is also present throughout the embryo. Aub and Ago3 are found in the cytoplasm and accumulated in discrete foci, a distribution similar to that of CCR4 and Smg. CCR4 and Smg were reported to partially colocalize in small cytoplasmic foci (Zaessinger, 2006). Aub and Ago3 also partially colocalize with Smg and CCR4 in the bulk of syncytial embryos, in both cytoplasmic foci and a diffusely distributed cytoplasmic pool. Importantly, the distributions of CCR4 and Smg depended on the piRNA pathway; they are strongly affected in aub and spn-E mutant embryos. Although global amounts of CCR4 and Smg do not decrease in mutant embryos, CCR4 foci strongly increase in size, whereas Smg foci decreases in size or disappears. This suggests that subsets of CCR4 and Smg foci have different functions and that deadenylation may take place diffusely in the cytoplasm. These results demonstrate a functional link between CCR4-mediated deadenylation and the piRNA pathway (Rouget, 2010).
Co-immunoprecipitation experiments showed that Aub co-precipitate Smg, CCR4 and Ago3 in the absence of RNA, indicating the presence of these proteins in a common complex. Smg also co-precipitates CCR4, Aub and Ago3; however, Piwi is not found to co-precipitate Smg or CCR4. Importantly, Smg, CCR4 and Ago3 also co-precipitate with Aub in osk54 mutant embryos that are defective in pole plasm assembly, indicating the presence of this complex outside the pole plasm. Next it was shown that nos mRNA co-precipitate with Aub in both wild-type and osk54 embryos. The amount of nos mRNA is similar in Aub and Smg immunoprecipitates (Rouget, 2010).
These findings show that the Argonaute proteins Aub and Ago3 associate with Smg and the CCR4 deadenylase complex to directly regulate nos mRNA in the bulk cytoplasm of early embryos (Rouget, 2010).
The nos 3'untranslated region (UTR) contains Smg-binding sites located in its 5'-most region [referred to as the translational control element (TCE)]. piRNAs sequenced from early embryos and presumed capable of targeting nos 3'UTR were sought based on their sequence complementarity. Notably, a specific region located in the 3'-most part of the 3'UTR could be targeted by over 200 copies of piRNAs originating from two transposable elements, 412 and roo. piRNAs complementary to nos 3'UTR were visualized by northern blots. In addition, piRNAs predicted to target nos 3'UTR co-immunoprecipitated with Aub. nos genomic transgenes deleted for different parts of the 3'UTR were used to address the requirement of the corresponding regions for nos mRNA deadenylation. It was shown previously that the TCE (nucleotides 1-184) is required for nos mRNA poly(A) tail shortening, consistent with the role of Smg in this process. Deletion of region 184-403 (nos(delta1)) had no effect, whereas poly(A) tails from the transgene deleted for the region 403-618 (nos(delta2)) were elongated in 3-4-h embryos. This could indicate regulation by the miRNA predicted by miRBase to target this region. Deletion of 618-844 in the nos 3'UTR (nos(delta3)) had a strong effect on nos deadenylation. Consistent with this, nos mRNA levels produced by this transgene remained mostly stable. This resulted in defects in embryo patterning: a total of 35% of embryos from nos(delta3) females did not hatch and among them 86% showed head skeleton defects. Next specific sequences complementary to 412 (15 nucleotides) and roo (11 nucleotides) retrotransposon piRNAs were deleted. These short deletions, either independently or in combination, affected nos mRNA deadenylation (Rouget, 2010).
To support further the role of retrotransposon piRNAs in nos mRNA regulation, 412 and roo piRNAs were blocked by injecting specific 2'-O-methyl anti-piRNA in embryos, and cuticles were recorded as a functional assay of Nos ectopic synthesis at the anterior pole. Injection of anti-piRNA(412) or anti-piRNA(roo) resulted in specific head development defects (Rouget, 2010).
Together, these results provide strong evidence that an interaction between piRNAs and nos mRNA is required for nos mRNA deadenylation and translational repression in the first hours of embryogenesis (Rouget, 2010).
This study has identified a new function of the piRNA pathway in the regulation of maternal mRNAs. Recently, piRNAs derived from the 3'UTRs of cellular transcripts have been identified in gonadal somatic cells, although their biological role has not been clarified. It is proposed that piRNAs, in complex with Piwi-type Argonaute proteins Aub and Ago3, target nos maternal mRNAs and recruit or stabilize the CCR4-NOT deadenylation complex together with Smg. These interactions induce rapid mRNA deadenylation and decay. Thus, activation of mRNA deadenylation represents a new direct mechanism of action for the piRNA pathway with an essential developmental function during the first steps of embryogenesis (Rouget, 2010).
