InteractiveFly: GeneBrief

TER94: Biological Overview | References

BIOLOGICAL OVERVIEW

The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. This study shows that mutation or inhibition of Valosin-Containing Protein (VCP; termed TER94 by FlyBase), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, it was found that VCP inhibition causes an altered splicing pattern of the large pruning gene Molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). These data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 (Effete) and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, these results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism (Rumpf, 2014).

To achieve specific connections during development, neurons need to refine their axonal and dendritic arbors. This often involves the elimination of neuronal processes by regulated retraction or degeneration, processes known collectively as pruning. In the Drosophila, large-scale neuronal remodeling and pruning occur during metamorphosis. For example, the peripheral class IV dendritic arborization (da) neurons specifically prune their extensive larval dendritic arbors, whereas another class of da neurons, the class III da neurons, undergo ecdysone- and caspase-dependent cell death. Class IV da neuron dendrite pruning requires the steroid hormone ecdysone and its target gene SOX14, encoding an HMG box transcription factor. Class IV da neuron dendrites are first severed proximally from the soma by the action of enzymes like Katanin-p60L and Mical that sever microtubules and actin cables, respectively. Later, caspases are required for the fragmentation and phagocytic engulfment of the severed dendrite remnants . Another signaling cascade known to be required for pruning is the ubiquitin-proteasome system (UPS). Covalent modification with the small protein ubiquitin occurs by a thioester cascade involving the ubiquitin-activating enzyme Uba1 (E1), and subsequent transfer to ubiquitin-conjugating enzymes (E2s) and the specificity-determining E3 enzymes. Ubiquitylation of a protein usually leads to the degradation of the modified protein by the proteasome, a large cylindrical protease that consists of two large subunits, the 19S regulatory particle and the proteolytic 20S core particle. Several basal components of the ubiquitylation cascade—Uba1 and the E2 enzyme ubcD1—as well as several components of the 19S subunit of the proteasome have been shown to be required for pruning, as well as the ATPase associated with diverse cellular activities (AAA) ATPase Valosin-Containing Protein (VCP) (CDC48 in yeast, p97 in vertebrates, also known as TER94 in Drosophila), which acts as a chaperone for ubiquitylated proteins. Interestingly, autosomal dominant mutations in the human VCP gene cause hereditary forms of ubiquitin-positive frontotemporal dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). A hallmark of these diseases is the occurrence of both cytosolic and nuclear ubiquitin-positive neuronal aggregates that often contain the RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). It has been proposed that ubcD1 and VCP promote the activation of caspases during dendrite pruning via degradation of the caspase inhibitor DIAP1. However, mutation of ubcD1 or VCP inhibit the severing of class IV da neuron dendrites from the cell body, whereas in caspase mutants, dendrites are still severed from the cell body, but clearance of the severed fragments is affected. This indicates that the UPS must have additional, as yet unidentified, functions during pruning (Rumpf, 2014).

This study further investigated the role of UPS mutants in dendrite pruning. vcp mutation was shown to lead to a specific defect in ecdysone-dependent gene expression, as VCP is required for the functional expression and splicing of the large ecdysone target gene molecule interacting with CasL (MICAL). Concomitantly, mislocalization of Drosophila TDP-43 and up-regulation of other RNA-binding proteins were observed, and genetic evidence suggests that these alterations contribute to the observed pruning defects in VCP mutants. Defects in MICAL expression and TDP-43 localization are also induced by mutations in ubcD1 and in the 19S regulatory particle of the proteasome, but not by a mutation in the 20S core particle, despite the fact that proteasomal proteolysis is required for dendrite pruning, indicating the requirement for multiple UPS pathways during class IV da neuron dendrite pruning (Rumpf, 2014).

