smaug
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).
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).
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).
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).
Asymmetric localization of mRNAs within cells promotes precise spatio-temporal control of protein synthesis. Although cytoskeletal transport-based localization during Drosophila oogenesis is well characterized, little is known about the mechanisms that operate to localize maternal RNAs in the early embryo. One such mechanism—termed “degradation/protection”—acts on maternal Hsp83 transcripts, removing them from the bulk cytoplasm while protecting them in the posterior pole plasm. The RNA binding protein, Smaug, previously known as a translational repressor of nanos, has been identified as a key regulator of degradation/protection-based transcript localization. In smaug mutants, degradation of Hsp83 transcripts is not triggered, and, thus, localization does not occur. Hsp83 transcripts are in an mRNP complex containing Smaug, but Smaug does not translationally repress Hsp83 mRNA. Rather, Smaug physically interacts with the CCR4/POP2/NOT deadenylase, recruiting it to Hsp83 mRNA to trigger transcript deadenylation and degradation. When Smaug is targeted to heterologous stable reporter transcripts in vivo, these are deadenylated and destabilized. A deletion that removes the gene encoding CCR4 exhibits dose-sensitive interactions with Smaug in both a loss-of-function and a gain-of-function context. Reduction of CCR4 protein levels compromises Hsp83 transcript destabilization. It is concluded that Smaug triggers destabilization and localization of specific maternal transcripts through recruitment of the CCR4/POP2/NOT deadenylase. In contrast, Smaug-mediated translational repression is accomplished via an indirect interaction between Smaug and eIF4E, a component of the basic translation machinery. Thus, Smaug is a multifunctional posttranscriptional regulator that employs distinct mechanisms to repress translation and to induce degradation of target transcripts (Semotok, 2005; full text of article).
The Drosophila Pan Gu (Png) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of Png activity, but Png is required for translation from egg activation. Although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for Png-mediated control of cyclin B translation and of the early embryonic cell cycles. Evidence is presented that Png functions instead as an antagonist of Pumilio-dependent translational repression. The data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that Png-mediated derepression of translation is an important alternative mechanism in Drosophila (Vardy, 2007).
These data indicate that in Drosophila translation of cyclin B can proceed in the absence of polyadenylation in ovaries and syncytial embryos. Polyadenylation is not required for the unmasking of the mRNA, but it may play a role in fine-tuning translation. A model is proposed in which Png has dual roles—poly(A) dependent and independent—in promoting cyclin B translation. At egg activation, Png is needed for full-length poly(A) tails, and this may augment translation. The data indicate, however, that Png also promotes translation independently of poly(A) tail length, most likely by overcoming repressive action by Pum. Removal of Pum in a png mutant therefore allows translation of the target mRNA even if the poly(A) tails are not fully elongated (Vardy, 2007).
SMAUG (SMG) protein levels and the poly(A) tail are decreased in the png mutant, and restoration of poly(A) tail length by overexpressing poly(A) polymerase is not enough to promote smaug translation in a png mutant. Thus, Png also appears to regulate smaug translation in a poly(A)-dependent and -independent manner. This indicates that multiple levels of translational regulation may be a common strategy in development (Vardy, 2007).
If the Png kinase complex functions ultimately to regulate the activity of Pum, one of the key questions concerns the nature of this regulation. Since NANOS is found predominantly at the posterior pole, Pum may be able to recruit different factors to the target mRNA. The defect seen in png is likely due to a failure to relieve translational repression as opposed to a failure in activation, because injection of excess amounts (1 μg/μl) of the 5′FF3′−cycB transcript into png mutant embryos allows translation to proceed at much stronger levels. This suggests that the repressor is limiting in this reaction, thus allowing translation to proceed, and is consistent with Pum being downstream of Png. It will be interesting to determine the nature of the Png kinase substrates and how they relate to Pum function (Vardy, 2007).
Png likely has multiple targets, because removal of Pum in a png mutant does not completely restore the embryonic divisions. While the early syncytial cycles can progress in a png;pum double mutant, mitosis still fails in the later S-M cycles. Smg protein levels are decreased in the png mutant due to a failure in translation. Smg protein levels are not restored in a png;pum double mutant, and given Smg's role in progression through cycle 11/12, it provides an explanation for why the embryos fail at this stage. An independent role for the Png kinase complex later in embryogenesis has been described in the maternal mRNA degradation pathway. Although these pathways appear to be independent, in each case translational regulation of a key component appears to be the mechanism: Cyclin B to control the S-M cycles and SMG to control maternal transcript destruction (Vardy, 2007).
