InteractiveFly: GeneBrief

trailer hitch: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. Drosophila trailer hitch (tral) has been identified as required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral reveals that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components, that regulate exit of proteins from the ER. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell (Wilhelm, 2005).

Localization of mRNAs is used by many polarized cells as a means of restricting the distribution of a protein to a particular cytoplasmic domain. This mechanism for protein targeting within the cytoplasm is critical for embryonic patterning, synapse formation, and cell migration. Three methods of localization have been described. Active transport of mRNA along either microtubules or actin filaments has been directly demonstrated for a number of transcripts, suggesting a role for cytoskeletal motor proteins in mRNA localization. Diffusion trapping involves the creation of a binding site for the message of interest at a particular subcellular location and allowing that site to passively trap target messages that it contacts by diffusion. Such a mechanism is used to localize nanos mRNA to the posterior pole of the developing Drosophila oocyte. Degradation protection involves selective stabilization of messages at the correct location while unlocalized messages are degraded. This type of mechanism is responsible for the posterior localization of a number of maternal messages during early embryogenesis in Drosophila (Wilhelm, 2005 and references therein).

One of the most extensively characterized systems for studying mRNA localization is the Drosophila oocyte. Oocytes develop as part of an egg chamber, which is composed of the oocyte and 15 nurse cells surrounded by a layer of somatic follicle cells. The oocyte is connected to the nurse cells by a network of cytoplasmic bridges called ring canals. Various mRNAs that are required for early embryogenesis are synthesized in the nurse cells and transported into the oocyte where some, such as oskar (osk) and gurken (grk), are localized to discrete subcellular locations (Wilhelm, 2005).

While genetic and biochemical approaches have identified an ever increasing group of proteins required for mRNA localization during Drosophila oogenesis, recent work in Drosophila and other systems has revealed unexpected connections between the factors that regulate mRNA localization, mRNA stability, and translation. For instance, Cup was identified as an eIF4E binding protein that is required to both translationally repress osk mRNA as well as to recruit the localization factor Barentsz. Similarly, the RNA helicase me31B has been shown to be required for translational repression of osk and Bicaudal-D in Drosophila, while its orthologs in yeast and humans are required for mRNA degradation. Most surprisingly, staufen, which is the archetypal mRNA localization factor in Drosophila and vertebrates, has recently been shown to regulate message stability via the same components that control nonsense-mediated mRNA decay. Consistent with these factors affecting multiple aspects of mRNA metabolism, the Drosophila proteins required for localization and translational control have long been known to reside in large cytoplasmic particles that are both static and exhibit microtubule-dependent transport. Furthermore, the yeast and human orthologs of one component of these particles, the translational repressor Me31B, have also been shown to be critical for the function of processing bodies (P bodies), large cytoplasmic particles that contain many of the regulators of mRNA stability (Cougot, 2004; Sheth, 2003). These results have suggested a model (Coller, 2004) in which mRNA localization, translational regulation, and stability are integrated functions of the P body (Wilhelm, 2005).

Because early patterning events in oogenesis are intimately connected to the proper regulation of cytoplasmic mRNA processing (i.e., translation, localization, and stability), existing insertional mutants were screened for defects in patterning in order to identify novel P body components. This screen identified a Drosophila gene, trailer hitch, that is required for efficient secretion of the TGF-α family member Grk and the vitellogenin receptor Yolkless (Yl). Biochemical and genetic analysis of Tral revealed an unexpected connection between cytoplasmic mRNA processing events and trafficking through the secretory pathway (Wilhelm, 2005).

A number of findings support a direct role for tral in promoting efficient trafficking through the secretory pathway: (1) tral is required for the efficient secretion of both Grk and Yl; (2) immunohistochemistry places the Tral complex on the ER in close association with a subset of ER exit sites in nurse cells, although further analysis at the EM level will be necessary to demonstrate this conclusively; (3) Tral protein is biochemically associated with the transcripts of two ER exit site components: sar1 and sec13; (4) tral is required for normal distribution of Sar1 protein, arguing that the sar1 message is a regulatory target of tral. Together, these results suggest that tral's role in secretion is to regulate the transcripts of ER exit site components on the surface of the ER (Wilhelm, 2005).

ER exit sites have traditionally been defined as localized foci of components of the COPII complex, which is required for ER exit. However, recent work has shown that proteins actually exit from the region of the ER surrounding these foci, rather than from the foci themselves (Mironov, 2003). Because these foci are quite stable, this result has led to a model where “ER exit sites” are actually storage sites to allow a high concentration of COPII components to be available to drive local vesicle formation (Wilhelm, 2005).

This model fits quite nicely with the current results. The observations that the Tral complex borders a subset of ER exit sites and is associated with sar1 mRNA suggest that the Tral complex regulates sar1 expression on the surface of the ER. Because recruitment of Sar1 is the first step in assembling the COPII complex on the membrane, Sar1 is an excellent target for regulating the size and distribution of ER exit sites. Consistent with this, tral mutants display abnormalities in both ER exit site morphology and secretion. Thus, the properly regulated assembly of COPII foci/ER exit sites is critical for normal ER-to-Golgi trafficking (Wilhelm, 2005).

Because many components of the secretory pathway are highly conserved, one might expect tral orthologs to also play a role in secretion. Genetic screens in Drosophila and C. elegans have suggested that this family of proteins does play a conserved role in the regulation of membrane trafficking. The yeast ortholog of tral, Scd6, was identified as a high copy suppressor of a deletion of the clathrin heavy chain locus, while the worm ortholog, car-1, was identified in RNAi screens for genes that are required for cytokinesis (Nelson, 1993 and Zipperlen, 2001). While there have been no additional studies of Scd6 to determine whether it acts directly or indirectly in the secretory pathway, abnormalities in membrane trafficking to the late cleavage furrow have been found in car-1-depleted embryos. Furthermore, it has also been found that disruption of car-1 leads to ER morphology defects similar to the foci of Grk and Yl that were observe in the Drosophila oocyte. The findings that Tral is associated with the ER and is required for efficient trafficking of secreted proteins are consistent with the phenotypes described in C. elegans and suggest that the role of the trailer hitch family in regulating membrane trafficking is conserved (Wilhelm, 2005).

Given these similarities, one might have anticipated that tral mutants would also have defects in cytokinesis. However, it is likely that none of the hypomorphic alleles of tral disrupts tral function sufficiently to display a cytokinesis defect. Consistent with this interpretation, it is clear that only small amounts of Tral protein are required for some of its functions. For instance, tral¹ homozygotes display no defect in Yl trafficking, but tral¹ hemizygotes do, even though tral¹ homozygous or hemizygous ovaries express undetectable amounts of Tral. The identification of stronger alleles of tral will likely be necessary to determine whether the role of tral in cytokinesis is conserved between Drosophila and C. elegans (Wilhelm, 2005).

One of the most unexpected results of these studies was the identification of Tral as a component of an RNA-protein complex that is required for efficient membrane trafficking. Because cup and me31B have been implicated in various aspects of mRNA localization, mRNA stability, and translational control, this finding provided an unexpected link between cytoplasmic mRNA processing and the secretory pathway. While previous studies of cup and me31B did not describe any defect in secretion, such a role would likely have been missed, since the strongest alleles of cup and me31B cause egg chamber degeneration before either Grk or Yl secretion could be examined. Because the Tral complex localizes to the ER and sar1 mRNA is associated with Tral, it is proposed that Tral promotes proper ER exit site formation by local regulation of sar1 expression on the ER (Wilhelm, 2005).

The idea of a link between ER function and cytoplasmic mRNA processing is consistent with recent work in a number of other systems. In rice endosperm, prolamine mRNA, which encodes a storage protein used to support growth of the developing embryo, is targeted to discrete domains of the endoplasmic reticulum via its 3'UTR (Choi, 2000). This targeting is believed to be necessary for the formation of a specialized protein storage body derived from the ER. In Saccharomyces cerevisiae, the mRNA for the membrane protein Ist2p is localized to the cortical ER of daughter cells and this localization event allows Ist2p to be transported to the plasma membrane independent of the classic secretory pathway (Juschke, 2004). Work on tral suggests that the connection between cytoplasmic mRNA processing and ER function may be broader than these two isolated examples and that cytoplasmic mRNA processing regulates exit from the ER in addition to its previously established role in the translocation of prolamine and Ist2p into the ER. These findings raise exciting new possibilities for how cytoplasmic mRNA processing could interface with the classical secretory pathway to promote efficient protein trafficking in the cell (Wilhelm, 2005).

Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition

The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. A previous paper (Hara, 2017), demonstrated PNG activity is under tight developmental control and restricted to this transition. This study's examination of PNG specificity showed it to be a Thr-kinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and PUM was shown to be a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors (Hara, 2018).

As an initial approach to identify substrates for the PNG kinase, predicted to be a Ser/Thr kinase, attempts were made to determine whether PNG phosphorylation occurs at consensus sequences. A positional scanning peptide library was treated with active PNG kinase complex or a complex with catalytically inactive PNG (KD: kinase dead) purified from Sf9 cells. Peptides were robustly phosphorylated by the active PNG kinase complex in contrast to the kinase-dead control. PNG exhibited a strong preference to phosphorylate threonine, because peptides whose phospho-acceptor site was fixed with threonine were strongly phosphorylated, whereas serine peptides were phosphorylated at reduced levels. Although no strong consensus sequence was identified, PNG was most strongly selective for hydrophobic amino acids at -3 relative to the phosphorylated residue, and it had some preferences for aromatic residues at position -2 and for arginine at position +2. Increased phosphorylation of peptides with threonine present outside of the intended phospho-acceptor position was likely an artifact resulting from the presence of two potential phosphorylation sites (Hara, 2018).

The peptide arrays did not yield a consensus sequence for PNG of sufficient specificity to be used to identify putative substrates. Because of the limitations of this approach, an unbiased biochemical screen was carried out. Purified recombinant PNG kinase was used to thio-phosphorylate substrates in embryonic extracts, identifying them by recovery of thio-phosphorylated peptides by mass spectrometry. A pilot screen was done with wild-type PNG kinase and 45 proteins were phosphorylated. A second screen was done in which extracts were treated in parallel with wild-type and kinase-dead PNG. In this second screen, the total representation of peptides in the extract was quantified by doing mass spec analysis of the peptides that did not bind to iodoacetyl agarose. In the second experiment, 36 proteins had at least two independent peptides phosphorylated by wild-type but not kinase-dead PNG. These included 27 of the proteins identified in the pilot experiment (Hara, 2018).

A high representation of phosphopeptides was recovered for the translational repressor Trailer Hitch (TRAL) with wild-type but not kinase-dead PNG. Other phosphorylated proteins were ribosomal proteins and translation factors, as well as the PLU activating subunit of the PNG complex. Out of 36 substrates identified, 19 were proteins known to be involved in mRNA translation (Hara, 2018).

79% of the identified unique peptides had threonine as the phospho-acceptor residue. The identified peptides showed an enrichment of hydrophobic residues at -3 position as in the peptide library, confirming that PNG tends to phosphorylate threonine three residues downstream of a hydrophobic amino acid. The threonine preference was also highly significant in the context of the Drosophila proteome. The correspondence with the peptide sequence preference of PNG is further confirmation that the observed phosphopeptides likely reflect direct phosphorylation by PNG. Although the substrates don't reveal a strong PNG consensus sequence, it is possible that interaction between substrates and the PLU or GNU activating subunits may provide specificity beyond that at the phosphorylation site (Hara, 2018).

Focus was placed on TRAL, because although there were many more abundant proteins in the extracts, a high number of PNG-phosphorylated peptides for TRAL were recovered. TRAL is a member of the (L)Sm protein family composed of RAP55 in vertebrates, CAR1 in C. elegans, and Sdc6 in yeast. Tests were performed to see whether PNG can phosphorylate TRAL in vitro. A powerful aspect of the thio-phosphate substrate screen is that the MS analysis identifies the phosphorylated amino acids. 15 amino acids (13 of them threonine), clustered in the C-terminal half of the protein, were phosphorylated by PNG in embryonic extracts. MBP fusions of purified full length TRAL, or the N- and C- terminal fragments were incubated with purified PNG and [γ 32P]-ATP and analyzed by autoradiography. The full-length protein and the C-terminal half, but not the N-terminal half, were phosphorylated by PNG in vitro. To determine whether PNG-dependent phosphorylation required the amino acids identified in the substrate screens, all 15 were changed to alanine. For both the full-length protein and the C-terminal half, the level of phosphorylation by PNG was reduced with the alanine-substituted forms. Residual phosphorylation of the alanine-substituted form of TRAL raises the possibility that there are other potential PNG phosphorylation sites in the C-terminus of TRAL that were not detected in the screen (Hara, 2018).

Whether phosphorylation of TRAL by PNG inhibits its activity was tested. RAP55 from Xenopus and Sdc6 from yeast are able to inhibit translation in vitro in yeast apparently by blocking the function of the eIF4G subunit of the eIF4F initiation factor. This study examined translation of an mRNA encoding Myc-tagged GFP in reticulocyte lysates and found that as for other family members, addition of Drosophila TRAL inhibited translation. Because in the in vitro reaction purified PNG does not phosphorylate TRAL to full stoichiometry, the effect of PNG phosphorylation was evaluated by generating a phosphomimetic form of TRAL in which aspartic acid was substituted for the fifteen PNG phosphorylation sites. Strikingly, the phosphomimetic mutations suppressed the translational repression by TRAL. The potential existence of additional PNG phosphorylation sites in the C-terminus of TRAL could account for why suppression of translational repression by the phosphomimetic form of TRAL was not complete. In contrast, TRAL in which these residues were replaced by alanine still inhibited translation of the reporter mRNA in the extracts. These results are consistent with phosphorylation of TRAL by PNG relieving its ability to repress translation. Together these results support the conclusion that TRAL is a PNG substrate, but they reveal that TRAL phosphorylation is developmentally dynamic and involves several kinases (Hara, 2018).

Therefore, genetic interactions were sought between png and tral mutants. The png gene was identified because mutant females produce eggs that complete meiosis but subsequently fail to initiate mitotic divisions. Nevertheless, DNA replication continues, resulting in embryos with giant, polyploid nuclei. In strong alleles of png there is no mitosis, whereas weaker alleles permit a few mitotic divisions but these nuclei ultimately also become polyploid. The absence of mitosis in png mutants is due to a failure to promote cyclin B mRNA translation at egg activation. Removal of one copy of some genes (such as the translational repressor pum,) can suppress the giant-nuclei png phenotype, resulting in embryos that undergo more mitotic divisions and thus have more nuclei. If the gene acts downstream of png, this suppression is consistent with png acting negatively on the gene. In contrast, removal of one copy of a gene such as cyclin B enhances the png phenotype, consistent with png having a positive effect on this gene (Hara, 2018).