Class IV da neurons have long and branched dendrites at the third instar larval stage. In wild-type animals, these dendrites are completely pruned at 16-18 h after puparium formation (h APF). VCP mutant class IV da neurons were generated by the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique for clonal analysis. Mutant vcp^26-8 class IV da neurons displayed strong pruning defects and retained long dendrites at 16 h APF. Expression of an ATPase-deficient dominant-negative VCP protein (VCP QQ) under the class IV da neuron-specific driver ppk-GAL4 recapitulated the pruning phenotype and also led to the retention of long and branched dendrites at 16 h APF. VCP inhibition also causes defects in class III da neuron apoptosis. This combination of defects in both pruning and apoptosis is reminiscent of the phenotypes caused by defects in ecdysone-dependent gene expression. Indeed, overexpression of the transcription factor Sox14, which induces pruning genes, led to a nearly complete suppression of the pruning phenotype caused by VCP QQ. This genetic interaction suggested that VCP might be required for the expression of one or several ecdysone target genes during pruning (Rumpf, 2014).

How could VCP be linked to Sox14? The suppression of the vcp mutant phenotype by Sox14 overexpression could be achieved in one of several ways. Sox14 could be epistatic to VCP -- that is, VCP could be required for functional Sox14 expression -- and this effect would be mitigated by Sox14 overexpression. However, VCP could also be required for the expression of one or several Sox14 target genes, and enhanced Sox14 expression could overcome this requirement either via enhanced induction of one or several particular targets or via enhanced induction of other pruning genes, in which case Sox14 would be a bypass suppressor of VCP QQ. To distinguish between these possibilities, the effects were assessed of VCP inhibition on the expression of known genes in the ecdysone cascade required for pruning in class IV da neurons. Class IV da neuron pruning is governed by the Ecdysone Receptor B1 (EcR-B1) isoform, which in turn directly activates the transcription of Sox14 and Headcase (Hdc), a pruning factor of unknown function. Sox14, on the other hand, activates the transcription of the MICAL gene encoding an actin-severing enzyme. In immunostaining experiments, VCP QQ did not affect the expression of EcR-B1, Sox14, or Hdc at the onset of the pupal phase. However, the expression of Mical was selectively abrogated in class IV da neurons expressing VCP QQ, or in vcp^26-8 class IV da neuron MARCM clones . These data indicated that VCP might affect dendrite pruning by regulating the expression of the Sox14 target gene Mical, indicating that Sox14 might act as a bypass suppressor of VCP QQ (Rumpf, 2014).

How could VCP inhibition suppress Mical expression? To answer this question, whether Mical mRNA could still be detected in class IV da neurons expressing VCP QQ was assessed. To this end, enzymatic tissue digestion and FACS sorting were used to isolate class IV da neurons from early pupae (1-5 h APF). Total RNA was then extracted from the isolated neurons, and the presence of Mical mRNA expression was assessed by RT-PCR, using control samples or samples from animals expressing VCP QQ under ppk-GAL4. The Mical gene is large (~40 kb) and spans multiple exons that are transcribed to yield a ~15 kb mRNA. To detect Mical cDNA, primer pairs spanning several exons were used for two different regions of Mical mRNA, exons 14-16 and exons 8-12. [MICAL is on the (-) strand, but the exon numbering denoted by Flybase follows the direction of the (+) strand. Therefore, exons 14-16 are upstream of exons 8-12, and the latter are closer to the 3' end of MICAL mRNA.] MICAL mRNA was detectable upon VCP inhibition in these extracts with a primer pair spanning exons 14-16. The second primer pair spanning exons 8-12 also detected MICAL mRNA in both samples, but the RT-PCR product from the VCP QQ-expressing class IV da neurons had a larger molecular weight. Sequencing of the PCR products indicated that MICAL mRNA from VCP QQ-expressing class IV da neurons contained exon 11, which was not present in Mical mRNA from the control sample. Exon 11 is absent from all predicted MICAL splice isoforms except for a weakly supported isoform designated 'Mical-RM'. It introduces a stop codon into MICAL mRNA that would lead to the truncation of the C-terminal 1,611 amino acids from Mical protein. This portion of Mical protein contains several predicted protein interaction domains such as a proline-rich region, a coiled-coil region with similarity to Ezrin/Radixin/Moesin (ERM) domains, and a C-terminal PDZ-binding motif, and is required for the interaction between Mical and PlexinA. In addition, the truncated region contains the epitope for the antibody used in the immunofluorescence experiments, thus explaining the observed lack of Mical expression upon VCP inhibition. Given that a mutant of Mical with a smaller C-terminal truncation (compared with the one induced by VCP inhibition) was not sufficient to rescue the class IV da neuron dendrite pruning defect of mical mutants, disruption of VCP function likely results in expression of a truncated Mical protein without pruning activity. Taken together, these data suggest that the observed defect in MICAL mRNA splicing contributes significantly to the pruning defects of VCP mutants (Rumpf, 2014).