This study has established a role for the Png kinase complex in the translational regulation of cyclin B in the early syncytial cycles of Drosophila. A role for the Png kinase complex in protein stability has been suggested, and it is possible that it could act to regulate Cyclin B levels by multiple mechanisms. These data, however, reveal it to be a key regulator of cyclin B translation, ensuring coordinated passage through these early cycles (Vardy, 2007).
Translational regulation plays an essential role in development and often involves factors that interact with sequences in the 3' untranslated region (UTR) of specific mRNAs. For example, Nanos protein at the posterior of the Drosophila embryo directs posterior development, and this localization requires selective translation of posteriorly localized nanos mRNA. Spatial regulation of nanos translation requires Smaug protein bound to the nanos 3' UTR; binding represses the translation of unlocalized nanos transcripts. While the function of 3' UTR-bound translational regulators is, in general, poorly understood, these regulators presumably interact with the basic translation machinery. Smaug is shown to interact with the Cup protein and Cup is an eIF4E-binding protein that blocks the binding of eIF4G to eIF4E. Cup mediates an indirect interaction between Smaug and eIF4E, and Smaug function in vivo requires Cup. Thus, Smaug represses translation via a Cup-dependent block in eIF4G recruitment (Nelson, 2004),
To understand the mechanisms that underlie Smg's ability to repress translation, attempts were made to identify Smg-binding proteins. Initial work focused on proteins that would interact with amino acids 583-763. This region contains the Smg SAM domain, which is the protein's RNA-binding domain. An affinity resin carrying covalently coupled GST-Smg583-763 was mixed with early embryo extracts. After extensive washing, bound proteins were eluted and detected via silver staining following SDS-PAGE. Several proteins were eluted from both the GST-Smg583-763 resin and a resin carrying covalently coupled GST-Smg179-307. However, an `80 kDa protein and an `140 kDa protein were specifically eluted from the Smg583-763 resin. Both proteins were subjected to MALDI-TOF mass spectrometry, and while the smaller protein was not identified the larger was identified as Cup, which plays an essential but ill-defined role during oogenesis and early embryogenesis. To confirm that Cup interacts with Smg583-763, Cup was generated via in vitro translation in rabbit reticulocyte lysate. This protein interacted with GST-Smg583-763, as assayed by capture of Cup on glutathione agarose in the presence of GST-Smg583-763 (Nelson, 2004),
Biochemical and genetic evidence is presented that is consistent with Cup functioning as an eIF4E-binding protein that mediates an interaction between Smg and eIF4E. Cup blocks the eIF4E/eIF4G interaction, suggesting that Smg-dependent translational repression of SRE-containing mRNAs results from a Cup-mediated block in the recruitment of eIF4G. Cup's role in Smg function is therefore similar to that played by Maskin in translational repression mediated by CPEB. Given that Maskin and Cup are not homologous, this suggests that other undiscovered adaptor eIF4E-binding protein/3' UTR-binding protein pairs will employ this mechanism to regulate translation (Nelson, 2004),
Cup interacts with eIF4E using both an eIF4E-binding motif and a second site that interacts with eIF4E through a distinct mechanism. Despite this difference, the second site is still able to inhibit the eIF4E/eIF4G interaction in vitro. Further work will be required to assess the significance of this site to Cup function in vivo (Nelson, 2004),
This model for Cup suggests that Smg represses translation at the level of initiation. However, the association of repressed nos mRNA with polysomes indicates that translational repression is achieved at a step after initiation. This apparent contradiction may reflect the fact that repression of nos translation is mediated by at least two trans-acting factors: Smg and a yet to be identified factor that functions through sequences in the nos 3' UTR that are distinct from the SREs. Thus, while Smg regulates translation at the level of initiation, additional factors may function at other levels. Similarly, Smg itself may utilize multiple mechanisms to repress nos expression, only one of which is Cup dependent (Nelson, 2004),