Smg is a general factor for mRNA decay during early embryogenesis. Because Aub and Ago3 are present in a complex with Smg in early embryos, a proportion of Smg mRNA targets could be regulated by the piRNA pathway. Consistent with this, other maternal mRNAs that are destabilized during early embryogenesis are targeted by abundant piRNAs and their deadenylation depends on the piRNA pathway (Rouget, 2010).
These piRNAs involved in gene regulation are generated from transposable element sequences. Although transposable elements have been described to be essential for genome dynamics and evolution, their immediate function within an organism has remained rather elusive. This study provides evidence for a co-evolution between transposable elements and the host genome and reveals the direct developmental function of transposable elements in embryonic patterning, through the regulation of gene expression (Rouget, 2010).
Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far (Jain, 2008). Through a sensitized genetic screen, the Argonaute family member Aubergine (Aub) has now been identified as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. These results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex (Becalska, 2011).
Localization of nos mRNA to the posterior of the Drosophila embryo is critical for patterning of the A-P body axis. Although a cis-acting nos mRNA localization signal has been identified, the complement of trans-acting factors required for assembly of a nos RNP complex competent for posterior localization has remained elusive. From a sensitized genetic screen, Aub was identified as a novel nos mRNA localization factor, Aub interacts with nos mRNA in vivo. Importantly, nos localization was shown to be affected by aub acting downstream of osk mRNA localization, implying independent roles for aub in regulating these two transcripts. Although the role of aub in osk localization appears to be indirect, through a requirement in oocyte microtubule organization, these results suggest that aub plays a more direct role in regulating nos mRNA (Becalska, 2011).
A decrease in aub activity leads to defects in nos mRNA localization and, consequently, in patterning of the A-P axis when nos localization signal redundancy is reduced by removing two localization elements. A similar behavior is observed in rump mutants, which exhibit only weak segmentation defects unless redundantly acting elements are removed from the nos localization signal (Jain, 2008). Presumably, elimination of individual localization signal elements compromises localization by stripping away the contributions of nos localization factors with overlapping functions in nos RNP assembly. Conversely, elimination of multiple nos localization factors should lead to a more severe defect than elimination of an individual factor. Consistent with this prediction, decreasing aub gene dosage in rump mutants also leads to more severe loss of abdominal segments (Becalska, 2011).
In addition to allowing a requirement for aub to be uncovered, the sensitized nos+1+3 background has facilitated the separation of an indirect requirement for aub in osk localization from a more direct requirement in nos localization. Defects in osk regulation and abdominal segmentation are observed only when females are homozygous mutant for aub and not when they are heterozygous. By contrast, defects in nos+1+3 localization are observed when females are heterozygotes for aub mutations. These results are further supported by previous data showing that the ability of ectopically expressed Osk to recruit nos mRNA is compromised in aub−/+ embryos (Becalska, 2011).
Aub has been implicated in the rasiRNA pathway that silences retrotransposons in the germline. However, mutation of squash, which encodes a rasiRNA pathway component that interacts with Aub, has no effect on the nos+1+3 transgene. Interestingly, another rasiRNA pathway component, piwi, has the opposite effect of aub on the nos+1+3 transgene, as heterozygosity for a piwi mutation results in increased segmentation in the sensitized background. Mutations that inactivate the rasiRNA pathway, including aub mutations, activate the DNA damage checkpoint, presumably due to unsuppressed transposon activity. Checkpoint activation disrupts microtubule organization and grk translation, resulting in a failure of axis specification that is thought to lead to subsequent defects in osk mRNA localization. However, the effect of aub mutation on nos+1+3 mRNA localization is independent of the DNA damage pathway, providing further evidence that Aub regulates nos independently of osk. Moreover, these results indicate that Aub function in nos localization is distinct from its function in RNA silencing (Becalska, 2011).