How is VCP linked to alternative splicing of MICAL mRNA? A plausible mechanism for the control of an alternative splicing event would be the modulation of specific (pre)mRNA-binding proteins. VCP has recently been linked to several RNA-binding proteins: human autosomal dominant VCP mutations cause frontotemporal dementia or ALS with inclusion bodies that contain aggregated human TDP-43; a genetic screen in Drosophila identified the RNA-binding proteins Drosophila TDP-43, HRP48, and x16 as weak genetic interactors of the dominant effects of VCP disease mutants; and HuR (a human homolog of the neuronal Drosophila RNA-binding protein elav) was recently shown to bind human VCP. Of these, TDP-43 and also elav have been linked to alternative splicing in various model systems, including Drosophila. Therefore this study used available specific antibodies to assess the levels and distribution of Drosophila TDP-43 (hereafter referred to as TDP-43) and elav. TDP-43 has previously been shown to localize to the nucleus in Drosophila motoneurons and mushroom body Kenyon cells. Surprisingly, TDP-43 was largely localized to the cytoplasm in class IV da neurons, where it was enriched in a punctate pattern around the nucleus, with only a small fraction also detectable in the nucleus, a localization pattern that could be reproduced with transgenic N-terminally HA-tagged TDP-43. Elav is a known nuclear marker for Drosophila neurons; in class IV da neurons, it was somewhat enriched in nuclear punctae. The effects of VCP inhibition on these two RNA-binding proteins was assessed. Elav localization did not change notably upon VCP QQ expression. Strikingly, TDP-43 became depleted from the cytoplasm of class IV da neurons and relocalized to the nucleus upon VCP QQ expression. Closer inspection revealed that TDP-43 in VCP-inhibited neurons was now enriched in nuclear dots that often also exhibited increased elav staining. The relocalization of TDP-43 from the cytoplasm to the nucleus was also observed in vcp^26-8 mutant class IV da neuron MARCM clones. Importantly, quantification and normalization of TDP-43 levels showed that VCP inhibition did not alter the absolute levels of TDP-43, suggesting that the observed effects were not a consequence of a defect in TDP-43 degradation. In fact, the only manipulation that resulted in a mild but significant increase in TDP-43 levels -- but without a change in localization -- was the expression of an RNAi directed against the autophagy factor ATG7, perhaps reflecting the degradation of cytoplasmic RNA granules through the autophagy pathway (Rumpf, 2014).

It was next asked whether manipulation of TDP-43 would affect class IV da neuron dendrite pruning. A previously characterized TDP-43 mutant, TDP-43 Q367X (28-128">28), did not display pruning defects, but overexpression of TDP-43 led to strong dendrite pruning defects at 16 h APF. In support of the hypothesis that TDP-43 acts in the same or a similar pathway as VCP during dendrite pruning, it was also found that a more weakly expressed TDP-43 transgene (UAS-TDP-43^weak) and VCP A229E, a weakly dominant-active VCP allele corresponding to a human VCP disease mutation, exhibited a synergistic inhibition of pruning when coexpressed. Interestingly, manipulation of elav gave very similar results as with TDP-43: elav down-regulation by RNAi did not affect pruning, but elav overexpression led to highly penetrant pruning defects (Rumpf, 2014).