A comparison was made of embryos laid by females with png¹⁰⁵⁸/png³³¹⁸ with one copy of tral mutated to sibling controls solely mutant for png. Reducing the dosage of tral (a heterozygous tral1 mutation, which has a P element insertion) suppressed the png phenotype, permitting additional mitoses and increased numbers of nuclei. This suppression is even more pronounced with a deletion that completely removes the tral gene. These genetic epistasis results complement the in vitro translation results with the phosphomimetic TRAL form. They are consistent with TRAL being a target of PNG and phosphorylation negatively affecting TRAL (Hara, 2018).

To test whether the genetic interactions between tral and png affect cyclin B mRNA translation, protein levels were examined by immunoblotting of extracts from the mutant and control embryos. Strikingly, Cyclin B protein levels were increased in the png transheterozygous embryos when the dosage of tral was reduced. Consistent with the suppression phenotypes, the amount of Cyclin B was restored more with the deletion than with the tral1 allele. Cyclin A, another PNG translational target, also was increased with reduced TRAL (Hara, 2018).

Taken together, the in vitro and in vivo phosphorylation results and the genetic interaction data indicate that phosphorylation of TRAL by PNG blocks its repressive effects on translation, permitting translation of cyclin B at egg activation to permit embryonic mitoses. This could be due to PNG phosphorylation directly repressing TRAL function or via an effect of phosphorylation on the localization of TRAL. TRAL is present in large cytoplasmic RNP granules in mature oocytes in both Drosophila and C. elegans, and these disperse on egg activation. Thus, one model for the effect of PNG on TRAL is that phosphorylation could affect the localization of TRAL to RNP granules. These large visible granules were examined using a GFP-Tral FlyTrap line with or without png mutations and following TRAL localization during in vitro egg activation. Early in activation, by about 10 min, TRAL granules became diminished. In png mutant eggs, the TRAL granules also disappeared with normal timing. It is concluded that PNG does not appear to be involved in this reorganization of TRAL granules. Indeed, dispersal of TRAL from granules occurs prior to when PNG becomes active at 30 min after egg activation. PNG phosphorylation may more directly affect the ability of TRAL to inhibit translation initiation, as indicated by the effect of the phosphomimetic form on translation in reticulocyte lysates (Hara, 2018).

Given the hundreds of mRNAs whose regulation at egg activation is dependent on PNG, it seemed probable that PNG affects translation through multiple mechanisms and may have multiple substrate targets. Previous work showed that the translational repressor pumilio (pum) dominantly suppresses png; a heterozygous mutation of pum restores both Cyclin B protein levels and mitosis in png mutant embryos. Even PUM nonphosphorylated peptides were not recovered in the substrate screen, therefore, the possibility of PUM being a PNG substrate could not be evaluated. Consequently, a direct interaction between png and pum was tested by asking whether PNG can phosphorylate PUM in vitro. A GST-PUM fusion protein is phosphorylated by purified wild-type PNG kinase but not by the kinase-dead form (Hara, 2018).

The ME31B RNA helicase acts as a translational repressor and is a binding partner to TRAL. The helicase was not recovered above the cut off in the substrate screen, although one ME31B phosphopeptide was present in the wild-type but not kinase-dead PNG sample. Given its interaction with TRAL, ME31B was directly tested in vitro, and PNG was able to phosphorylate it. Thus, PNG phosphorylation may affect both of these conserved proteins and their role as a complex in controlling translation (Hara, 2018).

Another translational regulator that is a potential PNG substrate is BICC. BICC binds to the GNU subunit of the PNG complex directly through its SAM domain, and BICC also is known to physically interact with TRAL. BICC was not, however, recovered from the substrate screen. Despite this, PNG readily phosphorylates BICC in vitro (Hara, 2018).

These results raise the possibility that PNG acts on a number of translational repressors. The two PNG substrate screens likely were not saturating to identify all potential translational repressor targets. The translational repressors Cup and Caprin were recovered in the first substrate screen but not by this study's criteria in the second. The dominant genetic suppression of png observed with mutation of tral or pum generates the hypothesis that PNG may inactivate multiple translational repressors by phosphorylation to promote translation of different sets of mRNAs at egg activation. It is also possible that PNG's effect on multiple repressors may target a single set of mRNAs localized to RNP granules. For example, ME31B is bound to TRAL. BicC genetically interacts with tral, the protein appears to localize to the RNP granules in which TRAL and ME31B reside, and it binds to GNU. From these observations, PNG might phosphorylate multiple targets on RNP granules to de-repress translational inhibition of maternal mRNAs at egg activation (Hara, 2018).

In addition to its effects at egg activation, PNG may indirectly affect translational repressors later in embryogenesis, at a developmental time when PNG appears to be inactivated. In the embryo the TRAL, ME31B, and Cup proteins form an inhibitory complex that represses the translation of maternal mRNAs. These proteins have been shown to be degraded during the maternal-to-zygotic transition, and functional PNG is a prerequisite for this degradation (Hara, 2018).

Previous work has shown that the PNG kinase is activated by a signal downstream of egg activation and thus controls massive changes in maternal mRNA translation. This study has now found that TRAL is a PNG substrate using a biochemical screen. Phosphorylation by PNG suppressed TRAL's ability to repress mRNA translation. This antagonism also was supported by genetic interaction between png and tral in fertilized embryos, suggesting that TRAL phosphorylation by PNG during the oocyte-to-embryo transition is a key to remodel maternal mRNAs' translation activity (Hara, 2018).

The PNG kinase functions as a signal transducer for the external egg activation signal to mRNA translation in the cytoplasm in the activated eggs. Similar strategies can be used in oocyte maturation, during which a hormonal signal leads to phosphorylation of translational regulators to control mRNA translation. In neurons, stimuli cause translocation of mRNA followed by translational activation. Understanding signaling pathways that transmit extracellular signals to translational controls thus is likely to provide insight into molecular mechanisms in fertility as well as synaptic plasticity and memory (Hara, 2018).

The E2 Marie Kondo and the CTLH E3 ligase clear deposited RNA binding proteins during the maternal-to-zygotic transition

The maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal to the zygotic genome. Although the MZT is typically considered from its impact on the transcriptome, previous work found that three maternally deposited Drosophila RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. This study shows that these proteins are degraded by the ubiquitin-proteasome system. Marie Kondo, an E2 conjugating enzyme (FlyBase: Ubiquitin conjugating enzyme E2H), and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Structure modeling of the Drosophila CTLH complex suggests that substrate recognition is different than orthologous complexes. Despite occurring hours earlier, egg activation mediates clearance of these proteins through the Pan Gu kinase, which stimulates translation of Kdo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT (Zavortink, 2020).

Proper embryogenesis is critical for animal development. Many of the earliest events occur prior to the onset of zygotic transcription, and they are instead directed by maternally deposited proteins and messenger RNAs (mRNAs). During the maternal-to-zygotic transition (MZT), genetic control of developmental events changes from these maternally loaded gene products to newly made zygotic ones. Thus, the MZT requires both the activation of zygotic transcription and clearance of maternal transcripts. Failure to mediate either of these processes is lethal for the embryo (Zavortink, 2020).

In contrast to understanding of the transcriptome during the MZT, much less is known about changes in the proteome. Despite the fact that the maternal dowry of proteins plays key roles in embryogenesis, there are only a handful of examples of cleared maternal proteins. Recently, three RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) were found to be rapidly degraded during the MZT in Drosophila melanogaster, at a time point coinciding with the major wave of zygotic transcription. ME31B, TRAL, and Cup form a complex that blocks translation initiation. All three proteins are required for oogenesis, and they appear to bind and repress thousands of deposited maternal mRNAs. The degradation of ME31B, TRAL, and Cup coincides with many of the hallmarks of the MZT, but explorations into this issue have been hindered by a lack of understanding of how their destruction is controlled (Zavortink, 2020).

An intriguing observation has been made that genetically linked the clearance of ME31B, TRAL, and Cup, to the Pan Gu (PNG) kinase (Wang, 2017). Composed of three subunits (PNG, Giant Nuclei [GNU], and Plutonium [PLU]), the PNG kinase is central to the oocyte-to-egg transition and mediates key aspects of embryogenesis, including resumption of the cell cycle, zygotic transcription, and maternal mRNA clearance. Unlike many animals, the oocyte-to-egg transition in Drosophila does not require fertilization but is instead triggered by egg activation. The PNG kinase is activated by mechanical stress as the oocyte passes through the oviduct, and then phosphorylation and degradation of the GNU subunit quickly inactivates the kinase, restricting its activity to the first half hour after egg activation (Hara, 2017). One way that PNG mediates the oocyte-to-embryo transition is by rewiring post-transcriptional gene regulation. Possibly by phosphorylating key RNA-binding proteins such as Pumilio, PNG activity leads to changes in the poly(A)-tail length and translation of thousands of transcripts during egg activation (Hara, 2018). Importantly, two targets induced by PNG activity are the pioneer transcription factor Zelda, which is responsible for initial zygotic transcription, and the RNA-binding protein Smaug, which is responsible for clearance of many maternal transcripts. The PNG kinase also phosphorylates ME31B, Cup, and TRAL (Hara, 2018), but it is unclear what effect phosphorylation has on these proteins. One possibility has been that PNG phosphorylation could lead to the degradation of ME31B, TRAL, and Cup, but this model has been thus far unexplored (Zavortink, 2020).

The ubiquitin-proteasome system is a major protein degradation pathway. A series of ubiquitin activating enzymes, conjugating enzymes, and ligases (E1, E2, and E3, respectively) lead to the post-translational addition of a polyubiquitin chain on a target protein, which then serves as a molecular beacon for degradation by the proteasome. E3 ligases are typically thought to recognize target proteins, while E2 conjugating enzymes provide the activated ubiquitin and in turn recognize the E3 ligase. There are hundreds of different E3 ligases and 29 annotated E2 conjugating enzymes in Drosophila, but most of the client substrates are unknown, and few have been implicated in the MZT (Zavortink, 2020).

Given the key roles of ME31B, Cup, and TRAL in oogenesis and embryogenesis, it was of interest to understand the mechanisms controlling their degradation. In particular, this study sought to answer how PNG activity at egg activation leads to the degradation of these three RNA-binding proteins several hours later, and how their degradation is coordinated with other elements of the MZT, including zygotic transcription and maternal mRNA clearance. To answer these questions, a selective RNAi screen was performed in Drosophila, and the E2 conjugating enzyme was identified as UBC-E2H/Marie Kondo (Kdo) and the E3 ligase as the CTLH complex. Interestingly, structural models based on the S. cerevisiae complex (Qiao, 2020) suggest that the Drosophila version is organized differently than its orthologous complexes. The CTLH complex recognized and bound ME31B and Cup even in the absence of PNG activity, strongly suggesting that phosphorylation is not required for the destruction of these proteins. In contrast, Kdo mRNA is translationally upregulated by more than 20-fold upon egg activation in a PNG-dependent manner. Thus, egg activation through PNG mediates translation upregulation of Kdo and so leads to ME31B, Cup, and TRAL destruction (Zavortink, 2020).

Kdo is conserved from yeast to humans and is known to work through the CTLH E3 ligase, a multicomponent complex. (Note that the S. cerevisiae complex is called the Gid complex.) Using BLAST for the human CTLH components, it was easy to identify putative D. melanogaster homologs: RanBPM (homologous to Hs RanBP9), Muskelin, CG3295 (homologous to Hs RMND5A/GID2), CG7611 (homologous to Hs WDR26), CG6617 (homologous to Hs TWA1/GID8), and CG31357 (homologous to Hs MAEA). Putative homologs for Hs GID5/ARMC8 or Hs GID4 were not found. Notably, none of these genes were annotated as putative E3 components in FlyBase, and thus none were included in an original screen (Zavortink, 2020).

ME31B, Cup, and TRAL are RNA-binding proteins that are degraded during the MZT. Despite occurring several hours after egg laying, degradation of these proteins is triggered by egg activation through the activity of the PNG kinase and appears to be mediated by the ubiquitin-proteasome system. Through a medium-scale RNAi screen, the E2 conjugating enzyme Kdo was identified as being required for the clearance of ME31B, TRAL, and Cup. Kdo is conserved from yeast to humans and, as in those systems, appears to work with the CTLH complex, which acts as the E3 ligase. Components of the CTLH complex physically interact with ME31B and Cup, and the CTLH complex is also required for the degradation of ME31B, TRAL, and Cup during early embryogenesis. Structure-based homology suggests that, despite its conservation from yeast to humans, the Drosophila CTLH complex has an unusual architecture, and it remains unclear how it recognizes its substrate. The association of CTLH with ME31B occurs in the absence of PNG activity, suggesting that, although ME31B (as well as TRAL and Cup) are phosphorylated by the kinase, phosphorylation may not be required for their destruction. Instead, translation of Kdo appears to be suppressed during oogenesis by its short poly(A) tail length and binding of ME31B. Its translation is dramatically upregulated at the oocyte-to-embryo transition, in a process that depends on PNG activity. Together, these data suggest a model that egg activation via the PNG kinase leads to translational activation and production of Kdo, which then allows the CTLH complex to ubiquitinate ME31B, TRAL, and Cup and ultimately leads to their destruction. Interestingly, based on RNA-seq data from FlyBase, Muskelin shows exquisite tissue-specificity and is only strongly expressed in the ovaries. This observation, together with the translational control of Kdo, may partly explain how ME31B, a ubiquitous protein, is specifically destabilized in the early embryo (Zavortink, 2020).

Although the CTLH complex is conserved, it has not yet been studied in Drosophila. The data point to this complex being composed of multiple components (Muskelin, RanBPM, Houki, Souji, and Katazuke), as in other organisms. However, due to a lack of available reagents, the stoichiometry of these components is unknown, and it remains possible that there are additional, Drosophila-specific components. Nonetheless, so far, the CTLH complex in Drosophila appears different than the human and yeast complexes. Although Gid7 and WDR26 are important in the yeast and human versions, respectively, and a Drosophila ortholog (CG7611) was identified, no evidence was found of its association with ME31B or requirement for ME31B degradation; the role of CG7611 in the Drosophila CTLH complex warrants further investigation. Orthologs of Gid4 and Gid5, which are critical for substrate recognition in S. cerevisiae, were not found. Intriguingly, the residues and domains important for the Gid1-Gid4 and Gid8-Gid5 interactions in budding yeast appear absent to be in RanBPM and Hou, raising the fundamental question of how the Drosophila CTLH complex recognizes and positions its substrate proteins. Answering this question will require future investigation and may shed light on other proteins targeted by the Drosophila CTLH complex and the extent to which ME31B, a ubiquitously expressed protein, is targeted outside of the MZT (Zavortink, 2020).

One unexpected result is the role of PNG in mediating the destruction of ME31B, TRAL, and Cup. PNG phosphorylates all three proteins, and so the initial hypothesis was that this modification also stimulated their destruction. However, contrary to expectations, ME31B and Cup interacted with the CTLH complex even in png50 embryos, demonstrating that phosphorylation by PNG was not required for binding of ME31B and Cup by the E3 ligase. An unresolved question, then, is how PNG phosphorylation affects the activities of ME31B, TRAL, and Cup. Intriguing observations from the Orr-Weaver lab suggest that the modification can impact the ability of these proteins to repress gene expression (Hara, 2018). It is tempting to speculate that phosphorylation may then contribute to the MZT by modulating the activities of ME31B, TRAL, and Cup, rather than their stability (Zavortink, 2020).