Regulation of translation during development often involves both translational repression and translational activation. The combination of these controls can spatially or temporally restrict the expression of an mRNA, thereby directing the proper development of a cell type or tissue. For example, nos translation is spatially regulated allowing for the proper development of the posterior of the Drosophila embryo. Smg plays an essential role in this process by repressing the translation of unlocalized nos mRNA, while nos mRNA localized to the posterior escapes this repression allowing for the accumulation of Nos protein specifically at the posterior. Given that Smg protein is distributed throughout the embryo, this suggests that Smg function must be over-ridden at the posterior. Cup is also distributed throughout the embryo, suggesting that spatial regulation of nos translation may involve disrupting Cup and/or Smg function specifically at the posterior. Osk protein, which is localized to the posterior, is required for nos translation and Osk interacts with Smg. Thus translational activation could involve Osk binding to Smg thereby blocking Smg function. Interestingly, Cup and Osk interact with the same region of the Smg protein. This might imply that Osk's interaction with Smg could disrupt the Cup/Smg complex and in so doing play a role in activating nos translation at the posterior (Nelson, 2004),
In Xenopus, temporal regulation of translation involves Maskin-mediated repression of target mRNAs in immature oocytes. Upon oocyte maturation, this repression is disrupted resulting in the activation of translation . This activation of translation involves a CPEB-mediated increase in the length of the transcript's poly(A) tail and subsequent recruitment of poly(A)-binding protein (PABP) to the message. PABP brings eIF4G to the mRNA, which in turn disrupts the Maskin/eIF4E complex resulting in translational activation. Measurement of the length of the nos poly(A) tail suggests that regulation of nos translation does not involve changes in poly(A) tail length. Thus, activation of nos translation does not likely involve disruption of the Cup/eIF4E complex through poly(A)-dependent eIF4G recruitment. Taken together, these results also suggest that the use of adaptor proteins such as Cup in translational regulation mediated by sequence-specific RNA-binding proteins is not restricted to mRNAs whose translation is regulated through their poly(A) tail (Nelson, 2004),
The data demonstrate that the same region of Smg that has previously been shown to function in sequence-specific RNA binding also interacts with Cup. The model therefore suggests that this region of the protein would be sufficient to repress translation. However, a transgene that expresses the Smg RNA-binding domain plus a short carboxy-terminal extension fails to rescue the smg mutant phenotype. These results would suggest that Smg has other essential functions in the early embryo in addition to Cup-dependent translational repression. Smg has been suggested to induce the degradation of target mRNAs in a process that may be distinct from its ability to repress translation. Perhaps this ability to induce mRNA degradation is essential and requires regions of Smg outside of amino acids 583-763 (Nelson, 2004),
Phenotypic analysis of several cup mutant alleles highlights Cup's involvement in a number of different biological processes during oogenesis and early embryogenesis, including oocyte growth, maintenance of chromosome morphology, and the establishment of egg chamber polarity. However, the molecular mechanisms that underlie Cup function have not been characterized. The demonstration that Cup is an eIF4E-binding protein suggests that at least some of the defects associated with mutations in the cup gene result from misregulation of translation. Consistent with this possibility is the fact that Cup has been previously shown to interact with Nos protein, which is itself a translational repressor. Genetic experiments suggest that Cup negatively regulates Nos activity during oogenesis, but the molecular mechanisms are not understood. This contrasts with Cup's positive effect on Smg-mediated translational repression. Thus Cup might utilize different molecular mechanisms to influence different translational repressors. The pleiotropic nature of the cup mutant phenotype suggests that Cup may serve as an adaptor protein that is utilized by multiple translational repressors to interact with eIF4E (Nelson, 2004),
Cup is homologous to 4E-T, a human nucleocytoplasmic shuttling protein that employs an eIF4E-binding motif to transport eIF4E into the nucleus (Dostie, 2000). The similarity between these proteins may suggest that Cup also functions to transport eIF4E into the nucleus. Thus some of the phenotypes associated with cup mutants may be related to a defect in eIF4E shuttling during oogenesis. The similarity between Cup and 4E-T also suggests that 4E-T might function in translational repression as an adaptor protein that mediates interactions between eIF4E- and 3' UTR-binding proteins. Specifically, 4E-T could function in translational repression mediated by the human Smg homolog. Similarly, additional RNA-binding proteins that interact with other eIF4E-binding proteins could function to regulate translation spatially or temporally. These protein pairs could control the translation of different mRNAs in various cell types throughout development (Nelson, 2004),
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, 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).