To exclude the possibility that the pruning defects induced by TDP-43 or elav overexpression were due to long-term expression and aggregation of RNA-binding proteins, TDP-43 and elav overexpression was also induced acutely (24 h before the onset of pupariation). Pruning was still inhibited in these cases. Also, overexpression of several other RNA-binding proteins did not cause pruning defects, with two exceptions: a UAS-carrying P-element in the promotor of the adjacent x16 and HRP48 genes caused a strong pruning defect when expression was induced in class IV da neurons, and levels of a GFP protein trap insertion into the x16 gene were also markedly increased in class IV da neurons expressing VCP QQ, possibly indicating a role for VCP in x16 degradation. In further support of an involvement of VCP with RNA-binding proteins during neuronal pruning processes, it was also found that VCP is required for mushroom body γ neuron axon pruning and induces the accumulation of Boule, an RNA-binding protein that had previously been shown to inhibit γ neuron axon pruning when overexpressed. Thus, the data suggest that VCP regulates a specific subset of RNA-binding proteins and that this regulatory role of VCP is associated with its role in pruning (Rumpf, 2014).

As VCP is an integral component of the UPS, it was next asked whether the role of VCP in MICAL regulation and TDP-43 localization was also dependent on ubiquitylation and/or the proteasome. To address this question, Mical levels and TDP-43 distribution was assessed in UPS mutants with known pruning defects. An ubiquitylation enzyme known to be required for pruning is the E2 enzyme ubcD1. When TDP-43 localization was assessed in larval ubcD1^D73 mutant class IV da neurons, TDP-43 was again localized to the nucleus in these cells. Furthermore, a pronounced reduction of Mical expression in ubcD1^D73 mutant class IV da neurons was noted during the early pupal stage, indicating that ubiquitylation through ubcD1 is involved in the regulation of TDP-43 localization and Mical expression (Rumpf, 2014).

TDP-43 localization and Mical expression were assessed in proteasome mutants. A previously characterized mutant in the Mov34 gene encoding the 19S subunit Rpn8 was used. TDP-43 was again relocalized to the nucleus in Mov34 mutant class IV da neurons, and Mical expression was absent from Mov34 mutant class IV da neurons at 2 h APF. To rigorously address whether proteasomal proteolysis was also required for TDP-43 localization and Mical expression, the effect was assessed of Pros26¹, a previously characterized mutation in the 20S core particle subunit Prosβ6. In contrast to Mov34 mutant class IV da neurons, Pros26¹ mutant class IV da neurons displayed cytoplasmic TDP-43 localization, and robust Mical expression was detected in these neurons at 2 h APF. Thus, although ubiquitylation and the 19S proteasome are both required for Mical expression and normal TDP-43 localization, proteolysis through the 20S core particle of the proteasome is not. Importantly, Pros26¹ MARCM class IV da neurons showed strong dendrite pruning defects at 16 h APF, as did expression of RNAi constructs directed against subunits of the 20S core particle (Rumpf, 2014).

These data indicate that there must be several ubiquitin- and proteasome-dependent pathways that are required for dendrite pruning: one pathway requires ubcD1, VCP, and the 19S regulatory particle of the proteasome, but not the 20S core particle. This pathway regulates MICAL expression. A second UPS pruning pathway does depend on proteolysis through the 20S core. In an E3 ubiquitin ligase candidate screen, cul-1/lin19 was identified as a pruning mutant. Cul-1 encodes cullin-1, a core component of a class of multisubunit ubiquitin ligases known as SCF (for Skp1/Cullin/F-box) ligases. Class IV da neurons mutant for cul-1 or class IV da neurons expressing an RNAi construct directed against cul-1 had not pruned their dendrites at 16 h APF. However, unlike with VCP, ubcD1, and Mov34, cul-1 mutation did not affect Mical expression at 2 h APF, indicating that cullin-1 is not a component of the VCP-dependent UPS pathway involved in splicing and might thus be a component of a proteolytic UPS pathway. In support of this notion, a recent report independently identified cul-1 as a pruning mutant and associated it with protein degradation (Rumpf, 2014).