The link between PNG and the destruction of ME31B, TRAL, and Cup instead appears to be mediated through the translational upregulation of Kdo. Although PNG may contribute through other, as-yet undiscovered, mechanisms as well (such as phosphorylating unknown CTLH adaptor proteins), this link is sufficient to explain the PNG requirement for ME31B degradation: in the absence of Kdo, ME31B is stable during the MZT, and in the absence of PNG, Kdo is not detectably expressed. An important question for the future will be to understand what elements in the Kdo mRNA are responsible for its translational repression during oogenesis. One hint may be that the 3'UTR of Kdo contains several putative Pumilio-binding sites, and translation of Kdo is upregulated in ovaries where Pumilio has been knocked down. Pumilio is also a target of PNG (Hara, 2018), and so a possible model is that translational repressors, such as Pumilio, are phosphorylated and inactivated at egg activation, leading to the production of Kdo (Zavortink, 2020).

PNG also mediates the translational upregulation of key MZT effectors: Zelda, the pioneer transcription factor, and Smaug, an RNA-binding protein that targets nearly two-thirds of the maternal transcriptome for degradation. Together with the current results, a picture is emerging that egg activation stimulates the production of multiple key factors that are important for clearing the maternal RNA and protein dowry and for producing zygotic gene products (Zavortink, 2020).

Although the MZT has typically been considered from the perspective of RNA, a role for maternal protein clearance is becoming clearer. Over the past few years, the list of proteins degraded during the Drosophila MZT has grown and now includes GNU, Matrimony, Cort, Smaug, ME31B, TRAL, and Cup. Unbiased mass spectrometry experiments also suggest that Wispy and Dhd are also robustly degraded. As this list of proteins in Drosophila and other developmental systems increases, a new question is emerging: how many maternally deposited proteins are degraded during the MZT? Understanding the mechanisms controlling protein degradation during the MZT as well as the impact of removing the maternal protein dowry will be key issues to explore in the future (Zavortink, 2020).

Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. The maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster's genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT (Cao, 2020).

This study has shown that, in Drosophila, an extremely small subset of its maternal proteome is cleared during the MZT. This contrasts with the massive degradation of the maternal mRNA transcriptome that occurs during the MZT of all animals. Previous studies in other animals have suggested that the maternal proteome may behave very differently from the maternal transcriptome during the MZT. For example, in C. elegans, a quarter of the transcriptome is downregulated, whereas only 5% of the proteome shows a similar decrease. In frog embryos, there is also a discordance between the temporal patterns of protein and mRNA (Cao, 2020).

The set of proteins cleared during the Drosophila MZT is enriched for RNP granule components. This is consistent with the importance of post-transcriptional processes during the first ('maternal') phase of the MZT and the possible need to downregulate these processes upon ZGA and the switch to zygotic control of development. By focusing on a subset of these RNP components, which function as post-transcriptional repressors, this study has uncovered precise temporal control of their clearance by two distinct E3 ubiquitin ligase complexes: the SCF E3 ligase governs the degradation of SMG, whereas the CTLH E3 ligase is responsible for the degradation of Cup, TRAL, and ME31B. Intriguingly, SMG is degraded later during the MZT compared with its co-repressors Cup, TRAL, and ME31B. This study also showed that clearance of SMG is essential for appropriate levels of re-expression of a subset of its targets during ZGA. The results raise questions about how temporal specificity of protein degradation is regulated, as well as why at least two temporally distinct mechanisms of protein degradation exist during the MZT (Cao, 2020).

Expression data support the hypothesis that timing of E3 ligase function might, at least in part, be determined by the timing of expression of one or more of their component subunits, notably Muskelin for CTLH and CG14317 for SCF. During the Drosophila MZT, most components of the CTLH complex display constant expression levels, but Muskelin protein is degraded with a similar profile to its target repressors. Mammalian Muskelin has been shown to be auto-ubiquitinated and targeted for degradation. Detection of a ubiquitinated peptide in Muskelin supports the possibility that the Drosophila CTLH complex may be negatively autoregulated through its Muskelin subunit during the MZT. In contrast, activation of CTLH function at the beginning of the MZT may not depend on changes in complex composition: previous studies have shown that going from stage 14 oocytes to activated eggs or early (0-1 h) embryos, there are no significant changes in either the levels of CTLH subunit proteins (including Muskelin) or the ribosome association of their cognate transcripts. Thus, it is speculated that post-translational modification of one or more CTLH subunits may activate CTLH function (Cao, 2020).

Modification of substrates may also play a role: the degradation of Cup, TRAL, and ME31B depends on the PNG kinase, which itself has temporally restricted activity coinciding with degradation of these repressors. PNG-dependent phosphorylation of Cup, TRAL, and ME31B may make them ubiquitination substrates. Furthermore, evidence suggests that temporal regulation of the E2 ubiquitin-conjugating enzyme, UBC-E2H, at this stage depends on the PNG kinase and may also contribute to the timing of ubiquitin ligase complex function during the MZT. Concomitant PNG-dependent activation of the CTLH complex, its cognate E2, and its substrates, coupled with subsequent self-inactivation of the complex through Muskelin degradation, would provide a precise time window for CTLH function and, therefore, for degradation of Cup, TRAL, and ME31B early in the MZT (Cao, 2020).

In contrast with these three co-repressors, degradation of SMG occurs near the end of the MZT and depends on zygotic gene expression. Although the levels of most SCF complex subunits are constant during the MZT, the F-box protein CG14317 displays a unique expression pattern: CG14317 protein and mRNA are absent at the beginning of the MZT, are zygotically synthesized, peak in NC14 embryos, and sharply decline shortly thereafter. Thus, CG14317 expression coincides with the timing of SMG protein degradation and, coupled with the zygotic nature of its accumulation, makes it a strong candidate to be a timer for SCF function. The fact that knockdown of SLMB stabilizes SMG protein suggests that both F-box proteins may be necessary for SMG degradation, with CG14317 serving as the timer. At present there are no forward or reverse genetic reagents available to test this hypothesis. Additionally, the function of SLMB in directing SMG-protein ubiquitination may itself be temporally restricted. Both Drosophila SLMB and its mammalian homolog are known to bind phosphorylated motifs. Phosphorylated residues have been detected in SMG in the embryo, including residues within its C terminus; one of these, S967, resides close to a ubiquitinated lysine, K965. In summary, despite the stable expression of SLMB during the MZT, temporal regulation of phosphorylation of its target proteins, including SMG, through yet uncharacterized mechanisms, may also contribute to temporal control of SMG protein degradation (Cao, 2020).

Because Cup, TRAL, and ME31B are known to function as co-repressors in a complex with SMG, why are the timing of degradation of Cup-TRAL-ME31B and SMG differentially regulated? Although the SMG-Cup-TRAL-ME31B-mRNA complex has been characterized to be extremely stable in vitro, it would be disrupted in vivo by the degradation of Cup, TRAL, and ME31B (or by the degradation of nos and other SMG-target mRNAs). SMG directs translational repression both through AGO1 and through Cup-TRAL-ME31B, as well as transcript degradation through recruitment of the CCR4-NOT deadenylase. CTLH-driven degradation of Cup, TRAL, and ME31B would abrogate SMG-Cup-TRAL-ME31B-dependent translational repression, but not AGO1-dependent repression, because AGO1 levels increase during the MZT. However, the relative contributions of AGO1 versus Cup-TRAL-ME31B to translational repression by SMG are unknown. That said, the CCR4-NOT deadenylase is present both during and after the MZT (Temme et al., 2004); thus, SMG-dependent transcript degradation would occur both before and after clearance of Cup, TRAL, and ME31B. 12% of SMG-associated transcripts are degraded, but not repressed, by SMG. Perhaps this subset is bound and degraded by SMG late in the MZT, after the drop in Cup, TRAL, and ME31B levels (Cao, 2020).

Another possible role for clearance of ME31B and TRAL derives from studies in budding yeast, where it has been shown that their orthologs, respectively, Dhh1p and Scd6p, have a potent inhibitory effect on 'general' translation. If this is also true in Drosophila, then degradation of ME31B and TRAL, which are present at exceedingly high concentrations in embryos, might also serve to permit high-level translation during the second phase of the MZT (Cao, 2020).

Previous work has shown that SMG has both direct and indirect roles in the MZT. SMG's direct role is to bind to a large number of maternal mRNA species and target them for repression and/or degradation. Two indirect effects have been shown in smg mutants. First, if maternal transcripts fail to be degraded and/or repressed, ZGA fails or is significantly delayed, likely because mRNAs encoding transcriptional repressors persist. Second, because zygotically synthesized microRNAs direct a second wave of maternal mRNA decay during the late MZT, in smg mutants, failure to produce those microRNAs results in failure to eliminate a second set of maternal transcripts late in the MZT (Cao, 2020).

This study has uncovered a role for rapid clearance of the SMG protein itself late in the MZT: to permit normal levels of zygotic re-expression of a subset of it targets. Notably, stabilized SMG (SMG767Δ999) rescues both clearance of its maternal targets and ZGA, excluding the possibility that lower-than-normal levels of re-expressed targets are a result of defective SMG function upon deletion of its C terminus. Indeed, in control experiments, SMG's exclusively maternal targets actually dropped to lower levels than normal, likely because SMG767Δ999 continues to direct their decay beyond when SMG normally disappears from embryos. Furthermore, in another control, strictly zygotic transcripts that lack SMG binding sites were expressed at higher levels in SMG767Δ999-rescued mutants than in full-length SMG-rescued mutants. This result is consistent with the hypothesis that clearance of transcriptional repressors by SMG permits ZGA; persistent SMG would clear these repressors to lower levels than normal, hence resulting in higher zygotic expression. The higher-than-normal expression of zygotic transcripts that lack SMG binding sites makes the lower-than-normal levels of SMG's zygotically re-expressed target transcripts by SMG767Δ999 even more striking. Together, these data support a model in which the timing of both SMG synthesis and clearance are important for orderly progression of the MZT (Cao, 2020).

Comparative Proteomics Reveal Me31B's Interactome Dynamics, Expression Regulation, and Assembly Mechanism into Germ Granules during Drosophila Germline Development

Me31B is a protein component of Drosophila germ granules and plays an important role in germline development by interacting with other proteins and RNAs. To understand the dynamic changes that the Me31B interactome undergoes from oogenesis to early embryogenesis, this study characterized the early embryo Me31B interactome and compared it to the known ovary interactome. The two interactomes shared RNA regulation proteins, glycolytic enzymes, and cytoskeleton/motor proteins, but the core germ plasm proteins Vas, Tud, and Aub were significantly decreased in the embryo interactome. Follow-up on two RNA regulations proteins present in both interactomes, Tral and Cup, revealed that they colocalize with Me31B in nuage granules, P-bodies/sponge bodies, and possibly in germ plasm granules. It was further shown that Tral and Cup are both needed for maintaining Me31B protein level and mRNA stability, with Tral's effect being more specific. In addition, evidence is provided that Me31B likely colocalizes and interacts with germ plasm marker Vas in the ovaries and early embryo germ granules. Finally, it was shown that Me31B's localization in germ plasm is likely independent of the Osk-Vas-Tud-Aub germ plasm assembly pathway although its proper enrichment in the germ plasm may still rely on certain conserved germ plasm proteins (McCambridge, 2020).

To summarize, although Me31B's localization to the posterior of an oocyte is likely independent of Osk, Aub, and Dart5, its proper enrichment at the site may still rely on Aub. Together with a previous report that Me31B's localization pattern is not affected in vas and tud mutants, it is speculated that Me31B's localization in a developing oocyte may be independent of the Osk-Vas-Tud-Aub assembly pathway, but its proper enrichment at the posterior germ plasm may still depend on certain conserved germ plasm proteins like Aub. (McCambridge, 2020).

This speculation, together with earlier conclusions in this study, led to the proposal of a hypothetical model for Me31B localization and enrichment process in the germline cells (see Hypothetical model of Me31B localization and enrichment into germ plasm). In this model, Me31B and conserved germ plasm proteins, Osk-Vas-Tud-Aub, exist in distinct granules in the germ plasm, Osk-Vas-Tud-Aub in germ plasm granules and Me31B (possibly associated with Tral and Cup) in separate granules but in close proximity. Me31B granules use an Osk-Vas-Tud-Aub-independent mechanism to localize to the cortex and the posterior of a developing oocyte, then the posteriorly localized Me31B granules interact with the germ plasm granules, which is necessary for proper Me31B granule enrichment in the germ plasm. In the early embryos, Me31B proteins begin to degrade rapidly and become dispersed in the cytoplasm (McCambridge, 2020).

REGULATION

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. This study shows that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity (Eulalio, 2007).

The first proteins found in P bodies are those functioning in the degradation of bulk mRNA. In eukaryotes, this process is initiated by removal of the poly(A) tail by deadenylases. There are several deadenylase complexes in eukaryotes: the PARN2-PARN3 complex is thought to initiate deadenylation, which is then continued by the CAF1-CCR4-NOT complex. Following deadenylation, mRNAs are exonucleolytically digested from their 3' end by the exosome, a multimeric complex with 3'-to-5' exonuclease activity. Alternatively, the cap structure is removed by the decapping enzyme DCP2 after deadenylation, rendering the mRNA susceptible to 5'-to-3' degradation by the major cytoplasmic exonuclease XRN1 (Eulalio, 2007).

Decapping requires the activity of several proteins generically termed decapping coactivators, though they may stimulate decapping by different mechanisms. In the yeast Saccharomyces cerevisiae, these include DCP1, which forms a complex with DCP2 and is required for decapping in vivo, the enhancer of decapping-3 (EDC3 or LSm16), the heptameric LSm1-7 complex, the DExH/D-box RNA helicase 1 (Dhh1, also known as RCK/p54 in mammals), and Pat1, a protein of unknown function that interacts with the LSm1-7 complex, Dhh1, and XRN1. In human cells, DCP1 and DCP2 are part of a multimeric protein complex that includes RCK/p54, EDC3, and Ge-1 (also known as RCD-8 or Hedls), a protein that is absent in S. cerevisiae (Eulalio, 2007).

The decapping enzymes, decapping coactivators, and XRN1 colocalize in P bodies. Additional P-body components in multicellular organisms include the protein RAP55 (also known as LSm14; Drosophila homolog - Trailer hitch), which has a putative role in translation regulation, and GW182, which plays a role in the microRNA (miRNA) pathway (Eulalio, 2007).

