Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The Plu protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, Plu does not perturb the cell cycle, suggesting that Plu regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. Functional png is needed for the presence of Plu protein. By analyzing png mutations of differing severity, it has been found that the extent of the png mutant phenotype inversely reflects the level of Plu protein. These data suggest that Plu protein is required at the time of egg activation and the completion of meiosis (Elfring, 1997).
The plu and png genes genetically interact: mutation of plu dominantly enhances the phenotype of weak mutations in png (Shamanski, 1991). It was of interest to test whether this genetic interaction could result from the Plu and Png proteins interacting physically by examining whether the proteins could be co-immunoprecipitated from embryo extracts. To immunoprecipitate each of the proteins, transformant lines carrying MYC epitope-tagged Plu or MYC epitope-tagged Png were generated. These tagged proteins are fully functional because they completely rescue the maternal effect lethality of plu - or png - homozygotes, respectively, such that the embryos laid by the mutant mothers hatch and develop into adults. The Png-MYC fusion protein was efficiently immunoprecipitated with the MYC monoclonal antibodies. Detection with antibodies against Plu demonstrates that Plu protein co-immunoprecipitates with Png-MYC. The immunoprecipitation of Plu is not the result of an association with the MYC tag, because neither Plu nor Png alone co-immunoprecipitated with an unrelated MYC-tagged protein under the same conditions. Additionally, neither Png-MYC, Png, nor Plu were precipitated from extracts prepared from wild-type embryos not expressing the Png-MYC tagged protein (Fenger, 2000).
A complex between Plu and Png could also be demonstrated by immunoprecipitating MYC-tagged Plu. Immunoprecipitations and immunoblots show that both Plu-MYC and Png are precipitated by MYC antibodies from extracts expressing Plu-MYC, but not from wild-type controls. In these experiments the extracts were prepared from unfertilized eggs, but Png and Plu-MYC also co-immunoprecipitate from fertilized embryonic extracts. To test if Png activity is required for Png and Plu association, the immunoprecipitation was performed using extracts from embryos laid by mothers mutant for png but expressing the Plu-MYC protein. These png mutant proteins, Png1058, Png158, and Png172 are stable because they are present at near-normal levels in the supernatants. Despite the fact that Plu-MYC was immunoprecipitated, none of the mutant Png proteins co-immunoprecipitated at levels detectable by immunoblotting. These Png mutant proteins are predicted to have diminished or no kinase activity. Thus Png and Plu association requires functional Png, or the mutations change the structure of Png such that it cannot interact with Plu (Fenger, 2000).
To test if a kinase activity is associated with Png and Plu, in vitro kinase assays were performed on immunoprecipitates. The proteins were separated by SDS-PAGE and immunoblotted. A 30 kDa protein can be phosphorylated after immunoprecipitation from plu-myc unfertilized egg extracts using anti-MYC antibodies. The phosphorylated protein is not present in an immunoprecipitate that does not contain Plu-MYC. The kinase activity is Png dependent, because the phosphorylation is not observed in immunoprecipitates of png1058;plu-myc, which contains no Png (Fenger, 2000).
Early embryonic cell cycles in Drosophila consist of rapidly alternating S and M phases. Three genes, pan gu (png), plutonium (plu), and giant nuclei (gnu) coordinate these early S-M cycles by ensuring adequate Cyclin B protein levels. Mutations in any of these genes result in unregulated DNA replication and a lack of mitosis ('giant nuclei' phenotype). png encodes a serine/threonine protein kinase, and plu and gnu encode small, novel proteins. Png, Plu, and Gnu constitute a novel protein kinase complex that specifically regulates S-M cell cycles. All three proteins are required for Png kinase activity and are phosphorylated by Png in vitro. Yeast two-hybrid screening reveals a direct interaction between Png and Plu, and their co-expression is required for physical association and activation of Png kinase. Artificial dimerization of Plu via fusion to either GST or FK506 binding protein (in the presence of dimerizing agent) abrogates the requirement for Gnu to activate Png kinase. A model is proposed in which Gnu normally regulates embryonic cell cycles by promoting transient dimerization of a core Png/Plu complex, thereby stimulating Png kinase activity (Lee, 2003).