In animals, egg activation triggers a cascade of posttranscriptional events that act on maternally synthesized RNAs. In Drosophila, the Pan Gu (Png) kinase sits near the top of this cascade, triggering translation of Smaug (Smg), a multifunctional posttranscriptional regulator conserved from yeast to humans. Although Png is required for cytoplasmic polyadenylation of smg mRNA, it regulates translation via mechanisms that are independent of its effects on the poly(A) tail. Analyses of mutants suggest that Png relieves translational repression by Pumilio (Pum) and one or more additional factors, which act in parallel through the smg mRNA's 3' untranslated region (UTR). Microarray-based gene expression profiling shows that Smg is a major regulator of maternal transcript destabilization. Smg-dependent mRNAs are enriched for gene ontology annotations for function in the cell cycle, suggesting a possible causal relationship between failure to eliminate these transcripts and the cell cycle defects in smg mutants (Tadros, 2007).
Gene expression profiling analyses have shown that, in Drosophila, a remarkably high fraction (55%) of encoded mRNAs is expressed and loaded into mature oocytes. An earlier estimate of 30% was derived from methods biased toward identification of RNAs that are strictly maternally expressed, whereas, in principle, the method used identifies all maternally expressed genes, including those also expressed at other stages or in other cell types. The predicted number of maternal RNAs is likely to increase as more sensitive in situ hybridization methods are used to determine the maternal versus nonmaternal cutoff (Tadros, 2007).
In Drosophila, elimination of a subset of maternal transcripts is accomplished through the joint action of two pathways: one is maternally encoded and active in unfertilized eggs; the second requires fertilization and zygotic transcription. This study shows that 20% of the maternal mRNAs (more than 1600) are destabilized by the 'maternal' pathway. The actual number of maternal transcripts that are destabilized in embryos is, thus, expected to be significantly larger (Tadros, 2007).
Maternal mRNA destabilization in zebrafish depends on miR-430, which is absent from the oocytes and is transcribed only after fertilization. miR-430 therefore functions in a zebrafish pathway equivalent to the Drosophila 'zygotic' pathway (Bashirullah, 1999). These analyses suggest that, in Drosophila, the earlier, 'maternal' destabilization pathway does not require miRNAs, as known miRNA binding sites are not enriched in the 3′ UTRs of unstable transcripts. Nonetheless, the fact that several miRNA target sites are enriched in the maternal class as a whole suggests that miRNAs may function in the translational regulation of these transcripts rather than their degradation. It also remains possible that miRNAs participate in the 'zygotic' pathway; because this pathway is expected to affect a significantly larger subset of maternal mRNAs than the 'maternal' pathway, this would explain the observed enrichment of miRNA targets in the maternal class as a whole (Tadros, 2007).
Quite unexpected was the discovery that Smg is a major regulator of maternal transcript destabilization, being required for elimination of two thirds of the mRNAs that degrade upon egg activation. Smg regulates translation through cis elements known as SREs. However, SREs do not mediate Smg-dependent degradation of endogenous transcripts. For example, although both nanos (Smg-independent) and Hsp83 (Smg-dependent) mRNAs contain SREs, degradation is SRE dependent only in the case of the former. Therefore, it is not surprising that SREs are enriched in the unstable class of transcripts but not further enriched in the Smg-dependent subclass. In summary, though Smg may trigger the degradation of endogenous transcripts through an SRE-independent mechanism, so also SREs may bind a degradation factor other than Smg (Tadros, 2007).
The discovery that the Png kinase complex coordinates translation of smg mRNA through its 3′ UTR is reminiscent of the role of the Aurora A kinase in translational unmasking of maternal mRNAs during Xenopus oocyte maturation. In the frog system, Aurora A phosphorylates CPE binding protein (CPEB), which is bound to a 3′ UTR element known as the cytoplasmic polyadenylation element (CPE). CPEB then promotes lengthening of the poly(A) tail, thus facilitating binding of poly(A) binding protein (PABP). PABP in turn binds eIF4G, bringing it into proximity with eIF4E, thus disrupting eIF4E's interaction with the repressor, Maskin. This permits recruitment of the 40S ribosomal complex and initiation of translation. CPEB-mediated regulation of polyadenylation and translation is also crucial later, during early embryogenesis, for cell cycle progression (Tadros, 2007).