The P-body marker GW182 localizes to cytoplasmic foci in Drosophila S2 cells together with the decapping enzyme DCP2 and the decapping coactivator DCP1, suggesting that these foci represent P bodies. To characterize D. melanogaster P bodies further, antibodies were raised to the Drosophla orthologs of two proteins found in human-cell P bodies. These correspond to Ge-1 and Tral (LSm15), which is closely related to human RAP55 (or LSm14) (see Tanaka, 2006). Both antibodies stained the cytoplasm diffusely and also stained discrete cytoplasmic foci with a diameter ranging from 100 nm to 300 nm. The antibody signals are specific, as they are lost in cells in which the cognate proteins were depleted. The foci are present in about 95% of the cell population and are readily detectable because the concentration of Tral or Ge-1 in these foci is significantly higher than that in the surrounding cytoplasm (Eulalio, 2007).

The distribution of green fluorescent protein (GFP)-tagged versions of proteins found in P bodies was examined in yeast and/or human cells. These include DCP1, DCP2, GW182, Me31B (the D. melanogaster ortholog of S. cerevisiae Dhh1 and vertebrate RCK/p54), CG5208 (the D. melanogaster homolog of S. cerevisiae Pat1, referred to as HPat hereafter), and EDC3 (also known as LSm16). All of these proteins formed cytoplasmic foci that costained with the anti-Tral or anti-Ge-1 antibodies. Importantly, the expression of the GFP-tagged proteins did not significantly alter the number and size of endogenous P bodies. Together, these results indicate that the localization of decapping enzymes and decapping coactivators into P bodies is evolutionarily conserved. The localization of GW182 in Drosophila P bodies is in agreement with the proposal that GW-bodies and P bodies overlap, as reported for mammalian cells (Eulalio, 2007).

The localization of proteins implicated in translational regulation was examined in Drosophila oocytes whose corresponding transcripts are detectable in S2 cells, in particular, Smaug and the dsRNA binding protein Staufen. Smaug is a translational repressor that also promotes deadenylation of bound mRNAs by recruiting the CAF1-CCR4-NOT1 complex (Zaessinger, 2006). Both proteins localized to P bodies with endogenous Tral. Strikingly, P bodies increased in size in cells expressing Staufen at high levels but not in cells overexpressing GFP fusions of Smaug, suggesting that Staufen promotes P-body formation. Drosophila Staufen, Tral, DCP1, DCP2, XRN1, and Me31B have also been detected in RNP granules in neuronal cells and/or in oocytes, indicating that P bodies and other RNP granules observed in neuronal cells or during development share common components (Eulalio, 2007).

P-body formation requires nontranslating mRNPs and/or mRNPs undergoing decapping. A conserved feature of P bodies in human and yeast cells is that their formation depends on RNA and is enhanced in cells in which the concentration of nontranslating mRNAs or of mRNAs undergoing decapping increases. These observations indicate that mRNAs must exit the translation cycle to localize to P bodies. In agreement with this, it was observed that Drosophila P bodies decline when cells are treated with RNase A or with cycloheximide (which inhibits translation elongation and stabilizes mRNAs into polysomes). In contrast, P-body sizes increase in cells treated with puromycin, which causes premature polypeptide chain termination and polysome disassembly. Both puromycin and cycloheximide inhibit protein synthesis in S2 cells, as judged by the reduction of F-Luc and R-Luc activities after the treatment of cells transiently expressing these proteins with these drugs (Eulalio, 2007).

The size of Drosophila P bodies also depends on the fraction of mRNAs undergoing decapping, in agreement with the results reported for yeast and human cells. Indeed, blocking mRNA decay at an early stage, for instance, by preventing deadenylation in cells in which NOT1 (a component of the CAF1-CCR4-NOT deadenylase complex) is depleted, leads to the dispersion of P bodies, whereas P bodies are on average more prominent in cells from which DCP2 or XRN1 is depleted (in which decapping and subsequent 5'-to-3' mRNA decay are inhibited) (Eulalio, 2007).

Several lines of evidence show that P bodies do not serve as storage sites for the effectors of posttranscriptional process but are sites where mRNA degradation and silencing can take place. For instance, P-body formation is RNA dependent, and decay intermediates, siRNAs, and miRNAs and their targets are detected in P bodies. Moreover, the size and number of P bodies depends on the fraction of mRNAs undergoing decapping. However, the question of whether mRNA decay and silencing require the environment of microscopic, wild-type P bodies to occur or whether these processes can also occur outside of P bodies in soluble protein complexes remains open. This study shows that formation of large P bodies visible in the light microscope as observed in wild-type cells is not required for several processes associated with P-body components, including NMD, mRNA decay, and RNA-mediated gene silencing (Eulalio, 2007).

The question addressed in this study was whether the environment of macroscopic P bodies is required for posttranscriptional regulation. P bodies are defined as the large cytoplasmic foci visible by light microscopy in wild-type cells. These foci are on average 100 to 300 nm in diameter and are readily detected as bright cytoplasmic dots because the concentration of proteins in these foci is significantly higher than in the surrounding cytoplasm. Nevertheless, most P-body components are also detected diffusely throughout the cytoplasm. For a limited number of examples that have been analyzed, it has been shown that P-body components are not confined to these structures but dynamically exchange with the cytoplasmic pool. Quantitative information regarding the fractionation of P-body components between P bodies and the cytoplasm is still lacking, but given the volume of P bodies relative to that of the cytoplasm, it is likely that the diffuse cytoplasmic fraction is significantly larger. This suggests that posttranscriptional processes are likely to occur and may even be initiated in the diffuse cytoplasm or in soluble protein complexes that aggregate to form P bodies. Whether these processes take place in submicroscopic aggregates or soluble protein complexes in the absence of detectable microscopic P bodies remains to be solved. However, it is considered that aggregates or large multiprotein assemblies that are not detectable by light microscopy cannot be defined as bodies (Eulalio, 2007).

Translation factors or ribosomes are generally not present in P bodies (with the exception of cap binding protein eIF4E), indicating that mRNAs leave the translation cycle prior to entering P bodies. Consistently, releasing mRNAs from polysomes leads to increases in P-body sizes and numbers, whereas the stabilization of mRNAs into polysomes disrupts P bodies. These observations suggest that a critical step in P-body formation is the release of mRNPs from a translationally active state associated with polysomes to a translationally inactive state. This paper has shown that releasing mRNAs from polysomes by puromycin treatment is not sufficient to elicit P-body formation and that functional silencing pathways or proteins generically termed decapping coactivators are required for P-body assembly. These proteins include Me31B (Dhh1 in yeast), HPat (Pat1 in yeast), Ge-1, and the LSm1-7 complex (Eulalio, 2007).

What could be the role of these proteins in P-body formation? Me31B is an RNA helicase which could facilitate rearrangements in mRNP composition upon release from polysomes. The role of HPat is unclear, but the yeast ortholog interacts with Dhh1, XRN1, and the heptameric LSm1-7 complex. Coimmunoprecipitation assays indicate that the interaction between Dhh1 and Pat1 orthologs (i.e., Me31B and HPat) is conserved in Drosophila. Finally, the LSm1-7 complex associates with deadenylated mRNAs and stimulates decapping. Clearly, many details regarding the precise molecular function of these proteins remain to be discovered, but their requirement for P-body assembly indicates that mRNAs that are not actively translated do not enter into P bodies by default: the activity of a defined set of proteins is required. Alternatively, nontranslating mRNAs may enter silencing pathways, and this would also lead to changes in mRNP composition due to the recruitment of Argonaute proteins and binding partners, which include P-body components such as GW182, decapping enzymes, and RCK/p54 (Eulalio, 2007).

Once P-body components are bound to an RNP, P-body formation may then be triggered by protein-protein interactions. Indeed, proteins required for P-body assembly are known to interact to form multimeric protein complexes. Consistently, in addition to the interactions mentioned above, DCP1, DCP2, Ge-1, RCK/p54, and EDC3 form a multimeric protein complex in human cells. The absolute requirement of RNA for P-body formation could be explained if affinities between these proteins increased upon RNA binding. Additionally, proteins like GW182 and Ge-1 are multidomain proteins that could bind more than one RNP simultaneously, bringing into close proximity several components and thus nucleating the formation of P bodies (Eulalio, 2007).

RNAs targeted by silencing pathways nucleate P bodies. In this study, it is shown that both the RNAi and miRNA pathways contribute to the generation of a pool of nontranslating mRNPs and/or of mRNPs committed to decay which are required for P-body formation. Nevertheless, silencing can occur in the absence of microscopic P bodies. The results provide support to previous models proposing that silencing is initiated in the cytoplasm and that the localization of the silencing machinery into P bodies is a consequence, rather than the cause, of silencing (Eulalio, 2007).

An unexpected observation from these studies is that AGO2 and Dicer-2, which function in siRNA-mediated gene silencing in Drosophila, are required for P-body integrity. The role of these proteins in P-body assembly is unlikely to be structural, because P bodies are restored upon puromycin treatment in cells from which AGO2 or Dicer-2 is depleted. The most likely explanation for the requirement of these proteins is, therefore, that silencing by siRNAs also generates RNPs that elicit P-body formation. The requirement for AGO2 could be at least partially explained by the observation that the expression levels of a small subset of endogenous miRNA targets are affected in AGO2-depleted cells, suggesting that some miRNAs may be loaded into AGO2-containing RNA-induced silencing complexes. Furthermore, the AGO1 and AGO2 genes interact, although it is unclear how this interaction affects the activities of these proteins (Eulalio, 2007).

The requirement for Dicer-2 in P-body assembly, however, suggests that endogenous siRNA targets also contribute to P-body formation. Because the levels of dsRNA synthesis from endogenous loci that could provide precursors for the production of endogenous siRNAs are currently unknown, the fraction and origin of transcripts regulated by endogenous siRNAs cannot be estimated. Nonetheless, a possible source of endogenous dsRNAs is the bidirectional transcription of pseudogenes and transposable elements, in agreement with the role of the RNAi pathway as a defense mechanism against RNA viruses and mobile genetic elements (Eulalio, 2007).

The essential role of silencing pathways in P-body formation in Drosophila, and presumably in human cells, raises the question of how P bodies are assembled in S. cerevisiae, which lacks silencing pathways. One possibility is that other posttranscriptional processes generate nontranslating mRNPs required to nucleate P bodies. For instance, the NMD pathway contributes to P-body assembly in yeast cells, because depletion of Upf2 or Upf3 leads to increases in P-body size and number in a Upf1-dependent manner, whereas similar experiments with Drosophila cells do not affect P bodies (Eulalio, 2007).

With the exception of the proteins involved in silencing, the composition of P bodies and the effects of drugs such as cycloheximide and puromycin on P-body size and number are strikingly similar in yeast, Drosophila, and human cells, raising the question of what the role of these structures accounting for their conservation in eukaryotic cells could be. The results show that the environment of microscopic P bodies is not essential for mRNA decay or silencing but do not exclude that the formation of P bodies confers a kinetic advantage. Moreover, the results do not rule out a role for large P bodies in sequestering a specific set of nontranslating mRNPs and reinforcing their repression by shielding them from the translation machinery (Eulalio, 2007).

Finally, the conservation of P bodies may reflect a role for these structures in other cellular processes that is not yet fully appreciated. A role in some steps of retroviral or retrotransposon life cycles is suggested by the localizations of the antiretroviral proteins APOBEC3G and APOBEC3F in human cell P bodies and of the protein and RNA components of the retrovirus-like element Ty3 in yeast P bodies. A link between P bodies and the regulation of retrotransposition would be consistent with the role of RNAi pathways in silencing the expression of transposable elements. Because all known essential P-body components play roles in decapping and/or silencing and proteins playing an exclusively structural role in P-body assembly have not yet been identified, it is currently not possible to evaluate the role of P bodies for cell, tissue, or organism survival (Eulalio, 2007).

Fragile X mental retardation protein controls trailer hitch expression and cleavage furrow formation in Drosophila embryos

During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. This study demonstrates that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition. dFMRP is observed to colocalize with the cytoplasmic RNP body components Maternal expression at 31B (ME31B) and Trailer Hitch (TRAL) in a punctate pattern throughout the cytoplasm of cleavage-stage embryos. Complementary biochemistry demonstrates that dFMRP does not associate with polyribosomes, consistent with their reported exclusion from many cytoplasmic RNP bodies. By using a conditional mutation in small bristles (sbr), which encodes an mRNA nuclear export factor, to disrupt the normal cytoplasmic accumulation of zygotic transcripts at the midblastula transition, the formation of giant dFMRP/TRAL-associated structures was observed, suggesting that dFMRP and TRAL dynamically regulate RNA metabolism at the midblastula transition. Furthermore, dFMRP associates with endogenous tral mRNA and is required for normal TRAL protein expression and localization, revealing it as a previously undescribed target of dFMRP control. It was also shown genetically that tral itself is required for cleavage furrow formation. Together, these data suggest that in cleavage-stage Drosophila embryos, dFMRP affects protein expression by controlling the availability and/or competency of specific transcripts to be translated (Monzo, 2006).

The data suggest that in cleavage-stage Drosophila embryos, dFMRP affects translational initiation of specific mRNA molecules within cytoplasmic RNP bodies by controlling their availability and/or modulating their competency to be translated. dFMRP does not measurably associate with polyribosomes under a wide range of conditions in cleavage-stage Drosophila extracts, similar to results obtained for Drosophila S2 cells, but in contrast to reports in other systems. Instead, dFMRP colocalize and cosediment was observed with TRAL and ME31B, known components of translationally quiescent cytoplasmic RNP bodies. Although dAGO2 cosediments with polyribosomes in cleavage-stage embryo extracts and could directly suppress translational elongation or termination, a similar role for dFMRP is unlikely. In fact, there is no indication that endogenous dFMRP directly interacts with dAGO2 in cleavage-stage Drosophila embryos, in contrast to their observed association in Drosophila S2 cell extracts. This discrepancy could result from a fundamental difference in RNA metabolism between S2 cells and cleavage-stage embryos undergoing the MBT (Monzo, 2006).

tral mRNA represents a previously undescribed in vivo target of dFMRP regulation. Although there is no direct evidence that dFMRP and TRAL form a stable complex in cleavage-stage embryos in vitro, dFMRP activity is clearly required for normal TRAL protein expression in vivo. Mislocalization of TRAL protein but not ME31B in both fmr1^- and sbr^ts148 mutant embryos suggests that a specific functional relationship exists between dFMRP and TRAL. In fmr1^- embryos, TRAL protein levels also are reduced 2-fold, indicating that TRAL does not simply get redistributed into abnormal structures, its rate of synthesis and/or degradation must also be affected. The co-IP of tral mRNA with dFMRP from WT embryo extracts demonstrates that dFMRP and tral mRNA form a stable RNP complex and suggests that dFMRP is involved in tral mRNA metabolism. Although it has not yet been determined whether dFMRP directly binds tral mRNA, this analysis of the tral mRNA sequence, by using the fast RNA motif/pattern searcher RNABOB identified a single G-quartet stem-loop structure within the tral 3' UTR, a motif that FMRP can bind with high affinity. Regardless of whether dFMRP binds tral mRNA directly, dFMRP could control the assembly of a translationally competent tral mRNP complex and/or its localized delivery for translation. The transient association of tral mRNA with cytoplasmic RNP bodies in a translationally quiescent state might be required for dFMRP to promote the assembly of a translationally competent tral mRNP. Alternatively, the restricted translation of tral mRNA, controlled by dFMRP-dependent localized release from cytoplasmic RNP bodies, might promote the normal assembly of a functional TRAL RNP complex. In either case, lower steady-state TRAL protein levels resulting from decreased synthesis and/or increased degradation in fmr1^- embryos could be related to abnormal TRAL RNP complex assembly, observed as large structures by IF. Interestingly, the higher steady-state level of tral mRNA observed in fmr1^- embryo extracts is reminiscent of the increased levels of another dFMRP target, pickpocket mRNA, observed in fmr1^- embryo extracts and may reflect a common feature of dFMRP mRNA processing (Monzo, 2006).