This study demonstrates a novel cell-cycle protein kinase complex composed of the Png kinase and its associated subunits Plu and Gnu. The Png kinase complex regulates the early embryonic S-M cycles in Drosophila, promoting mitosis and inhibiting DNA replication. Png, Plu, and Gnu proteins are present only in ovaries and early embryos, and null mutations in their genes reveal that they play essential developmental roles exclusively during the early embryonic divisions following completion of meiosis. The biochemical analysis of the Png kinase complex described here defines the requirement for png, plu, and gnu during the S-M cycles and explains their shared phenotypes and genetic interactions by establishing that all three proteins are essential for Png kinase activity (Lee, 2003).
Cell-cycle regulatory kinases must be precisely controlled to ensure the orderly progression of cell-cycle events. Parallels can be drawn between the Png kinase complex and CDK/cyclin complexes. CDK kinases require association of an activating cyclin subunit. Similarly, the Plu and Gnu proteins are activating subunits of the Png kinase. Each CDK molecule partners with a single cyclin subunit: the Plu subunit is stably associated with Png in a 1:1 stoichiometry. Gnu is also needed for Png kinase activity, but it is much less stably associated with the Png/Plu complex, possibly serving to control when and/or where Png kinase is active. Both CDK1/cyclin complexes and the Png kinase complex promote entry into mitosis and block DNA re-replication (Lee, 2003).
The Png and Plu subunits are tightly and directly associated. In addition to the interaction observed following their co-expression in baculovirus-infected cells, they directly bind each other in yeast cells in a two-hybrid assay. Furthermore, they form a complex in vivo and can be co-immunoprecipitated from Drosophila ovary or embryo extracts (Lee, 2003).
It is striking that Png and Plu must be co-expressed to associate with each other and to form an active kinase with Gnu. A similar observation has been reported for the ß and gamma subunits of heterotrimeric G proteins. Physical interaction between ß and gamma subunits to form a functional heterodimer requires co-expression. In both cases, the requirement for co-expression likely reflects the fact that the physical association between the two proteins is very tight, and folding of the proteins may be coordinated. Consistent with this explanation, attempts to dissect the interaction domains between Png and Plu using the two-hybrid assay failed, suggesting that multiple regions on each protein are needed for interaction and/or that the truncated proteins did not fold correctly. Interestingly, Png mutant proteins predicted to have diminished or no kinase activity fail to co-immunoprecipitate with Plu-Myc from Drosophila embryo extracts, despite their presence at nearly normal levels. The results demonstrate that Png and Plu association can occur in the absence of Gnu; thus, functional kinase activity is not required for this association. Instead, mutations that affect Png kinase activity also affect its interaction with Plu, possibly via alteration of its three-dimensional structure (Lee, 2003).
The intimate association between Png and Plu suggests that the sole function of Plu is to form a complex with and activate Png. Co-expression of Png and Plu may permit Png to fold into an active conformation. It is also possible that Plu provides substrate specificity to the Png kinase by analogy to cyclin subunits and their CDKs. It remains to be determined whether phosphorylation of Plu affects its activity or stability. It is only weakly phosphorylated by Png in IP kinase assays. Thus, it is possible that Plu is only 'incidentally' phosphorylated as a consequence of its proximity to Png in the complex. Density gradient analysis suggests that Png and Plu proteins are part of a larger-molecular-weight complex in vivo, representing either a multimeric complex of the Png/Plu heterodimer or a complex containing additional, as yet unidentified, proteins. Future identification of other regulators and/or substrates of the Png kinase complex will further understanding of how S-M cycling is regulated and help to identify the mechanism by which the Png kinase complex controls transcript stability (Tadros, 2003; Lee, 2003).
In contrast to Plu, Gnu is not tightly associated with Png. Gnu is present in active recombinant Png kinase complexes as assessed by 32P incorporation in Png-Myc IP kinase assays. A very low level of Gnu protein is detected in these Png-Myc immunoprecipitates by immunoblotting, and the interaction between Gnu and the Png/Plu core complex appears to be transient and/or weak. Png and Gnu fail to interact in a yeast two-hybrid assay. Furthermore, endogenous Gnu (in contrast to Png) does not co-immunoprecipitate with Plu-Myc from Drosophila embryo extracts (Lee, 2003).