Though frog Aurora A and fly Png are both Ser/Thr kinases that function through 3′ UTRs to translationally activate maternal mRNAs, their modes of action differ. Although Png is required for the polyadenylation of its target transcripts, the data suggest that its role in promoting translation is either 'downstream' of or runs 'in parallel' to polyadenylation. The distinction between these two mechanisms lies in the interpretation of the fact that lengthening smg poly(A) tails results in increased translation in wild-type but not in png embryos. A downstream role for Png could be to transduce a signal linking polyadenylation and translation. For example, in plants, phosphorylation of PABP increases its cooperative binding to poly(A) RNA. Alternatively, Png might function in a pathway independent of polyadenylation. For example, during Xenopus oocyte maturation, Ser/Thr phosphorylation of Maskin is crucial for its dissociation from eIF4E and subsequent translational activation of CPE-bearing transcripts. Png could phosphorylate and cause dissociation of an analogous eIF4E binding protein (there is no clear Maskin ortholog in Drosophila) (Tadros, 2007).
Png has been shown to promote translation of Cyclin B and together with these results on smg, it is suggested that the previously surmised independent regulation of destabilization and the cell cycle by Png lies at the level of its targets: smg mRNA in the case of destabilization, and cyclin B mRNA in that of the early embryonic cell cycle. Though Png regulates cyclin B mRNA translation through the proposed relief of Pum-mediated translational repression, for smg mRNA, Png acts to relieve repression by Pum and one or more proteins that act in parallel. Because there are no canonical Nanos response elements (NREs) in the smg 3′ UTRs, regulation of smg translation by Pum must be indirect or occur via noncanonical NREs. Consistent with either of these possibilities is the recent finding that smg mRNA is associated with a transgenic Pum protein fragment in embryonic extracts (Gerber, 2006). Also noteworthy is the fact that Pum's repression of one of its known target mRNAs, hb, occurs through both polyadenylation-dependent and -independent mechanisms (Tadros, 2007).
Expression of Smg protein in png mutants is not sufficient to restore instability to Hsp83, a Smg-dependent maternal mRNA. Thus, destabilization of Smg-dependent maternal mRNAs in eggs from png mutant mothers requires one or more additional proteins. Png may promote the translation of Y, an essential component of the destabilization machinery, or may phosphorylate Y, thus activating the degradation machinery. Global analyses of maternal RNA stability in png mutants expressing UAS-smg-bcd3′UTR will identify whether any of the Png-dependent transcripts that are Smg dependent are Y independent (Tadros, 2007).
It is noted that a third of the unstable maternal mRNAs are Smg independent. Png function is likely to be required to destabilize a subset of these Smg-independent maternal transcripts. This is suggested by the fact that nanos mRNA is fully stabilized in png mutants but is only partially stabilized in smg mutants. Global analyses of maternal RNA stability in png mutants will identify all Png-dependent transcripts (Tadros, 2007).
In Drosophila embryos, the transition from maternal to zygotic control of development has been hypothesized to require two processes: elimination of maternal mRNAs and synthesis of zygotic mRNAs. Zygotic transcription is required for cellularization, the hallmark of the Drosophila MBT. However, the functional significance of maternal transcript elimination has remained largely unexplored. smg mutants have been shown to fail to progress beyond nuclear cycle 12, never reaching the MBT, and computational analyses have shown that the Smg-dependent unstable maternal transcripts are enriched for GO terms related to mitosis and the cell cycle. This enables the presentation of a model in which elimination of maternal cell cycle mRNAs by Smg is essential for progression through the final syncytial nuclear divisions and, ultimately, the MBT. Detailed cellular and molecular analysis of the smg mutant phenotype will be required to test this hypothesis (Tadros, 2007).
Smg homologs exist from yeast to humans, where they function in posttranscriptional regulation. Furthermore, the budding yeast homolog Vts1 has been shown to interact with the same cis element as Smg. Since turnover of maternal mRNAs occurs prior to the MBT in all metazoa, Smg homologs may fulfill a conserved developmental function: targeting a subset of maternal mRNAs for elimination and thus permitting the MBT to occur (Tadros, 2007).
To determine when and where Smaug is expressed, polyclonal antibodies against different portions of the protein were prepared. In extracts prepared from early embryos, these antibodies specifically recognize a protein with an apparent molecular weight of 120 kDa that is encoded by smg. Analysis of Western blots reveals that Smaug protein is undetectable in ovaries. Translation of maternal SMG mRNA during early embryogenesis results in the accumulation of protein 0-3 hr postfertilization. As is the case for many other maternal mRNAs, SMG transcripts are degraded prior to formation of the cellular blastoderm, and Smaug protein levels fall dramatically at this point in development.