It has been proposed that the E2 enzyme ubcD1 and VCP would act to activate caspases during pruning. However, the dendrite pruning defects caused by those UPS mutants are much stronger than the phenotypes caused by caspase inactivation, which mostly causes a delay in the phagocytic uptake of severed dendrites by the epidermis. Although it cannot be excluded that ubcD1 and VCP contribute to caspase activation during pruning, the new mechanism proposed in this study -- control of RNA-binding proteins and MICAL expression -- likely makes a much stronger contribution to the drastic pruning phenotypes of UPS mutants (Rumpf, 2014).

How precisely do VCP, ubcD1, and the 19S proteasome contribute to MICAL expression? The data indicate that VCP inhibition causes missplicing of MICAL mRNA that likely leads to the expression of an inactive Mical protein variant. At the same time, VCP inhibition leads to the mislocalization of TDP-43, and possibly the dysregulation of a number of other RNA-binding proteins. The fact that these phenotypes correlate in the vcp, ubcD1, and Mov34 mutants gives a strong indication that they are related. TDP-43 had previously been identified as a suppressor of the toxicity induced by a weak VCP disease allele in the Drosophila eye. In class IV da neurons, reducing the amounts of TDP-43 (with a deficiency) or elav (by RNAi) did not ameliorate the pruning defect induced by VCP inhibition. Therefore, the possibility cannot be excluded that the two proteins act in parallel rather than in an epistatic fashion. As VCP has been shown to remodel protein complexes that contain ubiquitylated proteins and is structurally similar to the 19S cap, it is interesting to speculate that VCP and the 19S cap might alter the subunit composition of ubiquitylated TDP-43-containing complexes of RNA-binding proteins, and that this activity—rather than a direct action on TDP-43 (or maybe also elav) alone—might lead to both MICAL missplicing and TDP-43 mislocalization (Rumpf, 2014).

Interestingly, autosomal dominant mutations in human VCP cause frontotemporal dementia and ALS, a hallmark of which is the formation of aggregates that contain TDP-43. Most of these aggregates are cytoplasmic (and contain TDP-43 that has relocalized from the nucleus to the cytoplasm), but VCP mutations also induce TDP-43 aggregation in the nucleus, a situation that might be similar to the situation caused by VCP inhibition in class IV da neurons. Although human VCP disease mutations have been proposed to act as dominant-active versions of VCP with enhanced ATPase activity, both the disease allele and the dominant-negative ATPase-dead VCP QQ mutant cause class IV da neuron pruning defects and TDP-43 relocalization to the nucleus of class IV da neurons and therefore act in the same direction. It is thought that VCP can only bind substrates when bound to ATP, and will release bound substrates upon ATP hydrolysis. Thus, it is conceivable that the phenotypic outcome of inhibiting the ATPase (no substrate release) should be similar to that of ATPase overactivation (reduced substrate binding or premature substrate release): in both cases, a substrate protein complex would not be properly remodeled (Rumpf, 2014).

Taken together, these results indicate the existence of a nonproteolytic function of VCP and the UPS in RNA metabolism and highlight its importance during neuronal development (Rumpf, 2014).