Biochemical experiments indicate nos mRNA forms a complex with Aub in vivo, although whether Aub interacts directly with nos mRNA, or is recruited to the complex by other proteins that bind directly to nos, is not yet clear. Soluble recombinant Aub necessary to distinguish between these possibilities has not been obtained. However, the RNA-dependent co-purification of Aub and Rump, combined with evidence for genetic interactions between aub and rump further supports the contribution of Aub to the formation and/or function of a nos localization RNP complex. Whereas Rump is not concentrated at the posterior of the oocyte, Aub-GFP is localized to the posterior during midoogenesis and continues to accumulate at the posterior pole throughout the later stages of oogenesis when nos becomes localized. Thus, the contributions of Rump and Aub to nos RNP complexes may be dynamic, with both proteins accompanying nos as it is dispersed throughout the oocyte during ooplasmic streaming, but only Aub remaining associated with the nos RNP upon its entrapment at the posterior. Isolation and characterization of the full complement of nos localization factors will be essential to dissect the assembly pathway for nos localization complexes. The isolation of aub in a sensitized genetic screen validates the use of such an approach, in addition to biochemical purification strategies that proved successful for isolation of Rump, for achieving this goal (Becalska, 2011).
Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). This study describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. Smaug interacts with the Ago1 protein, and Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, these data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation (Pinder, 2013).
The data indicate that Smaug directly recruits Ago1 to nos mRNA in a miRNA-independent fashion. Interestingly, previous work suggests similarities between the mechanisms that Smaug and Ago use to regulate mRNAs. For example, translationally repressed nos mRNA is polysome associated similar to some transcripts that are repressed by Ago proteins. However, it is unclear if Smaug protein participates in repression of nos mRNA that is polysome associated or if this is mediated the RNA-binding protein Glorund, which is another regulator of nos mRNA. Another similarity between Smaug and Ago-mediated mechanisms comes from in vitro translation extracts showing that both Smaug and Ago1 repress translation through steps that function downstream of translation initiation and require ATP. While these similarities are consistent with Smaug-mediated translational repression functioning, at least to some extent, through Ago1, it is noteworthy that several other mechanisms have been shown to have roles in translational repression by Ago protein (Pinder, 2013).
The data also indicate that while Ago1 functions in nos translational repression it does not induce nos mRNA degradation. Thus, in the context of nos mRNA, Ago1 does not induce transcript decay. In contrast, Smaug has a role in both translational repression and degradation of nos message, indicating that some of Smaug's functions are Ago1-independent (Pinder, 2013).
The interaction of Smaug with both Ago1 and Ago2 could reflect a common binding site on Ago proteins that mediates their interaction with Smaug. However, no ectopic Nos protein was seen in Ago2 mutant embryos. This could suggest that while the Smaug/Ago1 interaction is functional, the interaction with Ago2 is not, perhaps because of the different mechanisms that are used by Ago1 and Ago2 to repress translation (Pinder, 2013).
The data presented in this study, along with previous work, indicate that Smaug uses several mechanisms to regulate its target mRNAs. In addition to translational repression mediated by Smaug/Ago1, Smaug interacts with the Cup protein, which in turn interacts with the cap-binding protein eIF4E. eIF4E bound to the 5' cap of an mRNA indirectly recruits the 40S ribosomal subunit to an mRNA through eIF4E's interaction with eIF4G. As Cup blocks the eIF4E/eIF4G interaction, formation of the Smaug/Cup/eIF4E complex on a target mRNA represses translation initiation. Smaug also recruits the Ccr4/Not deadenylase to target mRNAs resulting in removal of the transcripts poly(A) tail, thus repressing translation and/or inducing transcript degradation. Interestingly, Smaug has also been shown to interact with the Piwi-type Ago proteins, Aubergine and Ago3. That study proposed that a complex consisting of Smaug, Aubergine with associated piwiRNAs and the Ccr4/Not deadenylase is recruited to nos mRNA through binding of both Smaug to the nos SREs and piwiRNAs to complementary to sequences in the nos 3' UTR (Pinder, 2013).
These models of Smaug function raise the question as to why one RNA-binding protein uses several mechanisms to repress its target transcripts? One possibility is that by using several mechanisms, Smaug ensures that its targets are efficiently silenced. This might be especially important for a transcript, such as nos, where even low levels of inappropriate expression are lethal to the embryo. Alternatively, Smaug might use different mechanisms to regulate different target mRNAs. Indeed, while Smaug induces the degradation of Hsp83 mRNA, it does not repress Hsp83 translation. In contrast, while Smaug represses nos translation, it has only a modest role in destabilizing nos mRNA. This differential regulation could reflect the fact that the location of the SREs in the target mRNA (e.g., 3' UTR for nos versus open reading frame for Hsp83) and/or more cis-elements within these target mRNAs along with the trans-acting factors that recognize these influence the mechanisms of Smaug function (Pinder, 2013).