In conclusion, it is believed that a system of cytoplasmic RNP bodies exists in cleavage-stage embryos that associates with maternal and zygotic mRNAs to mediate their degradation or processing for subsequent release for translation during the MBT. A large proportion of these cytoplasmic RNP bodies contain dFMRP. It is likely that the cleavage furrow formation defect observed in fmr1^- mutants is the result of disrupting TRAL function. Indeed, tral^- embryos have a cellularization phenotype that resembles that of fmr1^- embryos. A similar requirement has also been found for the Caenorhabditis elegans homolog of tral, car-1, in cleavage furrow formation. However, as with Fragile X syndrome, it is possible that the altered expression of many targets is responsible for the full fmr1^- cellularization phenotype (Monzo, 2006).

Protein Interactions

In order to better understand the role of Tral in regulating membrane trafficking, the identification of Tral-associated proteins was attempted by immunoprecipitating Tral from Drosophila embryo extract using Tral antibody. By colloidal blue staining, three major bands were found that specifically coimmunoprecipitated with Tral: p147, p70, and p50. Using mass spectrometry, p147 was identified as the eIF4E binding protein Cup, and p70 as poly(A) binding protein (PABP). p50 was found to be a mixture of the RNA binding protein Ypsilon Schactel (Yps) and the RNA helicase Me31B. To confirm the identities of the Tral-associated proteins, Tral was immunoprecipitated from ovarian extracts and immunoblotted for Cup, Yps, and Me31B. Me31B, Yps, and Cup all specifically coimmunoprecipitate with Tral, indicating that these proteins are bona fide components of the complex. Because Me31B, Yps, and Cup have been previously shown to be part of an RNA-protein complex, the ability of each protein to coimmunoprecipitate with Tral was tested in RNase-treated ovarian extracts. It was found that while the association of Tral with Me31B, Yps, and Cup is RNase resistant, the association of Yps with Cup is sensitive to RNase treatment, indicating the presence of RNA in the complex (Wilhelm, 2005).

Previous work has shown that Me31B, Cup, and Yps colocalize in vivo. In order to demonstrate that Tral is part of the Me31B-Cup-Yps complex in vivo, egg chambers were immunoprecipitated for Tral and Me31B as well as Tral and Cup. The particulate staining in nurse cells showed a high degree of overlap for both the Tral/Cup and Tral/Me31B double-labeled egg chambers. Furthermore, the temporal-spatial pattern of Tral localization within the oocyte is identical to that previously described for Cup, Me31B, and Yps. These results, together with the previously demonstrated colocalization of Me31B, Cup, and Yps, indicate that Tral, Cup, Me31B, and Yps all exist as a complex in vivo (Wilhelm, 2005).

Because Tral is present on discrete domains of the ER, it was next asked whether other components of the complex were also present on the ER. Colocalization studies of GFP-KDEL with either Me31B or Cup showed that Me31B and Cup are both present on discrete ER subdomains. This observation, together with the biochemical analysis of the Tral complex, demonstrates that Tral is part of an RNA-protein complex that is associated with the ER (Wilhelm, 2005).

Because mutations in tral have such striking effects on morphology of COPII foci, attempts were made to define the relationship between these foci and components of the Tral complex. Using GFP-Sar1 as a marker for COPII complex formation, it was found that while some COPII sites are not associated with the Tral complex, a number of sites either colocalize with or are bordered by the Tral complex. These observations are highly suggestive of a direct role in regulating exit site function, as recent work has implicated the regions around COPII sites in exit from the ER (Wilhelm, 2005).

In order to explain how an RNA-protein complex might regulate ER exit site function, it was hypothesized that Tral regulates the transcripts for COPII components on the surface of the ER. If this were true, then Tral complexes should contain the messages of COPII components. To test this hypothesis, Tral was immunoprecipitated from ovarian extracts, RNA was isolated from the pellet, and the presence of a variety of transcripts were assayed for by RT-PCR. This experiment showed that the transcripts for the COPII components sar1 and sec13 were enriched in Tral immunoprecipitates, while bcd, grk, and sec23 messages were not. Because Tral is biochemically associated with the sar1 message and mutations in tral cause profound disruption of the distribution of Sar1 protein, sar1 mRNA is a likely regulatory target of the Tral complex (Wilhelm, 2005).

One trivial way that this regulation could occur is by general derepression of a maternal pool of the sar1 message causing overexpression of Sar1 protein and the accumulation of large Sar1 foci. However, the levels of GFP-Sar1 are equivalent in tral¹ heterozygotes and tral¹ hemizygotes. This argues against bulk changes in translation or stability of the sar1 message and suggests that any regulation of the sar1 message is likely to be restricted to a subset of the transcript pool (Wilhelm, 2005).

Similar modes of interaction enable Trailer Hitch and EDC3 to associate with DCP1 and Me31B in distinct protein complexes

Trailer Hitch (Tral or LSm15) and enhancer of decapping-3 (EDC3 or LSm16) are conserved eukaryotic members of the (L)Sm (Sm and Like-Sm) protein family. They have a similar domain organization, characterized by an N-terminal LSm domain and a central FDF motif; however, in Tral, the FDF motif is flanked by regions rich in charged residues, whereas in EDC3 the FDF motif is followed by a YjeF_N domain. This study shows that in Drosophila cells, Tral and EDC3 specifically interact with the decapping activator DCP1 and the DEAD-box helicase Me31B. Nevertheless, only Tral associates with the translational repressor CUP, whereas EDC3 associates with the decapping enzyme DCP2. Like EDC3, Tral interacts with DCP1 and localizes to mRNA processing bodies (P bodies) via the LSm domain. This domain remains monomeric in solution and adopts a divergent Sm fold that lacks the characteristic N-terminal alpha-helix, as determined by nuclear magnetic resonance analyses. Mutational analysis revealed that the structural integrity of the LSm domain is required for Tral both to interact with DCP1 and CUP and to localize to P-bodies. Furthermore, both Tral and EDC3 interact with the C-terminal RecA-like domain of Me31B through their FDF motifs. Together with previous studies, these results show that Tral and EDC3 are structurally related and use a similar mode to associate with common partners in distinct protein complexes (Tritschler, 2008).

This study shows that Tral and EDC3 are structurally related proteins that associate with common partners in distinct protein complexes. The N-terminal domains of Tral and EDC3 adopt a divergent Sm fold that mediates their interaction with the decapping activator DCP1 and is sufficient for P-body targeting. In Tral, this domain also mediates the interaction with the translational regulator CUP. Tral and EDC3 share an additional common partner, the RNA helicase Me31B. Both proteins interact with the C-terminal RecA-like domain of Me31B via their FDF motifs, suggesting that their binding is mutually exclusive. This agrees with the conclusion that Tral and EDC3 function in distinct protein complexes. EDC3-containing complexes are known to play a role in mRNA decapping. The localization of Tral in P bodies, its association with decapping activators, and translational repressors suggest that this protein functions in translational repression and/or mRNA degradation (Tritschler, 2008).

P bodies are cytoplasmic domains that accumulate a variety of proteins involved in mRNA degradation, translational repression, mRNA surveillance, and RNA-mediated gene silencing, together with their mRNA targets. The mechanisms leading to P-body assembly are not fully understood, but several P-body components, including RNA, are required for P-body integrity. In yeast cells, P-body assembly occurs through parallel redundant pathways, requiring either Edc3 or LSm4. In multicellular organisms, several P-body components are required for P-body formation, since depleting them disperses the remaining P-body components throughout the cytoplasm (Tritschler, 2008).

In vertebrates the Tral ortholog RAP55 is among the essential P-body components. In contrast, in Drosophila S2 cells, depleting either Tral or EDC3 does not affect P bodies , suggesting that these proteins are not essential for P-body assembly or that multiple redundant pathways lead to P-body formation. Similarly, CAR-1 is not required for P-granule formation in C. elegans. Still, Tral and EDC3 likely contribute to the assembly of RNP particles. Both proteins have a modular domain organization that allows them to interact with additional P-body components and probably with multiple RNPs, bringing them in close proximity and thereby facilitating the nucleation of P bodies (Tritschler, 2008).

Over the past few years, the number of proteins shown to localize to P bodies has increased dramatically. A question that remains open is what causes these proteins to accumulate into P bodies. Some proteins may passively accumulate in P bodies as components of mRNP complexes. In this case, RNA-binding domains or protein-protein interaction domains likely mediate their localization to P bodies. However, the aggregation of individual mRNPs into large granules detectable by light microscopy requires either (1) multidomain proteins that can bridge more than one mRNP or (2) specific proteins or protein domains that self-aggregate (Tritschler, 2008).

One such domain has been described in S. cerevisiae LSm4. LSm4 consists of a canonical LSm domain followed by a C-terminal extension rich in glutamine and arginine residues (Q/N-rich domain), which is not part of the Sm fold. This extension is required to localize LSm4 to P bodies). LSm4 is partially redundant with Edc3 for P-body assembly in yeast cells, but in cells lacking Edc3, P-body assembly relies on the Q/N-rich extension of LSm4. Indeed, a truncated version of LSm4 lacking the Q/N-rich region cannot sustain P-body assembly in the absence of Edc3 . It was proposed that the Q/N-rich extension of LSm4 has prion-like properties and promotes P-body formation by aggregating with itself or with additional Q/N-rich domains, similar to the assembly mechanisms for Q/N-rich domains in prions (Tritschler, 2008).

In contrast to LSm4, the LSm domains of Tral and EDC3 are sufficient to localize them to P bodies. These domains lack Q/N prion-like features and remain monomeric in solution, indicating that they are not self-aggregating domains. Furthermore, these domains do not bind RNA, suggesting that they accumulate in P bodies through protein-protein interactions. Nevertheless, in EDC3, mutations that disrupt DCP1-binding do not affect P-body localization, suggesting that interactions with additional P-body component(s) drive this protein into P bodies. In Tral, no mutations were identified that reduced its accumulation in P bodies without affecting the folding of the LSm domain (Tritschler, 2008).

RAP55 is the vertebrate ortholog of Tral and was originally identified in the salamander Pleurodeles waltl as a component of cytoplasmic RNP particles containing translationally repressed maternal mRNAs. Orthologous proteins have been described in several eukaryotic species, including C. elegans (CAR-1), X. laevis (xRAP55), and mammals (RAP55). Like Tral, these proteins localize in diverse cytoplasmic RNP granules that share components with P-bodies and serve as storage sites for translationally inactive mRNAs in germ cells. For instance, in young oocytes Tral, xRAP55 and murine RAP55 localize to the Balbiani body, a large organelle aggregate that includes mitochondria, endoplasmic reticulum, germinal granule proteins, and RNAs that become incorporated into germ cells in the developing embryos (Tritschler, 2008).

RNP granules containing Tral, CAR-1, or xRAP55 comprise additional proteins with roles in translational repression and/or mRNA decapping. These include, Me31B and its orthologs (C. elegans CGH-1 and X. laevis Xp54), the Y-box domain-containing proteins (D. melanogaster YPS, C. elegans CEY-2-4, and X. laevis FRGY2), which are major components of maternal RNP granules, as well as the eIF4E-binding proteins CUP and 4E-T. Moreover, the Drosophila fragile-X mental retardation protein (dFMRP), which is involved in translational repression, colocalizes with Tral in RNP-containing granules in both embryos and neuronal cells. Tral and CAR-1-containing granules in germ cells also include DCP1 and additional components of somatic P-bodies (Tritschler, 2008).

The localization of Tral and its orthologs in RNP granules in germ cells and young embryos and their association with proteins involved in translational repression, together with the conservation of these interactions in eukaryotes, suggest that these proteins play a fundamental role in regulating translation of maternal mRNAs during oogenesis and early embryogenesis. In agreement with this, xRAP55 represses translation both in vivo and in vitro. Nevertheless, the precise molecular mechanism by which Tral orthologs exert their regulatory functions remains to be established (Tritschler, 2008).

Depleting or mutating Scd6p, CAR-1, or Tral alters endoplasmic reticulum morphology and causes diverse developmental phenotypes. These phenotypes are likely due to the misregulation of specific mRNAs. However, only few mRNA targets of these proteins are known. It is also not yet clear whether these targets are conserved and whether Tral orthologs recognize specific cis-acting sequence elements on regulated mRNAs (Tritschler, 2008).

CAR-1 and xRAP55 expression is confined to the germ line and early embryos. In contrast, human RAP55 is ubiquitously expressed and localizes to P bodies in somatic cells at rest and in stress granules in cells exposed to heat shock or oxidative stress. Similarly, Tral expression is not confined to the germ line, the protein is detected in S2 cells and in neurons, where it is also a component of neuronal RNP granules and participates in neuronal translation regulation. The localization of human RAP55 and Tral in somatic P bodies and neuronal granules suggests that, in addition to their role in regulating maternal mRNAs, these proteins have acquired more general roles in mRNA metabolism (Tritschler, 2008).

Based on the structural similarity between Tral and EDC3 and their association with common partners, one might anticipate that these proteins represent alternative subunits in the assembly of DCP1- and Me31B-containing complexes. These complexes may perform dual and partially overlapping functions; they may repress translation (e.g., in oocytes) or enhance decapping (e.g., in somatic cells), depending on the additional partners with which they associate (Tritschler, 2008).

PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition

The nuage is a germline-specific perinuclear structure that remains functionally elusive. Recently, the nuage in Drosophila was shown to contain two of the three PIWI proteins - Aubergine and Argonaute 3 (AGO3) - that are essential for germline development. The PIWI proteins bind to PIWI-interacting RNAs (piRNAs) and function in epigenetic regulation and transposon control. This study reports a novel nuage component, PAPI (Partner of PIWIs), that contains a TUDOR domain and interacts with all three PIWI proteins via symmetrically dimethylated arginine residues in their N-terminal domain. In adult ovaries, PAPI is mainly cytoplasmic and enriched in the nuage, where it partially colocalizes with AGO3. The localization of PAPI to the nuage does not require the arginine methyltransferase dPRMT5 or AGO3. However, AGO3 is largely delocalized from the nuage and becomes destabilized in the absence of PAPI or dPRMT5, indicating that PAPI recruits PIWI proteins to the nuage to assemble piRNA pathway components. As expected, papi deficiency leads to transposon activation, phenocopying piRNA mutants. This further suggests that PAPI is involved in the piRNA pathway for transposon silencing. Moreover, AGO3 and PAPI associate with the P body component TRAL/ME31B complex in the nuage and transposon activation is observed in tral mutant ovaries. This suggests a physical and functional interaction in the nuage between the piRNA pathway components and the mRNA-degrading P-body components in transposon silencing. Overall, this study reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway (Liu, 2011).