Ter94 ATPase complex targets k11-linked ubiquitinated Ci to proteasomes for partial degradation

The Cubitus interruptus (Ci)/Gli family of transcription factors can be degraded either completely or partially from a full-length form [Ci155/Gli^FL] to a truncated repressor (Ci75/Gli(R)) by proteasomes to mediate Hedgehog (Hh) signaling. The mechanism by which proteasomes distinguish ubiquitinated Ci/Gli to carry out complete versus partial degradation is not known. This study shows that Ter94 ATPase and its mammalian counterpart, p97, are involved in processing Ci and Gli3 into Ci75 and Gli3^R, respectively. Ter94 regulates the partial degradation of ubiquitinated Ci by Cul1-Slimb-based E3 ligase through its adaptors Ufd1-like and dNpl4. Cul1-Slimb-based E3 ligase, but not Cul3-Rdx-based E3 ligase, modifies Ci by efficient addition of K11-linked ubiquitin chains. Ter94^{Ufd1-like/dNpl4} complex interacts directly with Cul1-Slimb, and, intriguingly, it prefers K11-linked ubiquitinated Ci. Thus, Ter94 ATPase and K11-linked ubiquitination in Ci contribute to the selectivity by proteasomes for partial degradation (Zhang, 2013).

Hh signaling plays important roles in metazoan development, and its malfunction is implicated in numerous human congenital disorders and cancers. Secreted Hh proteins bind Patched (Ptc)-iHog coreceptors to relieve an inhibitory effect of Ptc on Smoothened (Smo), which leads to activation of the Ci/Gli family of zinc finger transcription factors. Biochemical and genetic studies in Drosophila have revealed several important steps in the regulation of Ci/Gli activity. In the absence of Hh, full-length Ci, Ci155, is sequentially phosphorylated by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase I (CKI) and then ubiquitinated by Cullin 1 (Cul1)-Supernumerary limbs (Slimb, known also as β-TrCP)-based E3 ligase. This results in partial degradation by proteasomes, leaving the N terminus of Ci intact (Ci75) as a transcriptional repressor. In the presence of Hh, unphosphorylated Ci155 accumulates and enters nucleus to activate Hh target genes. As a feedback control of the pathway, active Ci155 induces the expression of roadkill (rdx)/Hib to form Cul3-Rdx-based E3 ligase and promotes complete proteasomal degradation of Ci155 (Zhang, 2013).

Although it is well established that Ci is ubiquitinated by Cul1-Slimb and Cul3-Rdx-based E3 ligases under different conditions, it remains unknown how proteasomes distinguish ubiquitinated Ci for partial versus complete degradation. As ubiquitinated proteins are transferred to proteasomes by different pathways, it is hypothesized that some specific components are involved in the recruitment of ubiquitinated Ci for partial degradation. Transitional elements of the endoplasmic reticulum 94 kDa (Ter94) was identified as the Drosophila homolog of yeast CDC48, which is a member of the ATPase associated with various cellular activities (AAA) family. In mammals, the CDC48/Ter94 homolog p97 (also known as VCP) mainly functions in endoplasmic reticulum-associated degradation (ERAD). Proteomic analysis revealed that p97 might play a broad role in regulating the turnover of ubiquitin proteasome system (UPS) substrates. This study has shown that Ter94 is a component of the Ci processing machinery and is critical for Ci75 formation (Zhang, 2013).

The control of partial versus complete proteasomal degradation of Ci and Gli3 is a major regulatory step in Hh signal transduction. How proteasomes distinguish ubiquitinated Ci to carry out either partial or complete degradation is not known. Based on the current findings, the following model is proposed. In the absence of Hh, Ci155 is phosphorylated and ubiquitinated by Cul1-Slimb-based E3 ligase to generate Ci75. In this process, K11-linked ubiquitin chains are added onto Ci155. Ter94Ufd1-like/dNpl4 forms a complex with Cul1-Slimb-based E3 ligase through Ufd1-like and Roc1a, and another component dNpl4 is bound to the K11-linked ubiquitin chains on Ci155. Through ATP hydrolysis, Ter94Ufd1-like/dNpl4 facilitates the delivery of ubiquitinated Ci155 to the proteasomes for processing (Zhang, 2013).