Next, antibodies that recognize Smaug were used to examine its distribution during early embryonic development. Smaug protein is distributed uniformly throughout the bulk cytoplasm and pole plasm of the syncitial embryo. At no point in early development is any asymmetry in the distribution of Smaug observed. Two lines of evidence demonstrate that these antibodies specifically recognize Smaug in situ: (1) embryos derived from females with extra copies of a smg+ transgene exhibit the same monotonous distribution of antigen, albeit at higher levels; (2) smg mutant embryos have no detectable protein (Dahnukar, 1999).
To examine the role of smaug in vivo, a loss-of-function mutation was isolated in smg by the following strategy. The gene was mapped to polytene band 66F, and deficiencies that uncover smg were identified by quantitative Southern blot analysis. Embryos from females bearing one of these Df(Scf) deficiencies have approximately one-half the amount of Smaug present as do embryos from wild-type females, collected under similar conditions, as measured by Western blot. Flies with a wild-type third chromosome were mutagenized with EMS and then crossed to flies bearing Df(Scf). Recessive mutations that cause lethal or female-sterile phenotypes in trans to Df(Scf) were identified by analysis of approximately 5000 crosses. One candidate from this screen proved to be a recessive, loss-of-function mutation in smg. While embryos from smg/+ heterozygous females are indistinguishable from wild type, embryos from smg/Df(Scf) females (hereinafter, smg mutant embryos) die. This embryonic lethality is rescued by maternal expression from a smg+ transgene but not by maternal expression from various smg deletion derivatives. In particular, expression of either a Smaug derivative lacking the RNA-binding domain or one consisting of the RNA-binding domain plus a short C-terminal extension does not rescue embryonic viability. Western blot analysis reveals that each derivative is stable in vivo, and, thus, Smaug appears to have separable essential functions in the N-terminal region and in the RNA-binding domain. smg mutant embryos contain approximately normal levels of full-length SMG mRNA, but essentially no Smaug protein is detected by Western blot using an antibody directed against the RNA-binding domain. The smg1 allele appears to be null (at least for early embryonic function), based on the criterion that the terminal phenotype of embryos from smg/Df(Scf) and from smg/smg females are indistinguishable (Dahanukar, 1999).
The embryonic lethality caused by loss of Smaug function is not a result of defects in NOS mRNA regulation, suggesting that Smaug has additional roles during early embryonic development. Unlike nos mutant embryos, which complete embryonic development and die with abdominal segmentation defects, smg mutant embryos arrest very early in embryonic development. To investigate the genesis of these early defects, development was monitored by preparing timed collections of wild-type and mutant embryos that were fixed and subsequently stained with the DNA-binding dye DAPI to reveal the distribution of nuclei. Smaug is required for normal progression beyond nuclear division cycles 11-12 in the precellular blastoderm stage. From fertilization through cycle 10, the nuclear divisions appear to be essentially indistinguishable in wild-type and smg embryos. However, beginning in cycle 11, the pattern of nuclei in smg mutant embryos becomes increasingly irregular, with a variety of defects including asynchronous mitoses and gaps in the otherwise regular array of nuclei at the surface of embryo. The proportion of mitotic nuclei in smg embryos suggests that progression through mitosis is retarded. The terminal phenotype appears to result from the aggregation of nuclei that fall from the surface into the middle of the embryo. smg mutant embryos never achieve the nuclear density at the surface of cycle 14 wild-type embryos and never cellularize (Dahanukar, 1999).
These phenotypes are similar in some respects to those associated with mutations in genes involved in regulating cell cycle progression, cytoskeletal integrity, or the coordination between the two. For example, mutations in grapes (grp) or nuclear-fallout (nuf) cause similar nuclear phenotypes and arrest development between cycles 10 and 14. grp encodes a S/T kinase that is thought to participate in a DNA damage checkpoint, and nuf encodes a protein that cycles between the cytoplasm and the centrosomes. While the genesis of the defects in smg mutant embryos has not been investigated further, Smaug evidently is required for normal progression through the late nuclear division cleavages in the syncitial blastoderm (Dahanukar, 1999 and references).
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smaug:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 30 May 2007
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