Although the nuage has long been discovered in the germline of diverse organisms, little is known about its function. In this study identified and molecularly characterized a novel nuage component, PAPI. PAPI is a TUDOR-domain-containing protein that recruits PIWI proteins, especially AGO3, to the nuage and stabilizes them. The interaction between PAPI and AGO3 in the nuage is mediated by sDMAs in the N-terminal domain of AGO3 but is RNA independent. Previous studies have suggested the nuage as the cytoplasmic loci where post-transcriptional silencing of transposons occurs. In addition, loss of Drosophila TUDOR protein has been shown to affect the localization of AUB to the nuage and to alter the piRNA profile. The new findings of this study indicate that TUDOR-domain-containing proteins might serve as a platform for the recruitment of PIWI proteins to the nuage and for the assembly of piRNA pathway components. A subset of transposons are de-repressed in papi deficient ovaries, suggesting that PAPI is involved in transposon silencing in the nuage, just like other piRNA pathway components. This study thus reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway (Liu, 2011).

Furthermore, a physical association of PAPI and AGO3 with the TRAL/ME31B complex has been identifie along with their colocalization in the nuage, and the role of these P body proteins in silencing the expression of some transposons. The TRAL/ME31B complex has been shown to interact with CUP, which also associates with the nuclear pore complex component NUP154 (Grimaldi, 2007). The current findings reveal an exciting physical and functional link between the piRNA machinery and the P body components in the nuage and a mechanism for nuage localization to the nuclear periphery. The P body proteins are well known for their function in mRNA processing and degradation, yet the piRNA machinery regulates transposon silencing by reducing the level of their mRNAs. The physical interaction between these two machineries, with the functional relationship among known components of these two machineries in the nuage illustrated in a working model, raises the intriguing possibility that these two pathways work together in the nuage as a post-transcriptional mechanism to degrade transposon mRNAs, leading to transposon silencing. In addition, these data implicate the interaction of between the TRAL/ME31B complex and NUP154 via CUP as a mechanism of nuage localization to the nuclear periphery (Liu, 2011).

Zfrp8 forms a complex with fragile-X mental retardation protein and regulates its localization and function

Fragile-X syndrome is the most commonly inherited cause of autism and mental disabilities. The Fmr1 (Fragile-X Mental Retardation 1) gene is essential in humans and Drosophila for the maintenance of neural stem cells, and Fmr1 loss results in neurological and reproductive developmental defects in humans and flies. FMRP (Fragile-X Mental Retardation Protein) is a nucleo-cytoplasmic shuttling protein, involved in mRNA silencing and translational repression. Both Zfrp8 and Fmr1 have essential functions in the Drosophila ovary. This study identifies FMRP, Nufip (Nuclear Fragile-X Mental Retardation Protein-interacting Protein) and Tral (Trailer Hitch) as components of a Zfrp8 protein complex. Zfrp8 is required in the nucleus, and controls localization of FMRP in the cytoplasm. In addition, Zfrp8 genetically interacts with Fmr1 and tral in an antagonistic manner. Zfrp8 and FMRP both control heterochromatin packaging, also in opposite ways. It is proposed that Zfrp8 functions as a chaperone, controlling protein complexes involved in RNA processing in the nucleus (Tan, 2016).

Stem cell maintenance is essential for the generation of cells with high rates of renewal, such as blood and intestinal cells, and for the regeneration of many organs such as the brain and skin. Previous work has shown that Zfrp8 is essential for maintaining hematopoietic, follicle, and germline stem cells (GSCs) in Drosophila melanogaster. Knockdown (KD) of Zfrp8 in GSCs results in the loss of stem cell self-renewal, followed by the eventual loss of all germline cells. Similarly in vertebrates, the Zfrp8 homolog, Pdcd2, is essential for embryonic stem cell maintenance and the growth of mouse embryonic fibroblasts; Pdcd2 mouse embryos die before implantation. PDCD2 is abundantly expressed and essential in highly proliferative cells including cultured cells and clinical isolates obtained from patients with hematologic malignancies. The function of Zfrp8 and PDCD2 is highly conserved, as expression of transgenic PDCD2 is sufficient to rescue Zfrp8 phenotypes. Zfrp8 directly binds to Ribosomal Protein 2 (RpS2), a component of the small ribosomal subunit (40S), controls its stability and localization, and hence RNA processing. Zfrp8 also interacts with the piRNA pathway, which is conserved throughout all metazoans and is also essential for the maintenance of GSCs (Tan, 2016).

The piRNA pathway functions in maintaining heterochromatin stability and regulating the expression levels of retrotransposons. Both processes are thought to occur through piRNA targeting of chromatin modifying factors to the DNA. Guided by piRNAs, the piRNA pathway protein Piwi and associated proteins can set repressive epigenetic modifications to block transcription of nearby genes. Levels of transposon transcripts are also controlled by cytoplasmic PIWI-piRNA complexes, which can bind complementary mRNAs and mark them for translational repression and degradation (Tan, 2016).

Fragile-X Mental Retardation Protein (FMRP) functions as a translational repressor involved in RNA silencing. FMRP is a Piwi interactor and part of the piRNA pathway. FMRP-deficient animals display phenotypes similar to piRNA pathway mutants including genomic instability and de-repression of retrotransposons. While FMRP is predominantly localized within the cytoplasm, FMRP complexes have also been demonstrated within the nucleus. In Xenopus, FMRP has been shown to bind target mRNAs co-transcriptionally in the nucleus. Like Zfrp8, FMRP has been shown to bind ribosomal proteins prior to nuclear export. In the cytoplasm, the FMRP-containing RNP complex controls mRNAs stability, localization, and miRNA-dependent repression. FMRP mRNA targets are not well defined, as different studies show low overlap of putative targets in neuronal tissues (Tan, 2016).

In Drosophila, FMRP is required to maintain GSCs, and loss of Fmr1 is associated with infertility and developmental defects in oogenesis and neural development. Fmr1, the gene encoding FMRP, is essential in both vertebrates and Drosophila for the maintenance of neural stem cells (NSCs). In humans, loss of FMRP is associated with Fragile X-associated disorders, which cover a spectrum of mental, motor, and reproductive disabilities. Fragile X-associated disorders are the most commonly inherited cause of mental disabilities and autism. In vertebrates, FMRP physically interacts in the nucleus with NUFIP1 (Nuclear FMRP-Interacting Protein 1), a nucleo-cytoplasmic shuttling protein involved in ribonucleoprotein (RNP) complex formation. NUFIP1 is found in the nucleus in proximity to nascent RNA, and in the cytoplasm associated with ribosomes. In the cytoplasm, FMRP co-localizes and associates with Trailer Hitch (Tral) to form a translational repressor complex. The Tral complex contains a number of translational repressor proteins, which together control the initiation of translation and the stability of mRNAs, such as gurken (grk). In Drosophila, loss of Tral causes ovary phenotypes similar to piRNA pathway mutants, including oocyte polarity defects and transposon activation (Tan, 2016).

This study has identified Zfrp8 interactors by performing a yeast-two hybrid screen, and also by analyzing the components of the Zfrp8 complex by mass spectrometry. The nature of the proteins in the Zfrp8 complex indicates that it is involved in mRNA metabolism and translational regulation. Zfrp8, Nufip, FMRP, and Tral are all part of the complex, and Zfrp8 interacts antagonistically with Fmr1 and tral, suppressing their oogenesis defects. Furthermore, it was determined that Zfrp8 is required within the nucleus, and controls FMRP localization within the cytoplasm. It was further confirmed that FMRP functions in heterochromatin silencing and that Zfrp8 is required in the same process, but has an opposite function of FMRP. It is proposed that Zfrp8 functions as a chaperone of the FMRP’ containing RNP translational repression complex and controls the temporal and spatial activity of this complex (Tan, 2016).

Zfrp8 is essential for stem cell maintenance, but its molecular functions have not yet been clearly defined. Two distinct approaches were taken to address this question. A yeast-two hybrid screen was performed to identify direct interactors of Zfrp8, and the components of the Zfrp8 complex were characterized by mass spectrometry (Tan, 2016).

Because of the high sequence and functional conservation of Zfrp8 (flies) and PDCD2 (mammals), and because no stem cell-derived cDNA library exists in Drosophila, a mouse embryonic stem cell cDNA library was screened using mammalian PDCD2 as bait. Forty-six initial positives were isolated, and 19 potential interactors were identified after re-testing of the positives (Tan, 2016).

In order to purify the Zfrp8 protein complex a transgenic line was established expressing NTAP-tagged Zfrp8 under the control of the general da-Gal4 (daughterless) driver. Two-step tandem affinity purification was performed on embryonic extracts and the purified proteins were separated by SDS-PAGE electrophoresis. The proteins were eluted and analyzed by mass spectrometry. Thirty proteins were identified as part of the Zfrp8 complex. The threshold for interactors was set to at least 5x peptide enrichment in Zfrp8 over vector control fractions. Eighteen of the proteins are predicted to function in ribosomal assembly or translational regulation, strongly suggestive of a function of Zfrp8 in mRNA processing (i.e. translation, localization, and stability). In the complex six ribosomal subunits were found (five 40S subunits and one 60S subunit); EF2 and eIF-4a, which are required for translation initiation and elongation; and FMRP, Tral and Glorund which function in mRNA transport and translational repression. While Zfrp8 interacts with several ribosomal proteins it does not appear to be part of the ribosome itself (Tan, 2016).

No overlapping interactors were found in the yeast-two hybrid screen and mass spectrometry assay. But interestingly, FMRP was identified as part of the Zfrp8 complex by mass spectrometry and NUFIP1 in a yeast-two hybrid assay. Most likely Nufip (estimated 57 kD) was not identified as part of the Zfrp8 complex in the TAP-purification approach, because proteins with similar size to tagged Zfrp8 (~55 kD) were excluded from the mass spectrometry analysis. To investigate whether these proteins could work together in the same molecular process, the interaction of both Zfrp8 and PDCD2 with Nufip (flies) and NUFIP1 (mammals) was confirmed in tissue culture cells. Immunoprecipitation of human HEK293 cell extracts expressing FLAG-tagged NUFIP1 pulled down endogenous PDCD2. Next whether this protein interaction also exists in Drosophila was examined. It was possible to co-purify endogenous Zfrp8 with NTAP-tagged Nufip from transfected S2 cells. An additional Western blot was performed on the purified NTAP-Nufip isolate and it was shown that FMRP is present in the protein complex, indicating that Nufip physically interacts with both Zfrp8 and FMRP. These results suggest that all three proteins function together in a molecular complex which regulates RNP processing/assembly and translation. Based on these results, and the requirement of both Zfrp8 and Fmr1 in stem cell maintenance, it was decided to characterize the genetic interaction between these genes (Tan, 2016).

To further characterize the connection between the two genes, whether the loss of Zfrp8 can modify oogenesis defects reported for Fmr1 females. Similar to what was previously reported, 100% of Fmr1^Δ50M/Df(3 R)Exel6265 and 80% of Fmr1^Δ50M/Fmr1³ ovaries displayed developmental defects. The ovarioles contained fused egg chambers, aberrant nurse cell numbers. Occasionally, egg chambers with oocyte misspecification/multiple oocytes were also observed. Interestingly, the loss of one copy of Zfrp⁸ suppressed the majority of Fmr1 ovary defects, restoring cell division in the germline, as well as egg chamber morphology and separation. In Zfrp8/+; Fmr1^Δ50M/Df(3R)6265, fusion of the first egg chamber is still observed in most germaria, but despite this, oogenesis appears to proceed normally resulting in normal looking ovarioles. Zfrp8/+; Fmr1^Δ50M/Fmr1³ ovaries appear almost completely normal even though these ovarioles contain no FMRP (Tan, 2016).

The loss of Fmr1 has also been associated with a strong reduction in egg production. This study found that similar to previous reports, Fmr1^Δ50M/Df(3R)Exel6265 and Fmr1^Δ50M/Fmr1³ mutants display a strong reduction in fertility; females laid on average of 1 and 6 eggs/day, respectively, as compared to 18 eggs/day for wild-type flies. The removal of one copy of Zfrp8 partially suppressed Fmr1 infertility and resulted in 8 eggs/day from Fmr1^Δ50M/Df(3R)Exel6265 and 15 eggs/day from Fmr1^Δ50M/Fmr1³ females. These results demonstrate that Zfrp8 and Fmr1 affect the same process and that even though they are found in the same complex, have opposing functions (Tan, 2016).

To investigate the nature of the Zfrp8 interaction with FMRP, the localization of the proteins within the ovary was examined. Zfrp8 displays ubiquitous distribution in all cells and cell compartments of the wild type ovary. No significant changes in Zfrp8 localization or levels are visible in Fmr1 ovaries. FMRP has a more varied distribution pattern, present in strong, cytoplasmic puncta in the cytoplasm of nurse cells and follicle cells, and also in high levels in the cytoplasm of the maturing oocyte. FMRP is also detectable in low levels in nurse cell nuclei at stage 8 egg chambers at an average of 9.76 puncta per nucleus. As expected, Fmr1 ovaries display no FMRP staining in either the cytoplasm or nucleus (Tan, 2016).

To determine whether Zfrp8 functions in FMRP regulation, Zfrp8 was depleted in the germline by expressing Zfrp8 RNAi under the control of the nos-Gal4 driver, and changes in FMRP expression were assessed. In control nos-Gal4 ovaries, FMRP levels and distribution were similar to that in wild-type ovaries. However, in Zfrp8 KD ovaries, aberrant FMRP localization is observed in the germline; FMRP is more uniformly distributed throughout the cytoplasm and puncta are strongly diminished. Remaining FMRP puncta appear fragmented, reduced in intensity, size and number (~10% of wild-type). These results indicate a Zfrp8 requirement for proper FMRP localization to the cytoplasm. FMRP normally functions by shuttling mRNA cargo from the nucleus to the cytoplasm, where it represses the translation of bound mRNA. The observed change of FMRP localization in Zfrp8 KD ovaries therefore may indicate a regulatory function for Zfrp8 in the nuclear export and localization of FMRP (Tan, 2016).