Gnu is required for Png kinase activity, and it is proposed that Gnu normally activates a core Png/Plu complex by facilitating its multimerization. This model was initially suggested by the observation that a GST-Plu fusion protein can fully activate Png kinase in the absence of Gnu. The role of dimerization in activating Png kinase has been directly demonstrated by using an FK506-binding protein fused to Plu and the AP20187 compound to dimerize the FKBP domain. Inducible dimerization of the FKBP domain greatly stimulates the kinase activity of the Png/FKBPPlu complex to levels comparable to that of the Png/Plu/Gnu and Png/GST-Plu complexes. There is precedence for the regulation of protein kinases via dimerization. Receptor tyrosine kinases dimerize upon ligand binding, and this dimerization activates their intracellular kinase domains. In another type of mechanism, dimerization of the MAP kinase ERK2 permits its entry into the nucleus (Lee, 2003).
The weak association between Gnu and the Png/Plu complex raises the possibility that Gnu has other as yet unidentified functions in addition to activation of Png kinase. Gnu could interact with other proteins in addition to the Png/Plu complex to potentially regulate other processes. However, the genetic phenotypes and developmental expression of Gnu show that, like Png and Plu, its activity is restricted to early embryogenesis. Ectopic expression of Gnu in ovarian nurse cells caused female sterility. This effect is dependent on having functional Png and Plu, suggesting that at least some of Gnu's in vivo activity is mediated through the Png kinase complex. This observation is consistent with biochemical results showing that Gnu is an activator of a core Png/Plu complex, and suggests that the female sterility associated with ectopic Gnu expression is a consequence of ectopic activation of Png kinase (Lee, 2003).
Gnu is strongly phosphorylated by Png in IP kinase assays using recombinant proteins. The results from a Png filter kinase assay indicate that endogenous Gnu (from embryo extracts) also is phosphorylated by recombinant Png kinase complex in vivo. Filter kinase assay results are consistent with the previous observation that an ~30-kD protein present in Drosophila embryo extracts is phosphorylated in a Png-dependent manner. Although not yet identified, this ~30-kD 32P-labeled band likely represents Gnu (Lee, 2003).
Phosphorylation of proteins on particular residues can sometimes result in an upward mobility shift on SDS-PAGE. Incubation of 35S-labeled Gnu (generated in a coupled in vitro transcription/translation reaction using reticulocyte lysate) with active recombinant Png kinase complex resulted in an easily detectable upward shift in its mobility on SDS-PAGE. Therefore, the mobility was compared of Gnu in embryo extracts from wild-type, png, and plu mutant females. No differences were observed in the mobility of Gnu from these different genetic backgrounds. Possible explanations for the failure to detect mobility differences in Gnu from wild-type versus png and plu mutant backgrounds is that only a small percentage of the total pool of Gnu is phosphorylated by Png, the modification is transient, and/or phosphorylated Gnu is unstable. Another possible explanation is that the in vivo Png phosphorylation sites differ from the in vitro phosphorylation sites, and they do not result in a mobility shift of Gnu (that is, Gnu is relatively 'hyperphosphorylated' by Png in vitro). In addition to possibly being a Png substrate in vivo, Gnu is phosphorylated by a different kinase during oogenesis, and this phosphorylation is removed at egg activation. The functional consequences of phosphorylation of Gnu await elucidation (Lee, 2003).
The giant nuclei class of genes is required to maintain adequate levels of Cyclin B protein normally needed to block DNA re-replication and to promote the onset of mitosis. png, plu, and gnu do not affect the steady-state level of cyclin B transcripts. Furthermore, Cyclin B protein does not appear to be a direct substrate of the Png kinase complex. In an effort to determine the mechanism by which the giant nuclei class of genes regulates Cyclin B protein levels, a genome-wide biochemical screen was performed to identify Png kinase substrates. Seven substrates of the Png kinase complex (including Gnu) were identified in this screen. Thus, regulation of Cyclin B protein levels may occur further downstream of these direct targets of the Png kinase complex (Lee, 2003).
png, plu, and gnu are the only genes known to be essential for S-M oscillations in the early embryo. Biochemical results suggest that activation of Png kinase is a major function of both Plu and Gnu. Archetypal cell cycles in developing eye imaginal discs were not perturbed by ectopic co-expression of Png and Plu. Likewise, misexpression of Gnu in polytene salivary glands and ovarian follicle cells has no obvious effects on the cell cycles in these tissues. Given the finding that Png kinase activity requires both Plu and Gnu, ectopic expression of only one or two components of the complex would likely have no effect on cell cycles in other tissues. In screens of maternal-effect lethal collections, png, plu, and gnu are the only mutants identified in which S phase is uncoupled from mitosis in the early embryo. Thus, the Png/Plu/Gnu kinase complex appears to be the crucial regulatory switch unique to the S-M cycles of early Drosophila embryogenesis (Lee, 2003).