While the data indicate that Smaug recruits Ago1 to nos mRNA via a miRNA-independent mechanism, it is still possible that a miRNA bound to Ago1, but not base-paired with nos mRNA, could be important for nos regulation. In this model, the miRNA would be required for Ago1 to repress nos translation at a step downstream of recruitment. For example, miRNA binding to Ago1 might be required to induce allosteric changes in Ago1 that facilitate its interaction with other factors that are required for Ago1-mediated repression. In this model, any miRNA would be sufficient for repression as it is not base-paired with the target transcript. Indeed, it has been suggested that miRNA binding might be involved in allosteric changes that are important for Ago-mediated repression, while other studies have shown that miRNA binding confers substantial structural stability to an Ago protein. It is noted that the Ago1 miRNA-binding mutant used in the present study also fails to interact with GW182, an Ago-binding protein that is essential for translational repression; therefore it could not be used to test for miRNA-independent translational repression of nos mRNA in vivo (Pinder, 2013).
A variety of RNA-binding proteins have been shown to function at numerous steps in miRNA/Ago-mediated post-transcriptional regulation. For example, both a mammalian and Caenorhabditis elegans Pum form a complex that includes an Ago protein, and in vitro experiments showed that the mammalian version of this complex represses translation of a reporter mRNA that carried only Pum-binding sites. The lack of miRNA-binding sites within this reporter suggests that in this in vitro system Pum is able to recruit Ago to an mRNA in a miRNA-independent manner. It is, however, unclear whether this Pum/Ago complex is able to recognize target mRNAs in vivo in the absence of miRNA binding. Indeed, it is suggested that in vivo targeting of the Pum/Ago complex might involve the presence of both Pum and miRNA-binding sites within a transcript. Thus, the data indicating that, in vivo, Smaug can recruit Ago1 to an mRNA in a miRNA-independent fashion, combined with other in vitro data suggesting that Pum can also directly recruit an Ago protein, suggests that other RNA-binding proteins might also function in a similar manner (Pinder, 2013).
Effect Nanos on posterior pole cells and pole plasm Posterior patterning in Drosophila embryos is governed by Nanos, which represses
the translation of maternal transcripts of the hunchback gene. Sites in HB mRNA that mediate this repression are termed Nanos response elements (NREs). One NRE-binding factor is Pumilio (pum). This suggests that PUM acts by recognizing the NRE and then recruiting NOS. Presumably, the resulting complex inhibits some component of the translation machinery (Murata, 1995).
Genetic as well as cytoplasmic transfer experiments have been used
to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. nanos can
restore normal abdominal development in posterior group mutants. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. The products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants (Lehmann, 1991).
Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. Maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 mRNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos
and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity (Ding, 1993).
licorne codes for a MAP kinase kinase exciting the p38 pathway in Drosophila. licorne mutant embryos are defined, for the purpose of this study, as hemipterous;licorne double mutants engineered to express a hemipterous transgene (see Licorne Effects of Mutation for more information about this genotype). lic mutant embryos show a segmentation phenotype that is
reminiscent of the one produced by mutations in the maternal
posterior-group genes, including oskar, vasa, and nanos. Most of the posterior-group genes can provoke both abdominal
segmentation defects and a loss of germ cells, a dual defect that is
due to the common localization of the posterior and germ cell
determinants in the posterior germ plasm.
Like several posterior-group genes, lic embryos lack or have a
strongly reduced number of pole cells, as shown using Vasa and Nanos as
markers. In most lic mutant embryos, Vasa
protein fails to be accumulated at the posterior pole, although in some cases weak staining is observed.
It is concluded that lic has a role in abdominal segmentation, proper Vasa protein
and Nanos mRNA localization at the posterior pole, and
formation of the pole cells. These results suggest that lic
also has a role in germ plasm assembly (Suzanne, 1999).
The assembly of the germ plasm takes place during oogenesis and
proceeds in several steps leading to the successive posterior localization of many different components (for review, see Rongo,1996). A pivotal step in this process is the
localization of the OSK mRNA to the posterior pole of the
oocyte in stage 8-9 egg chambers, which is the basis for the
recruitment and assembly of downstream components like Vasa and Nanos.