Zfrp8 protein is present in both the cytoplasm and nucleus and, as demonstrated above, controls the distribution of FMRP in the cytoplasm. It was decided to investigate the cell compartment in which Zfrp8 is required, in order to elucidate how Zfrp8 regulates FMRP. To do so, the capability of Zfrp8 deletion constructs to rescue mutant lethality was examined. Expression of human PDCD2 cDNAs driven by the general driver da-Gal4 is fully capable of rescuing Zfrp8 lethality. Mutated Zfrp8 constructs were created, removing either the two putative NLSs or the putative NES domains. These proteins were expressed under the da-Gal4 driver, and while clearly overexpressed on Western blots, failed to rescue mutant lethality, suggesting that the three domains are essential for the function of the protein (Tan, 2016).

In an alternative approach, the function of Zfrp8 proteins targeted to a distinct cell compartment was examined. Four N-terminal GFP-tagged transgenic proteins were expressed, encoding a wild-type Zfrp8, nuclear-localized NLS-Zfrp8, cytoplasmic-localized NES-Zfrp8, and cell membrane-localized CD8-GFP-Zfrp8. Transgenic Zfrp8 subcellular localization is visible when the proteins are strongly overexpressed. When the transgenes were expressed at lower levels, similar to the endogenous levels, with the hsp70-Gal4 driver at 25 ^oC, both wild-type and nuclear-localized Zfrp8 were able to rescue mutant lethality at similar rates, whereas the cytoplasmic- and membrane-localized proteins did not show rescue. These results show that Zfrp8 is required in the nucleus and suggest that like FMRP, Zfrp8 may function by shuttling between nuclear and cytoplasmic compartments (Tan, 2016).

This study has shown that FMRP and Zfrp8 are present in the same protein complex. In addition to FMRP, the mass spectrometry results have also identified other translational regulators, such as Tral. Tral has previously been shown to function in conjunction with FMRP to control the translation of mRNAs (Tan, 2016).

To determine whether Zfrp8 functions in Tral/FMRP-associated translational regulation, the genetic interaction between Zfrp8 and tral was investigated. Tral regulates dorsal-ventral (D/V) patterning through the localization and translational control of gurken (grk) mRNA. Eggs laid by tral females display ventralized chorion phenotypes, due to the aberrant Gurken morphogen gradient. If Zfrp8 functions to regulate the translational activity of FMRP/Tral, a suppression of the tral ventralized phenotypes should be apparent when Zfrp8 is reduced. Tral was depleted in the germline by expressing a TRiP RNAi lineunder the control of the nos-Gal4 driver. Tral KD resulted in similar ventralized egg phenotypes as previously observed in eggs laid by tral¹ females: 1% of eggs displayed two normal dorsal appendages (Wt), 36% had fused appendages, and 63% had no dorsal appendages. Removing one copy of Zfrp8 in the tral KD background suppressed the tral phenotypes. This genetic interaction suggests that in addition to controlling the localization of FMRP in the cytoplasm, Zfrp8 also influences the translational control by Tral, essential for formation of dorsal-ventral polarity in the egg (Tan, 2016).

Whether Zfrp8 regulates Tral localization as it does FMRP was investigated by examining the distribution of GFP-fusion Tral protein trap line. Tral protein was uniformly present in cytoplasmic compartments of germline and somatic cells, with stronger granules surrounding nuclei, and was highly enriched within the oocyte. Zfrp8 KD results in loss of oocyte identity, and the distribution of Tral was significantly altered in those cells. But in all other germline cells Tral distribution remained unaffected. Tral and its orthologs are cytoplasmic proteins and examination of the Tral protein sequence identifies no NLSs. Zfrp8 may therefore interact only indirectly with Tral and not regulate its localization (Tan, 2016).

Zfrp8 and Fmr1 control position effect variegation piRNA pathway genes have been shown to be essential for heterochromatin packaging in position effect variegation (PEV) experiments. PEV measures expression of endogenous or reporter genes inserted within or adjacent to heterochromatin. Fmr1 is specifically required for chromatin packaging as loss of a single copy of Fmr1 is sufficient to inhibit heterochromatin silencing of a white reporter inserted into the pericentric heterochromatin region 118E10 on the 4th chromosome (Tan, 2016).

PEV of Zfrp8 heterozygotes, Fmr1 heterozygotes and Fmr1, Zfrp8 transheterozygotes were examined using 118E10 (4th chromosome centromeric) and an additional white reporter, inserted into heterochromatin region 118E15 (4th chromosome telomeric). While thewhite+ reporters in Zfrp8null/+ eyes were expressed at levels comparable to those in wild-type controls, expression in Fmr1^Δ50M/+ of both white reporters was strongly enhanced. But, the removal of one copy of both Zfrp8 and Fmr1 decreased expression of the reporters back to the Zfrp8/+, near wild-type levels, indicating restored heterochromatin silencing of both 4th chromosomal insertions. These findings suggest that in normal eyes, Zfrp8 functions upstream of Fmr1 and controls Fmr1 effects on heterochromatin packaging (Tan, 2016).

A connection between regulation of heterochromatin silencing and Piwi has clearly been established and the current results show that Zfrp8 and FMRP are part of the mechanism that controls heterochromatin silencing. Heterochromatin is established at the blastoderm stage in Drosophila embryos and is subsequently maintained throughout development. Thus, FMRP and Zfrp8 function together in heterochromatin packaging in the early embryo in the same way as they do during oogenesis (Tan, 2016).

This study has shown that Zfrp8 is part of a complex that is involved in RNA processing, i.e. translation, localization, and stability. It is proposed that Zfrp8 likely forms a ribonucleoprotein complex with Nufip, FMRP and select mRNAs in the nucleus, and is required for localization of this complex in the cytoplasm. After nuclear export, mRNAs within the complex are targeted for translational control and repression by FMRP and Tral. The suppression of the Fmr1 and tral phenotypes in a Zfrp8 heterozygous background, occurs in the absence of Fmr1 and the strong reduction of tral. This suggests that Zfrp8 function is not protein specific, but rather that it controls the FMRP and Tral-associated complex, even in the absence of each of the two proteins. This hypothesis is consistent with Zfrp8 actively controlling the localization of FMRP to cytoplasmic foci, as this localization is affected in Zfrp8 germ cells (Tan, 2016).

Previous studies identified a piRNA pathway protein, Maelstrom (Mael), that is controlled by Zfrp8 in a similar manner as FMRP. Zfrp8 forms a protein complex with Mael, genetically suppresses the loss of mael, and controls Mael localization to the nuage, a perinuclear structure. But the Zfrp8 phenotype is stronger and appears earlier than that of mael, tral, Fmr1, or other piRNA pathway regulatory genes studied so far. Zfrp8 may therefore control a central step in the regulation of specific RNPs. Consistent with this hypothesis, the TAP purification and mass spectrometry analysis identified a number of Zfrp8-associated proteins, the majority of which function in ribosomal assembly or translational regulation, such as the ribosomal protein RpS2. And Zfrp8 KD in the germ line and partial loss of rps2 result in a similar "string of pearls phenotype", caused by developmental arrest in early stages of oogenesis. In addition, a recent study has shown that Zfrp8 and PDCD2 contain a TYPP (TSR4 in yeast, YwqG in E. coli, PDCD2 and PDCD2L in vertebrates and flies) domain, which has been suggested to perform a chaperone-like function in facilitating protein–protein interactions during RNA processing. These observations lead to a hypothesis that Zfrp8 functions as a chaperone essential for the assembly of ribosomes and the early recruitment and localization of ribosomal-associated regulatory proteins, such as FMRP, Tral and Mael (Tan, 2016).

Zfrp8 negatively controls the functions of Fmr1 and tral. In the absence of FMRP and Tral the temporal and spatial control of translation of their associated RNPs is lost. It is proposed that reducing the level of Zfrp8 diminishes the availability of these RNP-complexes in the cytoplasm resulting in suppression of the Fmr1 and tral phenotypes (Tan, 2016).

Zfrp8, Fmr1 and tral have all been shown to genetically and physically interact with components of the piRNA pathway, and to regulate the expression levels of select transposable elements. Transposon de-repression is often associated with the loss of heterochromatin silencing. The molecular mechanisms underlying heterochromatin formation appear to involve maternally contributed piRNAs and piRNA pathway proteins that control the setting of epigenetic marks in the form of histone modifications, maintained throughout development. But transposon expression can also be controlled post-transcriptionally by cytoplasmic PIWI-piRNA complexes, suggesting that transposon deregulation and heterochromatin silencing phenotypes seen in FMRP and Zfrp8 may be linked to translational de-repression. It is proposed that by facilitating the early assembly of ribosomes with specific translational repressors, Zfrp8 regulates several developmental processes during oogenesis and early embryogenesis including dorsal-ventral signaling, transposon de-repression, and position effect variegation (Tan, 2016).

DEVELOPMENTAL BIOLOGY

In order to better understand where Tral might be acting during oogenesis, The distribution of Tral was examined in the egg chamber. Within the nurse cells, Tral was present in discrete particles throughout oogenesis. However, within the oocyte, Tral showed a dynamic distribution -- first being localized to the posterior of the oocyte during stages 1-6, accumulating briefly at the anterior during stages 7-8, followed by weak accumulation along the oocyte cortex with substantial enrichment at the posterior pole during stages 9-10 (Wilhelm, 2005).

Live imaging of GFP-Tral in nurse cells demonstrated that while there are a number of motile Tral particles, a substantial fraction of the particles are immobile and appear to be tethered to a large reticular structure reminiscent of the ER. This result, together with the finding that tral mutations interfere with the ER-Golgi trafficking of Grk and Yl, suggested that Tral might function on the surface of the ER. To test this, the ER was visualized by using a transgenic Drosophila line that expresses GFP fused to the KDEL ER retention signal (GFP-KDEL) only in the germline. While it was found that Tral protein colocalizes with the ER of the oocyte, the dense accumulation of ER membranes within the oocyte made close examination of the sites of colocalization difficult. Therefore analysis focused on Tral particles within the nurse cells, where ER membranes do not completely fill the cytoplasm. Immunostaining for Tral and GFP-KDEL revealed that the numerous Tral particles within the nurse cells are all associated with subdomains of the ER. These subdomains are typically sites of concentrated ER and are often present at the end of ER tubules. Given the Grk and Yl secretion defects observed in tral mutants, the localization of Tral to discrete domains of the ER suggests that Tral acts directly to regulate ER exit site function (Wilhelm, 2005).

Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies: Me31B participates with an FMRP-associated, P body protein (Scd6p/Trailer hitch) in FMRP-driven, Argonaute-dependent translational repression in developing eye imaginal discs

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. This study shows that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic 'P bodies,' including the RNA-degradative enzymes Decapping protein 1 (Dcp1p) and Xrn1p/Pacman and crucial components of miRNA (Argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B, a DEAD-box helicases, is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/Trailer hitch) in FMRP-driven, Argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons (Barbee, 2006).

Several observations now indicate that P bodies, maternal granules, and a major subclass of neuronal RNP are similar in underlying composition and represent a conserved system for the regulation of cytoplasmic mRNAs. Known RNA transport and translational repressors shared between maternal and neuronal staufen granules now include, Stau, Btz, dFMR1, Pum, Nos, Yps, Me31B, Tral, Cup, eIF4E, Ago-2, and Imp. Strikingly, in human cells, the Me31B homolog RCK/p54, the Tral homolog RAP55, the four human argonaute proteins, eIF4E, and a eIF4E-binding protein analogous to Cup, 4E-T, are all found in P bodies. In yeast, homologs of Me31B (Dhh1p) and Tral (Scd6p) are also known to be in P bodies, and Dhh1p in particular plays a role in recruiting RNA-decapping proteins and exonucleases to these RNPs. Consistent with the above observations in yeast, the enzymes involved in mRNA hydrolysis including the 5′ to 3′ RNA exonuclease Xrn1p/Pcm and the RNA-decapping enzyme DCP1 are present on Drosophila neuronal staufen RNPs and maternal RNA granules. These data unequivocally demonstrate tight spatial proximity of components mediating various RNA regulatory processes in Drosophila neurons (Barbee, 2006).

The large collection of proteins and processes common to P bodies, staufen granules, and likely maternal RNA granules suggests that they share an underlying core biochemical composition and function, which would then be elaborated in different biological contexts. For example, one anticipates that proteins involved in mRNA transport will be more prevalent in maternal and neuronal RNPs, which need to be transported for their biological function (Barbee, 2006).

An interesting aspect of neuronal staufen RNPs described in this study is the diversity of translational repression systems that are present within them. (1) In Me31B, these RNPs contain a protein that works in general translation repression of a wide variety of mRNAs and can also affect miRNA-based repression. (2) In Ago-2, they contain a component specific to miRNA/RNAi-dependent repression. (3) Neuronal staufen granules also contain UPF1, which was originally thought to be solely involved in mRNA degradation. However, because UPF1 can act as a translation repressor and physically interacts with Stau, a reasonable hypothesis is that UPF1 might work in neuronal granules, in conjunction with Stau, to repress the translation of a subset of mRNAs. The presence of multiple mechanisms for translation repression colocalizing in granules in Drosophila neurons may allow for differential translation control of subclasses of mRNA in response to different stimuli (Barbee, 2006).

Evidence accumulating in the literature suggests that there is a potential diversity of RNA granule types in neurons. Observations in Drosophila neurons are most consistent with a model in which a major subclass of neuronal RNP, in which various translational repressor and mRNA turnover proteins colocalize, is related to other compositionally distinct, diverse RNPs. A major subclass of staufen-containing RNP is indicated by data showing substantial colocalization among various proteins analyzed. Diversity is indicated by the lack of 100% colocalization: for instance, 55% of staufen-positive particles in wild-type neurons do not contain detectable dFMR1 (Barbee, 2006).

Two types of observations suggest that the apparent subclasses of particles containing Stau or dFMR1, but not both, are related to the particles in which they colocalize: (1) these two types of RNPs are clearly compositionally related to particles that contain both proteins; (2) this is supported by the observation that colocalization can be substantially increased under some conditions. Overexpression of either dFMR1 or Stau:GFP increases colocalization between Stau and dFMR1 from 45% in wild-type neurons to more than 80%. Concurrent with increased frequency of colocalization, Stau:GFP or dFMR1 induction increases apparent particle size (or brightness) and reduces the total number of particles. The increase in colocalization and brightness, as well as reduction in particle number, is most easily explained by growth and/or fusion of related RNPs. Significantly, similar effects on mammalian neuronal granule size and number have been reported following overexpression of Stau or another granule protein, RNG105. Thus, the underlying regulatory processes appear conserved between Drosophila and mammalian neurons (Barbee, 2006).

While it remains unclear how FMRP, Stau, or RNG105 enhance granule growth or fusion, it is conceivable that individual mRNAs first form small RNPs whose compositions reflect specific requirements for translational repression of the mRNAs they contain. These small RNPs exist in dynamic equilibrium with larger RNPs in which multiple, diverse translational repression complexes are sequestered. Induction of factors that promote granule assembly could push the equilibrium toward mRNP sequestration within large granules. A requirement of this dynamic model, which postulates interactions among different types of RNP, is that the RNPs themselves can change in composition during transport to synaptic domains. This is supported by FRAP analyses showing rapid exchange of Stau:GFP between cytosol and granule (Barbee, 2006).