Although traditional organism-based mutational analysis is powerful in identifying genes involved in specific biological processes, limitations include incomplete coverage and time required for gene identification. Biochemical screens using cell transfection or yeast two-hybrid methods are rapid, but they are limited by cDNA library quality. The recent establishment of 'uni-gene sets' has made it feasible to biochemically screen an organism's entire genome. Radiolabeled protein pools prepared from the Drosophila Gene Collection were used in a Drosophila in vitro expression cloning ('DIVEC') screen for substrates of PAN GU kinase, which is crucial for S-M embryonic cell cycles. Ablation of one identified substrate, Mat89Bb, by RNAi produces a polyploid phenotype similar to that of pan gu mutants. Xenopus embryos injected with Mat89Bb morpholinos arrest with polyploid nuclei, and Mat89Bb RNAi in HeLa cells gives rise to multinucleated cells. Thus, Mat89Bb plays an evolutionarily conserved role as a crucial regulator of both cell cycle and development (Lee, 2005).
Seven genes were identified whose protein products had altered mobility (shifted upward) after incubation with active Png kinase. One of these seven genes is gnu, which encodes both an activator and substrate of Png kinase. Other than gnu and CG5739, a gene of unknown function, the other five genes are evolutionarily conserved with vertebrate homologs. Of particular interest are the genes encoding Mat89Bb and the small ribosomal protein subunit mRpS22. Mat89Bb shares a maternal expression pattern with Png, Plu, and Gnu, and the Png kinase complex regulates Cyclin B levels posttranscriptionally, possibly via regulation of its translation. Given that Mmp2, which shifts weakly after Png treatment, is a membrane bound matrix metalloproteinase that localizes to the cell surface, it is unlikely to be a bona fide Png substrate. Except for the guanyl-nucleotide exchange factor CG9139, the other genes identified as Png substrates have not been previously characterized (Lee, 2005).
To confirm that the decreased SDS-PAGE mobility of candidate Png substrates is actually due to phosphorylation, samples were treated with Lambda Phosphatase after Png kinase incubation. Treatment with Lambda Phosphatase completely reverses the mobility shifts of Mat89Bb and four other candidate Png substrates, thereby confirming that these shifts are a consequence of phosphorylation. In contrast, the mobility shifts of Gnu and mRpS22 observed after incubation with Png kinase were not altered by subsequent treatment with Lambda Phosphatase. This result, however, does not rule out the possibility that these mobility shifts are due to phosphorylation by Png (as opposed to another modification) because not all phosphoproteins are susceptible to Lambda Phosphatase (Lee, 2005).
The type I phosphatase PP1 87B has been identified as a dominant suppressor of png in a sensitized screen for genes that suppress or enhance png. One explanation for this genetic interaction is that PP1 may dephosphorylate substrates of Png, thereby counteracting its activity. Whether PP1 could dephosphorylate Gnu after incubation with the Png complex was tested. Treatment with PP1 completely reversed the effects of Png kinase on Gnu mobility; a similar result was obtained for mRpS22. Thus, the altered mobilities of all candidate Png substrates identified in the DIVEC screen represent Png-phosphorylated forms of the proteins (Lee, 2005).
Of the seven in vitro substrates for the Png kinase identified by DIVEC screening, loss-of-function mutations have been described for only gnu and Mmp2. Because mutation of any of the members of the Png kinase complex (png, plu, or gnu) results in a giant nuclei phenotype (DNA replication uncoupled from mitosis, leading to polyploidy in embryos from mutant females), the Png substrates identified by DIVEC screening might similarly be required for normal embryonic cell cycles. Given the small number of Png substrates identified in the DIVEC screen and the lack of pre-existing mutations in the genes encoding these proteins, RNA interference was used to rapidly assess the consequences of 'knocking down' the gene function of each identified Png substrate. RNA interference was performed by injection of double-stranded RNA corresponding to each candidate substrate into syncytial embryos and unfertilized eggs from wild-type Drosophila females, followed by DAPI staining to visualize the DNA (Lee, 2005).