In lic germ-line clones, both OSK mRNA expression and early posterior localization appear normal until stage 8 of oogenesis. However, in stage 9 and older egg chambers, the
OSK mRNA is mislocalized, diffusing in the whole oocyte in a
gradient from the posterior to the anterior pole. In later stages, OSK transcripts are
barely detectable, indicating that diffusion proceeds continuously in
mutant egg chambers. A similar phenotype is observed in some
osk missense mutants, suggesting a role for Osk
protein in the anchoring of its own mRNA at the posterior pole.
Staining of lic mutant egg chambers using an anti-Osk antibody
did not allowed detection of any reduction in Osk protein accumulation,
indicating that lic affects OSK mRNA
localization independent of Osk translation (Suzanne, 1999).
In some lic egg chambers, the mislocalized OSK
mRNAs also seem to partly accumulate in a more central position, reminiscent of the position of OSK
transcripts in mutants that have not reorganized the microtubules, as in
EGFR pathway mutants. This result suggests that lic
oocytes are not completely repolarized. However, no defect in the positioning of the nucleus, or in the
localization of a kinesin-lacZ microtubule-associated motor
protein fusion is observed, suggesting that
OSK mRNA mislocalization is a more sensitive assay and lic
defects are weak. The correct localization of osk RNA at stage 8 and its later
diffusion indicate that lic affects the maintenance of
OSK mRNA asymmetric localization in the oocyte (anchoring) rather than the mechanism of localization per se, most likely as a result of incomplete polarization along the AP axis (Suzanne, 1999).
Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. This study addresses the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. This dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior (Chau, 2012).
Sxl has a pivotal role in the cell fate switch from a self-renewing GSC to a differentiation-competent CB. This study addressed the underlying cellular mechanism by identifying nanos as a Sxl target gene. nanos is a conserved translational repressor that is thought to maintain GSC fate by silencing a set of as-yet-unidentified differentiation-promoting mRNAs. Cell fate switching occurs as one of the daughter cells initiates a differentiation program that includes significant accumulation of the Bam protein and rapid down-regulation of a set of GSC specific markers, including Nanos protein. Previous studies have shown that Nanos protein expression is dynamically regulated at the level of translation such that Nanos protein levels are high in GSCs but undetectable in all Bam-expressing daughter cells. This study found that Sxl is responsible for this dynamic expression pattern. Moreover. regulation is direct. nanos RNA is bound by the Sxl protein in ovarian extracts, and nanos silencing is dependent on the presence of Sxl binding sites located in the nanos 3' UTR. Together these studies point to a mechanism in which Sxl promotes the GSC/CB cell switch by lowering Nanos protein levels. The use of a female-specific factor to control nanos expression is consistent with earlier studies showing that the mechanism regulating GSC differentiation differs significantly between the sexes; for example, neither bam nor nanos is required for this process in males, although they are expressed. Thus, the Sxl-mediated posttranscriptional regulatory mechanism described in this study provides a direct link between sexual identity and execution of the appropriate differentiation pathway (Chau, 2012).
Sxl is expressed in both GSCs and their progeny, raising the question of how its role in Nanos down-regulation is restricted to Bam-expressing cells. bam itself may confer cell-type specificity, as it too is required for lowering Nanos protein levels, although there are, as yet, no physical data to support direct regulation. Nevertheless, taken together with the genetic studies that reveal that bam requires Sxl activity to promote differentiation, these observations suggest that cell-type specificity could be achieved by a regulatory complex containing Sxl and Bam. Both Sxl and Bam have been shown to repress translation in other contexts, further suggesting that the two proteins might function together to directly repress nanos translation. Biochemical studies will be required to test this model (Chau, 2012).
It was previously shown that Sxl is required for germ cells to progress from a stem cell to a differentiation-competent CB fate, and that if this pathway is blocked, the mutant germ cells form a tumor. Although this study shows that inappropriate nanos expression is necessary for tumor growth, genetic epistasis experiments indicate that nanos expression is not responsible for malignant transformation because the majority of surviving double-mutant germ cells continue to resemble a tumor cell. This conclusion is supported by studies showing that forced expression of nanos in GSC progeny is not sufficient to block differentiation. Even in a genetically sensitized background where an accumulation of extra stem cell–like germ cells has been observed, germ cells proceed through oogenesis. Therefore, these studies suggest that although forced expression of nanos is responsible for expanding the number of mutant germ cells, aberrant regulation of other genes and/or pathways under Sxl control elicit malignant transformation. The identification and analysis of additional Sxl targets genes will offer insights into how the failure to successfully exit the stem cell state is connected to the genesis of germ cell tumors (Chau, 2012).