Besides Ci and Gli3, the best example of partial degradation is the processing of human nuclear factor-κB (NF-κB) and its yeast homologs, SPT23 and MGA2. Previous studies have suggested that some common features of processing determinant domain (PDD) are involved in the partial proteasomal degradation of Ci and NF-κB. However, Ci also undergoes complete proteasomal degradation by Cul3-Rdx-based E3 ligase. Why such 'degradation stop signals' fail to work in such instances and how proteasomes make the decision for partial or complete degradation are unknown. Based on the current results and previous studies, it is proposed that Ter94/p97 complex may specifically target 'partial-degradation-proteins' to proteasomes through K11-linked ubiquitin chains. Further investigation is needed to provide direct evidence to support this hypothesis. As many similarities are shared between Ci and NF-κB precursors in partial degradation, it will be interesting to test whether p97 and K11-linked ubiquitination are also involved in the partial degradation and/or maturation of p100 in NF-κB signaling (Zhang, 2013).

This study found that K11-linked chains are added onto Ci by Cul1-Slimb-based E3 ligase in the absence of Hh pathway activity, whereas Cul3-Rdx-based E3 ligase mainly adds K48-linked chains on Ci when the pathway is active. This illustrates a phenomenon that the same protein can be modified with different types of ubiquitin chains by distinct E3 ligases. Although K11-linked chains added on APC substrates lead to complete degradation, the data demonstrate that K11-linked chains are involved in the partial degradation of Ci. These findings also raise the interesting possibility that the topology of ubiquitin chains may be recognized as a selective signal for proteasomal degradation. As mixed or heterologous ubiquitin chains may exist, further investigation is essential to determine whether mixed ubiquitin chains are formed by Cul1-Slimb-based E3 ligase on Ci (Zhang, 2013).

Protection of Armadillo/beta-Catenin by Armless, a novel positive regulator of Wingless signaling

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. This study identified a novel Wg/Wnt pathway component, Armless (Als; CG5469) that is required for Wg target gene expression in a cell-autonomous manner. Genetic and biochemical analyses showed that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced.Biochemical and in vivo studies showed that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. It is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, and it is proposed that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated (Reim, 2014).

The wingless (wg) gene was found nearly forty years ago with the characterization of aDrosophila mutant without wings. The gene encodes a secreted glycoprotein, the founding member of the Wnt family of signaling proteins. In the decades following its discovery, Wg/Wnt signaling has been shown to be essential during embryogenesis. Indeed, it is important throughout an organism's life, controlling also the homeostasis of different organs, for example, regeneration of epithelial cells in the intestine - the aberrant behavior of these cells in cancer is caused by constitutive Wg/Wnt signaling, which is consequently a key focus of medical and translational research (Reim, 2014).

The relay of the Wg signal is controlled at different levels. However, the pivotal step is the regulation of the levels of Armadillo (Arm)/β-Catenin, the key transducer of the Wg/Wnt pathway. A multiprotein complex consisting of the scaffold proteins Axin and APC and the kinases Shaggy/GSK3β and Casein kinase I (CKI) recruits and phosphorylates Arm/β-Catenin. This marks Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 ubiquitin ligase and subsequent degradation by the ubiquitin-proteasome system (UPS). When Wg/Wnt binds its receptors at the cell membrane, degradation of Arm/β-Catenin is prevented, presumably by protein interactions that lead to the dissociation of the E3 ubiquitin ligase from the Arm/β-Catenin destruction complex. As a consequence, Arm/β-Catenin translocates into the nucleus, where it adopts its role as a transcriptional effector of Wg/Wnt signaling. Although this step is crucial, and is a potential point of regulation, little is known about the players involved in the processing of Arm/β-Catenin and its ultimate degradation (Reim, 2014).