Additional types of RNPs have also been described in neurons. For example, polysomes apparently arrested in translation have been observed near dendritic spines, and these RNPs show no obvious similarity to large, ribosome-containing particles, termed neuronal RNA granules. In addition, a potentially distinct RNP containing Stau, kinesin, and translationally repressed RNAs, but not ribosomes, has been purified from the mammalian brain. More recently, it has been shown that RNPs containing stress-granule markers TIA-1 and TIA-R as well as pumilio2 are induced by arsenate-treatment of mammalian cultured neurons. Interestingly, as previously shown for somatic cells, these large stress granules appear tightly apposed to domains containing DCP1 and Lsm1, markers of P bodies. Determining the temporal and compositional relatedness of such varied RNPs, their pathways of assembly as well as their functions, is a broad area of future research not only in neuroscience but also in cell biology (Barbee, 2006).

These diverse types of biochemical compartments for individual mRNAs suggest that neural activity or other developmental signaling events would influence translation in two steps: first, by desequestering mRNPs held within large granules and, then, by derepressing quiescent mRNAs in individual mRNPs. Thus, RNPs described in this study could have a complex precursor-product relationship with other RNPs, including polysomes discovered by now-classical studies at dendritic spines (Barbee, 2006).

Despite the complexity revealed by the diversity of neuronal RNPs, the importance and significance of the observed colocalization of Me31B, Tral, argonaute, and dFMR1 in staufen-positive neuronal RNPs is most clearly demonstrated by functional analyses revealing biological pathways in which these proteins function together (Barbee, 2006).

Several independent lines of evidence are consistent with a function for Me31B in neuronal translational repression as part of a biochemical complex that includes dFMR1. (1) Subcellular localization studies indicate that Me31B and Tral localize to dFMR1-containing RNPs especially prominent at neurite branch points in cultured Drosophila neurons. (2) Me31B, Tral, and dFMR1 coimmunoprecipitate from Drosophila head extract, thus confirming the physical association of three proteins. (3) Loss-of-function alleles of either Me31B or Tral suppress the rough eye phenotype seen when dFMR1 is overexpressed in the sev-positive photoreceptors. (4) Overexpression of Me31B in sensory neurons leads to altered branching of terminal dendrites, a phenotype also seen with overexpression analyses of Nos, Pum, and dFMR1. (5) Reduction of Me31B expression in sensory neurons by RNAi results in abnormal dendrite morphogenesis and tiling defects, phenotypes similar to that observed following loss of nanos, pum, or dFmr1 function. Significantly, the effect of Me31B on dendritic growth is correlated with its ability to function in translational repression. These five independent lines of evidence provide considerable support for Me31B (and Tral) function in neuronal translation control processes. While the site of functional interaction between dFMR1, Me31B, and Tral (soma or neuronal processes) is not identified here, the importance of the physical interactions is clearly demonstrated (Barbee, 2006).

Several observations also argue that Me31B acts, at least in part, within neurons to promote translation repression and/or mRNA degradation in response to miRNAs. This possibility was first suggested by the physical and genetic interactions of Me31B with dFMR1, a protein that has been implicated in the miRNA-mediated repression. Using direct assays for miRNA-mediated function in vivo, this study shows that Me31B is required for efficient repression by the bantam miRNA in developing wing imaginal discs. This identifies Me31B as a protein required for efficient miRNA-based repression (Barbee, 2006).

Recently, miRNA-based regulation has been shown to be important for the control of spine growth in hippocampal neurons and to be a target of protein-degradative pathways involved in long-term memory formation in Drosophila. Thus, the data predict that Me31B will be important in modulating miRNA function pertinent to development of functional neuronal plasticity. More generally, because Me31B homologs in yeast and mammals have been shown to function in P body formation in somatic cells, the requirement for Me31B in miRNA function provides evidence to support a model in which formation of P bodies is required for efficient miRNA-based repression in varied cell types and biological contexts (Barbee, 2006).

The conclusion that staufen- and dFMR1-containing neuronal RNPs are similar in organization and function to P bodies has several implications for neuronal translational control. (1) The presence of diverse translational repression systems on these RNPs suggests that, like in P bodies, different classes of mRNAs will be repressed by different mechanisms. This may allow specific RNA classes to be released for new translation in response to different stimuli. Such diversity of control may allow synapses to remodel themselves differently, depending on the frequency and strength of stimulation (e.g., LTD or LTP). (2) FRAP experiments indicate that both P bodies and staufen granules are dynamic structures. This argues that, like P bodies, staufen granules are in a state of dynamic flux, perhaps in activity-regulated equilibrium with the surrounding translational pool. (3) The presence of mRNA-degradative enzymes on staufen granules suggests regulation of mRNA turnover may play an important role in local synaptic events. For example, if synaptic signaling were to induce turnover of specific mRNAs at a synapse, then stimulated synapses could acquire properties different from unstimulated ones that retain a 'naive' pool of stored synaptic mRNAs. Finally, these observations imply that the proteins known to function in translation repression within P bodies will play important roles in modulating translation in neurons. Thus, it is anticipated that proteins of mammalian or yeast P bodies such as Edc3p, Pat1p, the Lsm1-7p complex, GW182, and FAST will be present on and influence assembly and function of neuronal granules (Barbee, 2006).

EFFECTS OF MUTATION

While screening P element insertions generated by the Berkeley Drosophila Genome Project (BDGP) gene disruption project for uncharacterized genes required for embryonic axis formation, a female sterile P element insertion, KG08052, was identified that exhibited defects in the dorsal-ventral patterning of the eggshell. The KG08052 insertion site lies within the first intron of CG10686, suggesting that disruption of this gene, trailer hitch, is responsible for the dorsal-ventral patterning defect. Quantitation of the dorsal-ventral patterning defect in eggs laid by females homozygous for the KG08052 insertion (tral¹) revealed that 80% of eggs have either no dorsal appendages or display a single fused appendage -- a phenotype indicative of ventralization of the eggshell. Females hemizygous for tral¹ showed an enhancement of the dorsal appendage phenotype, with 100% of eggs showing either 0 or 1 dorsal appendages, indicating that tral¹ is a strong hypomorphic allele (Wilhelm, 2005).

In order to further characterize the tral locus, two additional insertions were obtained in the tral locus: e03082 (tral²), a PiggyBAC transposon insertion in the 5′UTR of tral and d09277 (tral³), a P element insertion in the first intron of tral. Ninety-three percent of eggs laid by tral¹/tral² mothers display a ventralized eggshell phenotype consistent with tral² being a strong hypomorphic mutation. In contrast, while tral³/tral¹ mothers are sterile, only 12% of their eggs had ventralized eggshells, indicating that tral³ is a weak hypomorphic allele of tral. Because the tral locus is quite close to the citron kinase gene (dck), complementation tests were performed between tral and dck to determine whether the phenotype was due to the insertions affecting both genes. The lethal allele, dck¹, fully complements tral¹, indicating that eggshell ventralization in tral mutants is not due to disruption of dck. Thus, tral¹, tral², and tral³ constitute an allelic series with respect to the strength of the ventralization phenotype and do not disrupt the closest neighboring gene, dck (Wilhelm, 2005).

To confirm that tral¹, tral², and tral³ disrupt Tral expression, antibodies were raised to the first 130 amino acids of Tral and immunoblots of ovaries derived from various tral allelic combinations for Tral protein were probed. tral¹ and tral² in combination with either each other or with the deficiency Df(3L)ED4483 decreased tral expression to an undetectable level, consistent with the disruption of tral expression being responsible for the observed ventralization of the eggshell. Immunoblots of ovaries from tral³/tral¹ or tral³/Df(3L)ED4483 females showed a decrease in Tral protein expression as compared to tral³ heterozygotes or a yw control but did not completely eliminate expression, consistent with tral³ being a weak hypomorphic allele of tral. Although rescue of the mutant phenotype would be necessary to rule out the possibility that a second site mutation is the cause of the observed phenotypes, the fact that tral¹, tral², and tral³ constitute an allelic series with respect to both strength of phenotype and expression of tral strongly argues that the observed phenotypes are due to decreases in tral expression (Wilhelm, 2005).

One of the key events in dorsal-ventral patterning is the localization of grk mRNA to the dorsal-anterior region. The localization of grk mRNA in turn causes the trafficking of Grk protein to be confined to dorsal-anterior endoplasmic reticulum (ER)-Golgi units. It is this localized secretion of Grk that instructs the dorsal follicle cells to assume a dorsal cell fate. These dorsal follicle cells then secrete the proper eggshell components to generate a dorsal appendage. The dorsal-ventral patterning defect of the tral mutants suggests that tral might regulate some aspect of the localization or secretion of Grk. In wild-type egg chambers, Grk protein is expressed homogeneously throughout the oocyte during stages 6–7 and then is found only in small puncta near the plasma membrane in the dorsal-anterior region of the oocyte during stages 8-10. These small Grk puncta are known to coincide with sites of exit from the ER (Herpers, 2004). In both tral¹ homozygotes and tral¹ hemizygotes, abnormally large Grk puncta were observed in 48% of homozygotes and 63% of hemizygotes during stages 6-8. This suggests that mutations in tral disrupt some aspect of Grk trafficking through the secretory pathway (Wilhelm, 2005).

Conceivably, tral mutants could affect Grk trafficking either by interfering with the proper localization/translational control of the grk message, by disrupting the microtubule cytoskeleton, or by blocking the normal trafficking of Grk protein through the secretory pathway. To test these possibilities, the localization of grk mRNA was assayed in tral mutant egg chambers by in situ hybridization. The localization of grk mRNA to the dorsal-anterior region of the oocyte during stages 8-10 is normal in tral mutant egg chambers. This result argues against defects in grk mRNA localization being responsible for the Grk trafficking defect observed in tral mutants (Wilhelm, 2005).

The fact that grk mRNA is correctly localized argues that the normal polarity of the microtubule cytoskeleton is intact in tral mutants; a polarized microtubule network is essential for grk mRNA localization. To confirm that the microtubule polarity is intact, whether the localization of Osk protein to the posterior is normal in tral mutant egg chambers was examined. Because the correct localization of Osk protein to the posterior requires both normal microtubule polarity and the proper localization of osk mRNA, this assay should reveal any functional defects in either the microtubule polarity or the transport of osk mRNA. Whereas large Grk puncta accumulate in the oocytes of tral mutants, Osk protein is present at the posterior of the oocyte. Consistent with this result, mutations in tral do not affect the normal anterior-posterior gradient of microtubule density in stage 9 egg chambers. Thus, mutations in tral do not affect either microtubule polarity or the transport of the grk and osk messages. This result may seem paradoxical, since grk signaling early in oogenesis is required to establish the microtubule polarity of the oocyte. However, the establishment of microtubule polarity is less sensitive to changes in the level of grk signaling than dorsal appendage formation. Because none of the tral alleles cause complete ventralization of the eggshell, it is not surprising that it has been possible to selectively affect dorsal appendage formation without altering the microtubule polarity of the oocyte (Wilhelm, 2005).

To rule out that large Grk puncta are due to a defect in Grk translational control, the distribution of large Grk puncta was examined during stages 8-10. If there were a defect in translational repression of grk mRNA, Grk protein should accumulate broadly throughout the oocyte. While some large Grk puncta are mislocalized to the side of the nucleus facing away from the oocyte cortex, both normal sized and large Grk puncta are restricted to the dorsal-anterior region of the oocyte. Because a defect in translational control would be expected to yield high levels of Grk protein throughout the oocyte, this result argues that the large Grk foci are not due to a loss of translational repression of the grk message. Because the polarity of the microtubule cytoskeleton and the localization/translation of grk mRNA appear normal in tral mutant egg chambers, the hypothesis was tested that the formation of large Grk puncta in tral mutants is due to a defect in the trafficking of Grk (Wilhelm, 2005).

In a variety of systems, ER exit sites are closely associated with Golgi units, presumably due to the role of ER trafficking in establishing and maintaining the Golgi. Because previous work established that small Grk puncta are coincident with ER exit sites, also known as the transitional ER, the effects of tral mutants on the distribution of Grk and its association with the Golgi were examined (Herpers, 2004). In wild-type egg chambers, the majority of Grk protein is present in small puncta that are closely associated with an individual Golgi complex that is positive for the Golgi marker Lava lamp. However, in tral mutants, the large Grk puncta have lost their intimate association with the Golgi. This suggested that the formation of large Grk foci might be due to a defect in ER exit (Wilhelm, 2005).

The COPII complex, which is required for ER-to-Golgi trafficking, is known to label discrete sites on the ER. Furthermore, a number of experiments have implicated these COPII sites and the regions surrounding them in exit from the ER (Bevis, 2002; Mironov, 2003). Using GFP-Sar1 as a marker for COPII complex formation, the distribution of ER exit sites was examined in wild-type and tral hemizygous egg chambers. GFP-Sar1 is distributed in small puncta throughout the nurse cells and oocyte in wild-type egg chambers. However, this organization is severely disrupted in tral¹/Df(3L)ED4483 egg chambers. In these egg chambers, the GFP-Sar1 is found in abnormally large puncta similar to those observed for Grk protein. Thus, tral is required for normal ER exit site distribution and morphology. The accumulation of Grk in large foci that are not correctly associated with the Golgi, together with the role of tral in organizing ER exit sites, argues that the disruption of ER exit sites in tral mutants leads to a functional defect in ER-Golgi trafficking. It is this disruption of ER-Golgi trafficking that likely underlies the failure in dorsal-ventral patterning observed in tral mutants (Wilhelm, 2005).

If tral plays a general role in ER exit site function, one would expect to observe defects in the trafficking of other secreted proteins. In order to test this, the effects of tral mutants on the trafficking of the vitellogenin receptor Yl were examined. Previous work on Yl has shown that in wild-type egg chambers, Yl protein is distributed homogeneously throughout the ER of the oocyte with an occasional small puncta until stage 8, when all of the Yl protein is transported to the plasma membrane (Schonbaum, 2000). Homozygous tral¹ oocytes showed no obvious disruption of Yl trafficking. However, 75% of hemizygous tral¹ oocytes showed Yl foci within the oocyte during stages 6-9. Therefore, tral is required for the trafficking of proteins besides Grk and likely plays a general role in promoting exit from the ER (Wilhelm, 2005).

It was next asked whether the Grk and Yl foci are distinct in tral¹ hemizygous oocytes. Immunostaining for both Grk and Yl revealed that the large foci for each protein are separate. This suggests that the two proteins use separate trafficking pathways that both require tral. The observation that the trafficking of Grk is more sensitive to decreases in tral function than the trafficking of Yl is consistent with this idea (Wilhelm, 2005).

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Search PubMed for articles about Drosophila trailer hitch

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date revised: 12 December 2020

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