BIOLOGICAL OVERVIEW

The regulation of protein activity in the Drosophila oocyte is essential for the establishment of anterior/posterior axis asymmetry in the Drosophila embryo. The initial steps of pattern formation along the anterior/posterior axis involve repression of maternally synthesized mRNAs by regulators produced at each pole of the Drosophila embryo. Posterior patterning is governed primarily by a cascade of translational control. A central aspect of this patterning is the selective repression or activation of Nanos (NOS) mRNA in different compartments of the syncitial embryo. Generation of Nos in the posterior of the embryo is essential to allow abdominal segmentation, and preventing Nos accumulation in the anterior is essential for normal head and thoracic segmentation. Smaug, the subject of this overview, 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 (Gavis, 1994). In particular, the 184 nt translational control element (TCE) contains all of the 3' UTR signals that are necessary and sufficient for NOS function (Dahanukar, 1996).

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 (Dahanukar, 1996 and Smibert, 1996) as well as Oskar-dependent activation in the pole plasm (Dahanukar, 1996). 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).

Analysis of point mutations in the TCEs reveals a strong correlation between Smaug binding and translational repression; mutants unable to effect Smaug binding in vitro are not repressed translationally in vivo, whereas mutants where Smaug does bind remain repressed translationally. These results strongly suggest that Smaug acts in translational repression of unlocalized NOS mRNA. Translational repression is essential, because embryos expressing a NOS mRNA with mutated SREs develop with anterior body patterning defects and die, despite the correct localization of RNA (Smibert, 1996),

At fertilization, NOS mRNA is present throughout both the bulk cytoplasm of the embryo and the pole plasm, the thin crescent at the posterior pole that contains the germline determinants. Only the NOS mRNA at the posterior pole is translated, giving rise to a gradient of Nos protein emanating from the posterior pole; the mRNA in the bulk cytoplasm is translationally repressed (Dahanukar, 1996 and Smibert, 1996), allowing for proper abdominal, head and thorax segmentation.

TCE-mediated activation in the pole plasm is dependent genetically on Vasa and Oskar (Dahanukar, 1996), two of the 'posterior group' gene products known to be required for assembly of the pole plasm. Of the posterior group proteins, Oskar (Osk) has been shown to play a critical role: ectopic expression of Osk induces formation of germline progenitor cells and activates translation of NOS mRNA (and thereby abdominal development). Thus, Osk appears to be a limiting embryonic component for both events. Activation of NOS mRNA requires, in addition to Osk, the activities of two other pole plasm constituents -- Tudor (Tud) and Vasa. The biochemical function of Tud is not yet known. Vasa is an RNA helicase that interacts weakly with Osk (Breitwieser, 1996), and this interaction could serve to recruit Vasa to NOS mRNA. Alternatively, since Osk, Vas, and Tud are all components of the polar granules, a robust interaction between Smaug and Osk could simply increase the probability of a productive interaction between NOS mRNA and other polar granule components. Further experiments will be required to dissect the biological significance of the interactions between Osk, Smaug, and Vasa (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 (Dahanukar, 1996 and Smibert, 1996). 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).

If Smaug binding mediates activity of the TCE in vivo, then loss of smg function and inactivation of the TCE should have similar consequences on the regulation of NOS mRNA. In the absence of the TCE, otherwise normal NOS mRNA is derepressed in the bulk cytoplasm, and ectopic Nos activity accumulates in the anterior of the embryo (Dahanukar, 1996 and Smibert, 1996 ). During the initial nuclear division cycles of embryogenesis, this ectopic Nos protein is not detected by available reagents. The ectopic Nos activity is readily detectable by monitoring the translational repression of Hunchback (HB) mRNA in the anterior of the embryo (Dahanukar, 1996). The repression of HB mRNA constitutes a sensitive assay for Nos activity. In wild-type embryos, for example, Nos blocks the accumulation of Hb near the middle of the embryo, even though Nos protein itself is not detected (Dahanukar, 1999).

The distributions of Nos and Hb proteins were examined in early smg embryos. The posterior gradient of Nos protein arising from translational activation in the posterior pole appears to be normal during early nuclear division cycles in smg mutant embryos. Ectopic Nos activity is readily detectable in smg mutant embryos during nuclear cycles 10-11. Hb is readily apparent in the anterior of wild-type embryos at this stage of development, whereas essentially no Hb is detected in smg embryos during cycles 10-11. Subsequent development of smg embryos is grossly abnormal, compromising the analysis of later events. It is concluded that Smaug function is required for TCE-mediated repression of NOS mRNA in the bulk cytoplasm, at least prior to nuclear cycle 12 (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 (nos^DeltaBX) contains essentially nothing other than the TCE and a polyadenylation signal (Dahanukar, 1996). Regulation of nos^DeltaBX and wild-type NOS mRNAs is indistinguishable. In particular, translation of nos^DeltaBX is dependent on the activities of pole plasm components such as Vasa and Osk, as is the case for nos⁺ mRNA. However, nos^DeltaBX 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 nos^DeltaBX 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 nos^DeltaBX 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 (Dahanukar, 1999), a domain of the protein that may also mediate interactions with the pole plasm constituents Vas and Staufen (Breitwieser, 1996).