In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. This study presents data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam (Li, 2013).
This study presents data that Mei-P26 cooperates with Bam, Bgcn and Sxl to control the translation of nanos mRNA in the Drosophila female germline. Co-immunoprecipitation experiments indicate Mei-P26 physically associates with the differentiation factors Bam, Bgcn and Sxl and yeast 2-hybrid assays suggest the interaction between Mei-P26 and Bgcn may be direct. Disruption
of mei-P26, or snf, which disrupts sxl expression in the germline, results in the upregulation of Nanos protein expression in early differentiating cysts. Both Mei-P26 and Sxl protein associate with nanos mRNA (Chau, 2012). In light of the recently published study that shows mutating Sxl binding sites within the 3′UTR of nanos mRNA leads to mis-regulation of the gene (Chau, 2012), these results suggest that Mei-P26 may be part of a Sxl, Bgcn and Bam complex that serves to promote cyst development by directly repressing the expression of Nanos. However, despite repeated attempts, direct interactions between Bam and Bgcn with nanos mRNA could not be detected. While various technical issues may prevent the detection of these specific interactions, the inability to observe direct association between Bam/Bgcn and nanos mRNA leaves open the possibility that interactions between the components of the Mei-P26, Sxl, Bam and Bgcn complex and its target mRNAs may be dynamic in nature. For instance, Bam and Bgcn may help to prepare Sxl and Mei-P26 for mRNA binding but do not themselves directly interact or only transiently interact with these targets. Further experiments will be needed to clarify the more specific molecular mechanisms that underlie Bam/Bgcn function with respect to the translational repression of nanos mRNA (Li, 2013).
Two other recent studies investigated the role of mei-P26 during germline development. Liu (2009) showed that the RNA helicase Vasa directly regulates the translation of mei-P26 mRNA through poly (U) elements within its 3' UTR. Mutations in each gene strongly enhance the phenotype of the other, resulting in the formation of cystic germline tumors. Neumuller (2008) focused on the function of Mei-P26, showing that it negatively regulates the activity of the miRNA pathway. It is now proposed that Mei-P26 functions in both GSCs and early differentiating germ cells. Within GSCs, Mei-P26 is in a complex with miRISC proteins and enhances miRNA-mediated silencing. In addition, Mei-P26 associates with Nanos protein and promotes BMP signaling within GSCs by repressing the expression of the negative regulator Brat. GSC daughters displaced away from the cap cell niche experience less BMP signaling, allowing for the expression of Bam (Li, 2013).
It is speculated that upon Bam expression, Mei-P26 switches its activity and/or its mRNA targets. This switch allows Mei-P26 to promote germline differentiation by both negatively regulating the miRNA pathway and cooperating with Bam, Bgcn and Sxl to repress the translation of specific mRNAs such as nanos. However the complex functional relationships between Mei-P26, Sxl, Bam and Bgcn remain incompletely understood. While evidence is provided that these factors can physically associate with each other under certain conditions, disruption of these genes results in two discrete phenotypes. mei-P26 and snf mutants exhibit a cystic tumorous phenotype marked by the accumulation of undifferentiated cysts that do not express A2BP1, a molecular marker present in 4-, 8- and 16-cell cysts in wild-type samples. In contrast, disruption of bam or bgcn results in the formation of single cell germ cell tumors. These phenotypic differences suggest that Bam and Bgcn carry out additional functions independent of Mei-P26 and Sxl. A more complete characterization of the regulatory networks that govern the very early steps of germline cyst differentiation will have to await a better biochemical characterization of Bam and Bgcn function (Li, 2013).