RNA interference of Mat89Bb gave rise to a phenotype similar to that of the giant nuclei mutants, and this phenotype is consistent with excessive DNA replication in both fertilized and unfertilized eggs. In unfertilized eggs from wild-type females, the four meiotic products are maintained in a condensed state to form distinctive rosette-like structures known as polar bodies. In unfertilized eggs from giant nuclei mutants (png, plu, or gnu), the meiotic products are decondensed and undergo inappropriate DNA replication to become polyploid. Remarkably, RNA interference of Mat89Bb in unfertilized Drosophila eggs yields a giant nuclei phenotype in roughly one-third of injected eggs that is essentially identical to that of png-derived eggs. Considering the lapse of time between the deposition of unfertilized eggs and injection of double-stranded RNA in these experiments (at least 38 min), these results suggest that the continued presence of Mat89Bb is required to maintain the characteristic rosette structure of polar bodies in unfertilized eggs (Lee, 2005).
Aberrant embryonic cell cycles also occur as a consequence of mutations in png, plu, or gnu, and uncoupling of S and M phases leads to cell cycle arrest with highly polyploid nuclei. RNA interference of Mat89Bb in Drosophila syncytial embryos similarly results in intensely DAPI-stained nuclei in approximately one-fourth of injected embryos. These effects of decreasing Mat89Bb gene function on DNA content in both unfertilized eggs and embryos are consistent with a role for Mat89Bb in the Png/Plu/Gnu pathway and suggest that it is a true in vivo substrate of the Png kinase complex (Lee, 2005).
Although decreasing Mat89Bb gene function by RNA interference in Drosophila syncytial embryos results in increased DNA content relative to buffer-injected control embryos, the degree of polyploidy was less than that observed in embryos from weak png mutants. One possible explanation for this difference is that Mat89Bb is only one of several in vivo substrates of the Png kinase complex that mediates its effects on the cell cycle. Another consideration is the experimental constraints that limit how soon after egg collection that injections of double-stranded RNA can be performed (at least 38 min). The giant nuclei genes (and presumably substrates of the Png kinase complex) are required as early as the first embryonic mitotic division following fertilization. In order for RNA interference to have an effect on a cellular process over a narrow window of time, such as during the early syncytial divisions, the targeted protein must be turned over rapidly. In attempts to knock down the function of the giant nuclei genes png or gnu by RNA interference in syncytial embryos or unfertilized eggs as a positive control, no effect was observed on cell cycles or polar body structure, as assessed by DAPI staining. RNA interference of the remaining five Png kinase substrates identified by DIVEC screening in syncytial embryos or unfertilized eggs similarly resulted in no obvious defects. Thus, negative RNA interference results do not rule out the possibility that these other in vitro substrates of Png kinase may be bona fide substrates required for normal embryonic cell cycles and maintenance of polar bodies (Lee, 2005).
The Drosophila Mat89Bb gene encodes a 76 kDa protein with a maternal expression pattern. A comparison of the sequences of Drosophila and human Mat89Bb revealed that they are 36% identical and 61% similar at the amino acid level. While homologs of the Png kinase complex have not yet been identified in organisms other than dipteran flies, identification of the evolutionarily conserved protein Mat89Bb as a substrate of Png kinase combined with RNA interference data suggests that this complex impinges on molecules that have a conserved cell cycle function in diverse organisms. Therefore, whether decreasing Mat89Bb function in Xenopus embryos or in cultured mammalian cells would similarly disrupt their cell cycles was tested (Lee, 2005).
As in Drosophila, Xenopus embryos undergo rapid S-M cycles prior to the midblastula transition. Based on
RNA interference results in Drosophila embryos, it was hypothesized that morpholino-mediated downregulation of Mat89Bb in Xenopus embryos would similarly affect both cell cycle and development. Injection of Mat89Bb morpholinos (200 ng) into one-celled Xenopus embryos results in a developmental delay at gastrulation in 100% of injected embryos and subsequent defects in which the embryos were shortened along the anterior-posterior axis; complete arrest occurred in all (50/50) embryos following injection of higher concentrations (>200 ng) of Mat89Bb morpholinos. A similar delay in Xenopus embryogenesis has been reported following morpholino-mediated downregulation of Cyclin E, which is normally required for proper initiation of DNA replication. Unlike Cyclin E, however, Xenopus embryos with decreased Mat89Bb levels do not develop normally and have defects consistent with abnormal gastrulation. Both the developmental delay and shortened axis phenotype observed following injection of Xenopus embryos with Mat89Bb morpholinos were rescued by coinjection of human Mat89Bb-GFP mRNA, indicating that the morpholino effects were specific (Lee, 2005).