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).

In the scheme described above, NOS mRNA regulators appear to be borrowed from other, fundamental processes that have no direct role in body patterning. Smaug is essential for normal progression through the cortical nuclear division cycles, and Osk is required for germline specification. This supports the view that the role of Nos in governing abdominal segmentation is a relatively recent evolutionary event (Dahanukar, 1999 and references).

Analysis of mutant embryos demonstrates that Smaug is necessary for the repression of NOS mRNA in the bulk cytoplasm of the early embryo. Smibert and Macdonald have independently identified Smaug by purification of a TCE-binding activity from embryonic extracts (C. A. Smibert and P. M. Macdonald, personal communication to Dahanukar, 1999). Thus, both their biochemical approach and the three-hybrid screen described have identified a single TCE-binding protein. Whether other such factors exist is currently unclear; however, all the regulatory events known to be mediated by the TCE can be accounted for by the activities of Smaug and its interaction with Osk (Dahanukar, 1999).

For some time, it has been apparent that development of the germline precursors and abdominal segmentation are mechanistically linked. Both events depend on assembly of the pole plasm, which is blocked by mutations in any of the posterior group of maternal effect genes, such as osk. A generally held view has been that one or more of the factors sequestered in the pole plasm activates translation of NOS mRNA, thereby generating the Nos protein gradient that governs abdominal segmentation. Based on the experiments described in the Dahanukar (1999) study, it is suggested that Osk itself may constitute the activator of NOS mRNA and that it functions by interacting directly with Smaug. The idea that Osk acts, at least in part, by binding to Smaug appears to explain most easily the antagonistic activities of these two proteins. Overexpression of Osk and overexpression of Smaug have opposing effects on translation of NOS mRNA and development of the embryonic body plan. Furthermore, the direct interaction observe between Osk and Smaug provides a simple molecular mechanism of Osk function (Dahanukar, 1999).

Although it has marked effects in two different sensitized backgrounds, overexpression of Smaug has no detectable effect on the translation of NOS mRNA in the pole plasm of wild-type embryos. In particular, excess Smaug inhibits the synthesis of Nos at the anterior but not at the posterior of BicD embryos. The modest degree of overproduction achieved in these experiments may simply be insufficient to titrate the Osk present in the normal pole plasm. Consistent with this idea, increasing the number of Smaug-binding sites in a chimeric reporter mRNA blocks its translation in the pole plasm (Bergsten, 1999). Presumably, the likelihood of forming a single TCE-Smaug complex is increased more by multimerization of the binding site in cis than by a modest increase in the concentration of the protein in trans.

Between the TCE and the poly(A) tail are other signals that mediate Osk-dependent activation of nos mRNA translation (Bergsten, 1999). While these signals suffice to generate a posterior gradient of Nos protein in the absence of either the TCE (Dahanukar, 1996) or Smaug, they are dispensable for nos function, and so their biological significance is unclear. Both the TCE and these redundant signals also mediate localization or concentration of NOS mRNA in the pole plasm (Dahanukar, 1996 and Bergsten, 1999). If Osk can form a ternary complex with Smaug and the TCE, it would appear to be sufficient to account for TCE-dependent localization of nos mRNA (Dahanukar, 1999).

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. This study shows that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates; and transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT (Benoit, 2009).

High-level zygotic expression of miR-309 cluster microRNAs (miRs) directs destruction of a subset of maternal mRNAs at the MZT (Bushati, 2008). Hybridization of total RNA purified from staged eggs or embryos to a microarray carrying probes for 68 known Drosophila miRs confirmed that high-level miR-309-cluster transcription occurs only in embryos and not in activated unfertilized eggs. Northern blot analysis demonstrated that expression of three of the miRs - miR-3, miR-6 and miR-286 - is disrupted in smg mutants. Four-hundred and ten maternal mRNAs appear to require zygotic expression of the miR-309 cluster for destabilization at the MZT (Bushati, 2008). Consistent with these observations, ~85% of these maternal transcripts are stabilized in smg mutants. Smaug-dependent expression of the miR-309-cluster thus leads to further destabilization of a subset of maternal mRNAs (Benoit, 2009).

This study shows that Smaug is required to slow the cleavage divisions, activate the DNA replication checkpoint and initiate high level expression of the vast majority of genes that are transcribed during the final stages of the MZT. Furthermore, Smaug is essential for blastoderm cellularization, which marks the MBT. Significantly, Smaug protein begins to accumulate during the early cleavage divisions, when maternal transcript destruction is initiated, and Smaug levels peak during the syncytial blastoderm stage, when the replication checkpoint is activated and high-level zygotic transcription begins. In addition, expression of Smaug in an anterior-to-posterior gradient triggers an anterior-to-posterior gradient in the timing of transcript destruction, checkpoint activation, cellularization and gastrulation. Smaug is therefore the first maternal or zygotic factor shown to control the timing of the MZT (Benoit, 2009).