Together these data suggest that Mei-P26 has a variety of molecular functions inside and outside of the germline. It remains unclear whether Mei-P26 exhibits the same biochemical activity when complexed with different proteins or whether its function completely changes depending on context. Based on the presence of a RING domain, Mei-P26 may act as an ubiquitin ligase. However this specific enzymatic activity has not been demonstrated nor have any direct in vivo substrates been identified. In regards to the translational repression of specific mRNAs, a model is favored in which Mei-P26 exhibits the same molecular activity within GSCs and their early differentiating daughters. It is further speculated that association of Mei-P26 with different mRNA binding proteins modulates its targeting of specific mRNAs, and/or the degree to which these different targets are repressed. The expression of Bam correlates with changes in the development role of Mei-P26 but the manner in which Bam alters the composition or activity of the Mei-P26 complex remains unknown. Regardless, the findings that Bam can associate with Mei-P26 and Sxl provide further support for the hypothesis that Bam regulates the translation of specific mRNAs to promote the early steps of differentiation within the Drosophila female germline (Li, 2013).
Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).
Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).
Quantitative analysis of germ plasm-localized mRNAs has revealed several intriguing features about the localization process and the coordinate regulation of their integration into the pole cells. nos, pgc and cycB mRNAs are transported within the oocyte as single mRNAs and are co-packaged into granules specifically at the posterior cortex concomitant with localization. Thus, localization serves not only to concentrate these transcripts at the posterior but also generates large, multi-copy polar granules to coordinate the efficient incorporation of these transcripts into the pole cells (Little, 2015).
Polar granules are heterogeneous with respect to both the amount of a particular mRNA and the combination of different mRNAs. Although nos mRNA content of polar granules varies over a large range, there is a tendency towards higher values fitting a log-normal distribution. Log-normal distributions are often associated with exponential growth processes, whereby the rate at which an object grows is proportional to the size of the object. Thus, a log-normal distribution suggests that large granules grow at faster rates compared with small ones as assembly is accelerated through positive feedback. In addition, this study found that for granules containing both nos and pgc or nos and cycB, the quantities of the two different mRNAs are correlated, and there is a greater fraction of granules completely lacking one species of mRNA entirely than granules containing just a few copies of that mRNA. Together, these data suggest that for each type of mRNA, cooperative interactions generate homotypic RNPs that then 1) accelerate the recruitment of additional mRNAs of the same type, and 2) facilitate granule assembly by promoting interactions with similarly sized homotypic clusters of other mRNAs. These results also predict the existence of dedicated molecular pathways, one to form homotypic clusters, and another to assemble homotypic clusters of many different transcripts into higher-order granules. These higher-order granules may form by fusion of smaller homotypic granules and indeed fusion of granules labelled with GFP-Vas is observed by live imaging. Alternatively, clusters of different mRNAs may grow alongside each other on a predefined granule scaffold. The localization of Caenorhabditis elegans germ granules -- P granules -- occurs through a phase transition in which soluble RNP components condense at the posterior of the embryo. It is interesting to consider whether formation of homotypic clusters occurs by a condensation of single transcript RNPs mediated by RNA-binding proteins (Little, 2015).
In contrast to other posteriorly localized RNAs, which travel as single molecules, osk forms oligomeric complexes beginning in the nurse cells. Previous studies indicated that reporters containing the osk 3'UTR can hitchhike on wild-type osk mRNA by 3'UTR-mediated dimerization. Hitchhiking is not required for osk transport, but co-packaging may be important for translational repression of osk before localization. Consistent with this, multi-copy RNPs are competent for localization by both kinesin-dependent transport during mid-oogenesis and diffusion/entrapment during late stages of oogenesis (Little, 2015).
Previous ISH-immuno-electron microscopy analysis of stage 10 oocytes showed co-localization of osk with Stau, but not with Osk protein in polar granules and the results indicate that osk/Stau RNPs are continuously segregated from the germ plasm granules. This physical separation has functional consequences. Whereas co-packaging of nos, pgc and cycB coordinates their transport to posterior nuclei and consequent segregation to the pole cells, osk is specifically excluded. Although it is not known why targeting of Osk to polar granules seems to alter their function, it is clearly detrimental to germline development. It will be interesting to determine whether osk/ Stau granules contain other mRNAs whose functions are required specifically during oogenesis or early embryogenesis but must be excluded from pole cells (Little, 2015).
Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. This study, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, shows that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into 'homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules (Trcek, 2015).
This study combined single-molecule FISH with structured illumination microscopy (SIM), a super-resolution technique, to gain a high-resolution view of the mRNA-bound germ granule. This combinatorial approach allowed the determination that germ granule-localized mRNAs occupy distinct positions within the granule and relative to each other, while germ granule proteins are homogeneously distributed within the granular space. Multiple localized mRNAs group to form homotypic cluster, which gives the germ granule its structure. This structure does not change through early embryonic development and does not correlate with the translational onset of localized mRNAs or with the ability of germ granules to protect bound mRNAs from decay (Trcek, 2015).