Given that RNA interference of Mat89Bb in Drosophila syncytial embryos results in polyploidization, DNA staining of the Mat89Bb morpholino-injected Xenopus embryos was performed to determine whether they were similarly affected. 100% of the Mat89Bb morpholino-injected embryos stained more intensely with propidium iodide than embryos injected with control morpholinos, suggesting that they have an increased DNA content. Because DNA of Xenopus embryos is difficult to visualize by microscopy due to the large amount of yolk protein, animal cap explants were isolated from embryos injected with either control or Mat89Bb morpholinos for DNA staining and confocal microscopy. Nuclei of animal cap cells from Mat89Bb morpholino-injected embryos have an average area greater than 1.6-fold larger than that of controls; assuming a spherical shape, this increase in area represents a 2-fold increase in nuclear volume. The average intensity of DNA staining per unit area was identical, however, confirming that the Mat89Bb morpholino causes polyploidy. Cell size is increased, also consistent with mitotic failure and early developmental arrest. These results indicate that the biological function of Mat89Bb in cell cycle control and development during Drosophila embryogenesis has been conserved in vertebrates such as Xenopus (Lee, 2005).
RNA interference of Mat89Bb in cultured human (HeLa) cells with lentiviral-based shRNA gives rise to multinucleated cells. This observed increase in the DNA content of HeLa cells following downregulation of Mat89Bb is remarkably similar to results with Drosophila embryos and eggs and Xenopus embryos and is a phenotype not commonly observed in cultured mammalian cells treated with shRNA. The differences in phenotypes observed between cultured mammalian cells versus Drosophila and Xenopus early embryos following downregulation of Mat89Bb function (i.e., multinucleated cells versus polyploid nuclei, respectively) may reflect inherent differences in the nature of their cell cycles (canonical G1-S-G2-M versus modified S-M cycles, respectively). For example, Mat89Bb appears to be required in HeLa cells for later stages of mitosis such as cytokinesis, which does not occur in the Drosophila syncytial embryo. Finally, these differences may also reflect the fact that downregulation of Mat89Bb transcripts in HeLa cells appears to be incomplete, with greater than 18% of control levels remaining, as determined by RT-PCR; assessment of Mat89Bb transcript levels following RNA interference in Drosophila was technically difficult due to the relatively low numbers of embryos that could feasibly be injected. Despite the observed differences in phenotypes, these studies clearly demonstrate that Mat89Bb plays a fundamental role in the regulation of mitosis in mammalian cells as well as Drosophila and Xenopus embryos (Lee, 2005).
Previous attempts to identify additional genes in the Png/Plu/Gnu pathway by screening large maternal-effect lethal collections and by yeast two-hybrid analysis were unsuccessful. This study has demonstrated that by applying IVEC screening methodology to the Drosophila Gene Collection (DIVEC screening), substrates of the cell cycle kinase Png have been sucessfully demonstrated. This study highlights the general applicability and advantages of using such an approach. The IVEC approach allows one to utilize a wide variety of biochemical assays for screening purposes (as opposed to two-hybrid approaches in which one is solely relying on protein-protein interactions), and the Drosophila Gene Collection allows for rapid and complete coverage of an organism's entire genome with equal representation of all genes. Furthermore, annotation of the Drosophila Gene Collection makes it feasible to identify positive clones within pools rapidly because the identity of all clones in each pool is known. Finally, in vivo verification of the biological roles of candidate genes identified by DIVEC screening can be rapidly achieved in Drosophila by analysis of existing mutants and/or RNA interference. As demonstrated by studies of Mat89Bb function in diverse organisms, the ability to perform rapid in vivo analysis of candidate genes is enhanced when homologs in other organisms can be readily identified (Lee, 2005).
One limitation of the DIVEC screen for Png kinase substrates is that not all phosphorylation events result in a detectable mobility shift by SDS-PAGE. As an alternative, future DIVEC screens for kinase substrates could be designed to directly detect phosphorylation of in vitro-translated proteins by measuring 32P incorporation. The DIVEC approach has recently been modified to allow high-throughput screening for proteins that physically interact with affinity-tagged 'bait' protein immobilized on beads. This variation of DIVEC screening is expected to be more broadly applicable because it does not require a priori knowledge of the biological function of a given protein. Identification of Mat89Bb-interacting proteins by using this approach should provide important biological insight into its role in the cell cycle and development of metazoan organisms (Lee, 2005).
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).
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).
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