Over-expression of Smaug does not accelerate maternal transcript destruction or cellularization, indicating that an additional factor or process becomes limiting under these conditions. Smaug triggers mRNA destruction by recruiting the CCR4/POP2/NOT complex, which catalyzes poly-A tail removal, and an additional factor ('Y') has been proposed to act with Smaug to trigger decay. Factor Y or a component of the CCR4/POP2/NOT complex could become limiting for transcript destruction and the MBT when Smaug is overexpressed. Alternatively, Smaug expression at wild-type levels could saturate the binding sites on target transcripts and the deadenylation machinery could be present in excess. Under these conditions, wild-type levels of Smaug would lead to transcript destruction at maximal rates, which in turn would determine the minimum time for the MZT. Smaug also represses translation of specific targets (e.g. nanos), and this function could also have a role in coordinating the MZT (Benoit, 2009).

Based on the present studies and earlier work, the simple hypothesis is favored that Smaug-dependent maternal transcript destruction triggers the MZT by coordinately downregulating a suite of maternal proteins that suppress zygotic transcription and the replication checkpoint. Consistent with this speculation, Smaug is required for maternal cyclin B transcript destruction, and overexpression of Cyclin B suppresses checkpoint activation and leads to additional rapid syncytial divisions (Crest, 2006). Similarly, Smaug triggers destruction of maternal mRNA encoding Tramtrack, which represses transcription of a subset of genes until near the end of the MZT (Benoit, 2009).

An initial wave of Smaug-dependent zygotic gene expression appears to trigger a series of positive and negative feedback loops that drive completion of the MZT. For example, Smaug is required for zygotic transcription of fruhstart (frs), which promotes destruction of maternal string mRNA. String activates Cyclin-B-Cdk1, and string mRNA destruction in response to Frs expression could cooperate with Smaug-dependent cyclinB mRNA destruction to terminate the rapid cleavage stage divisions. Smaug is also required for zygotic expression of transcriptional activators, including cyclin T, cdk7 and cdk9, and CyclinT-Cdk9 complex catalyzes phosphorylation of serine 2 on the RNAPII CTD repeat, which is linked to Smaug-dependent transcription at the MZT. Transcription of cyclin T and cdk9 concomitant with destruction of maternal RNAs encoding transcriptional repressors, could therefore accelerate activation of the zygotic gene expression. Finally, Smaug is required for zygotic expression of the miR-309 cluster, which promotes a second, more rapid phase of maternal transcript destruction that terminates maternal genetic control of embryogenesis. A recent study has shown that a maternally deposited transcription factor, Zelda, is also required for expression of the miR-309 cluster and number of other early zygotic genes. There is no evidence that Zelda regulates the timing of the MZT, but Zelda and Smaug could function cooperatively to promote transcription and maternal transcript destruction during this transition (Benoit, 2009).

GENE STRUCTURE

Bases in 5' UTR - 451

Bases in 3' UTR - 959

PROTEIN STRUCTURE

Amino Acids - 969

Structural Domains

The full-length Smaug protein is encoded by a 4.4 kb maternal mRNA. Its sequence bears no significant homology to other proteins of known function, nor does it have any of the previously characterized RNA-binding motifs (Dahanukar, 1999).

Anteroposterior patterning in Drosophila is dependent on the sequence-specific RNA-binding protein Smaug, which binds to and regulates the translation of nanos mRNA. The sterile-alpha motif (SAM) domain of Smaug functions as an RNA-recognition domain. This represents a new function for the SAM domain family, which is well characterized for mediating protein-protein interactions. Using homology modeling and site-directed mutagenesis, the RNA-binding surface of the Smaug SAM domain has been localized and the RNA consensus sequence required for binding has been elaborated. Residues that compose the RNA-binding surface are conserved in a subgroup of SAM domain-containing proteins, suggesting that the function of the domain is conserved from yeast to humans. The SAM domain of Saccharomyces cerevisiae Vts1 binds RNA with the same specificity as Smaug and Vts1 induces transcript degradation through a mechanism involving the cytoplasmic deadenylase CCR4. Together, these results suggest that Smaug and Vts1 define a larger class of post-transcriptional regulators that act in part through a common transcript-recognition mechanism (Aviv, 2003).

The Nanos protein gradient in Drosophila, required for proper abdominal segmentation, is generated in part via translational repression of its mRNA by Smaug. The crystal structure of the Smaug RNA binding domain, which shows no sequence homology to any previously characterized RNA binding motif, is reported. The structure reveals an unusual makeup in which a SAM domain, a common protein-protein interaction module, is affixed to a pseudo-HEAT repeat analogous topology (PHAT) domain. Unexpectedly, through a combination of structural and genetic analysis it has been found that it is primarily the SAM domain that interacts specifically with the appropriate nanos mRNA regulatory sequence. Therefore, in addition to their previously characterized roles in protein-protein interactions, some SAM domains play crucial roles in RNA binding (Green, 2003).

date revised: 5 December 2023

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