This analysis of the organizational structure of germ plasm focused on core germ granule protein components, Vas, Osk, Tud and Aub, and on cycB, nos, pgc, gcl and osk mRNA. cycB, nos, pgc, gcl and osk serve as prototypes for mRNA localization to the germ granules because their localization to the germ plasm, their regulation in the germ plasm and biological significance for germ cell biology are understood best. While mRNA localization studies suggest that up to 200 mRNAs may be localized to the posterior pole of the early embryo, it is assumed that regulatory mechanisms revealed by the study of cycB, nos, pgc and gcl are shared among other germ plasm-localized mRNAs. The study of germ plasm-localized mRNA regulation revealed that only localized mRNAs translate, while their unlocalized counterparts are translationally silent, that localized mRNAs are protected from mRNA decay, and that the 3′ UTRs of localized mRNAs are necessary and often sufficient to localize mRNAs to the posterior and render them translationally competent. The experiments demonstrate that cycB, nos, pgc and gcl mRNAs concentrate in homotypic clusters, assume specific positions within the germ granules, and can organize into separate granules. The results make it unlikely that cycB, nos, pgc and gcl clusters contain more than one type of mRNA. If clustering between heterotypic mRNAs was a common organizational strategy, the pairwise analysis with cycB, nos, pgc and gcl would have not yielded the distinct volumes observed. Thus, despite the fact that only a limited number of localized RNAs were sampled, it is anticipated that germ granule organization observed for cycB, nos, pgc and gcl is also shared by other germ granule-localized mRNAs, which are similarly regulated (Trcek, 2015).
Given that the core germ plasm proteins Osk, Vasa, Aub and Tud recruit other germ granule components and are themselves homogeneously distributed within the granule, it is unlikely that the germ granule structure is dictated by proteins alone. Homotypic clustering could also be driven by intramolecular RNA–RNA interactions, similar to those found in the localized bicoid mRNA at the anterior pole and in the co-packaged osk mRNA during transport to the oocyte posterior. The dramatic increase in mRNA concentration in the granule compared with rest of the embryo may raise the likelihood for two mRNAs to interact or even induce RNA–RNA interactions by altering mRNA conformation thus driving homotypic clustering (Trcek, 2015).
In yeast, the movement of mRNAs in and out of stress granules and processing bodies determines their translatability and stability and in Drosophila oocytes the position of bicoid and gurken mRNA within the sponge body correlates with their translational activity. This study found, however, that the mRNA position within the germ granule is independent of translational or degradation activity of localized mRNAs. Some translational and decay regulators found in germ granules are also found in sponge bodies, stress granules and processing bodies. Thus the data imply that in germ granules these proteins may regulate transcripts differently to allow for the dynamic regulation of different mRNAs. Alternatively, sorting of mRNAs into distinct granules could specify their activity. For example, pgc co-localizes with core germ-granule components as well as with osk mRNA. Thus, the pool of pgc associated with osk could be functionally different from the one that associates with Vasa, Osk, cycB, nos and gcl. Indeed, in older embryos just before pgc becomes translated, pgc moves away from osk, but not from VasaGFP, cycB, nos and gcl. Speculatively, this could be the mechanism that determines the onset of pgc translation (Trcek, 2015).
mRNA clustering could also enhance biochemical reactions locally either by enabling protein complex formation, by quick re-binding of a regulator to a neighbouring mRNA or by increasing the concentration of a regulator of the cluster RNAs. For example, it has been proposed that the repression of cycB translation by Nanos protein (Nos) depends on a high local concentration of Nos in the germ plasm. Multiple nos mRNAs within the cluster could increase the local concentration of Nos thus counteracting the loss of the unbound Nos due to diffusion into the embryo. Once bound to cycB, Nos could also be quickly re-bound by the neighbouring cycB mRNAs thus maintaining high Nos concentration and ensuring efficient cycB repression. In this way each mRNA cluster in the granule would resemble a biochemical territory, consistent with the recent observations showing that germ granules in Caenorhabditis elegans, which behave like liquid droplets, are also not homogeneous. It is proposed that an mRNA-protein granule organization similar to the one described in this study for Drosophila germ granules could be a conserved feature of larger ribonucleoprotein granules (Trcek, 2015).
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