In a genome-wide RNA interference (RNAi) screen Armless (Als) was isolated as a regulator of proximodistal growth of Drosophila limbs, and has been shown in subsequent analyses to exert its function in the Wg pathway. Detailed genetic studies demonstrate that Als acts downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. Cells depleted for Als exhibit strongly reduced Arm protein levels. Importantly, the activity of a constitutively active form of Arm, which cannot be phosphorylated and hence escapes ubiquitination and proteasomal degradation, is insensitive to depletion of Als. Using immunopurification and mass spectrometry analysis this study found that Ter94 interacts with Als. Ter94 is an AAA ATPase associated with protein turnover and proteasomal degradation. In sum, these data suggest that Als acts downstream of the Arm/β-Catenin destruction complex to positively regulate Arm protein levels, possibly by rescuing Arm from ubiquitination via Slimb. The human ortholog of Als, UBXN6, can substitute for Als in Drosophila, and Wnt target gene expression was impaired upon knock-down of UBXN6 in HEK-293 cells. It is thus infered that Als and UBXN6 represent regulators of a conserved mechanism that ensures appropriate levels of Armadillo/β-Catenin by antagonizing its entry into the UPS (Reim, 2014).

A prevalent mechanism for controlling information flow in signaling pathways is the alteration of the protein levels of key components. In the Wg/Wnt pathway, the Arm/β-Catenin destruction complex targets Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 Ub ligase, resulting in proteasomal degradation and low cytoplasmic levels of Arm/β-Catenin in the Wnt pathway off state. If the pathway is turned on, Slimb-mediated ubiquitination is prevented, thus rescuing Arm from its proteasomal fate and causing a concomitant increase in Arm protein levels. This study describes Als as a new component of this control system; Als was found to be required to prevent the degradation of Arm/β-Catenin (Reim, 2014).

This study has identified als in a genome-wide in vivo RNAi screen in Drosophila. Because no EMS- or P-element-induced null allele was isolated, and because another gene overlaps with als, particularly thorough evidence validating als gene function was obtained. (1) The als phenotypes could be reproduced by nine different UAS-RNAi transgenes encoding independent RNA target sites. Together with an extended off target analysis, unintentional RNAi was ruled out as a cause for the als phenotypes. (2) RNAi-mediated inhibition of als expression was ascertained by monitoring als mRNA expression via real-time PCR and antisense mRNA in situ hybridization. (3) Expression of Als with different RNAi-insensitive rescue transgenes, as well as with its human ortholog UBXN6, rescued als phenotypes (Reim, 2014).

These analyses show that als encodes an essential positive Wg signaling component. This conclusion is based on the following evidence. als depletion caused wings with notched wing margins and loss of sensory bristles, which is characteristic of impaired Wg signaling. The distal wing region is most sensitive to als levels, as is the case for other positive components of Wg signaling. In agreement with this, increased als expression was found in the central wing pouch, at least in earlier L3 larval stages. Stimulation of the Wg pathway in wing imaginal discs or Kc-167 cells caused higher als expression, suggesting that als can be positively controlled by Wg signaling. However, Als levels must be precisely controlled since already mild overexpression of UAS-als elicits a dominant-negative effect on Wg signaling. The function of als for Wg signaling is not restricted to the wing: also in other tissues, such as the thorax, eyes, legs, and the embryo, als phenotypes are identical to those seen when Wg signaling is disturbed. Also in human HEK-293 cells UBXN6/UBXD1, the ortholog of Als, was found to be required for Wnt signaling, and human UBXN6 largely rescues the als phenotypes in Drosophila, which suggests their functional conservation. Depletion of als also enhanced Wg-sensitized phenotypes, further supporting the notion that its product is a Wg pathway component. Moreover, the expression of positively regulated Wg target genes is reduced or abolished upon loss of als function, while Wg-repressed target gene expression is ectopically activated. Importantly, while interfering with als function suppressed Wg signaling, it did not affect other pathways, such as Notch and Hh, Jak/Stat, or EGFR signaling. However, it cannot be ruled out that als is not required in another pathway in a different biological context. In humans, UBXN6 is reported to play a role in diverse scenarios: for example, it was shown to play a role in Caveolin turnover in human osteosarcoma U2OS cells. This might indicate a broader role of UBXN6 in mammalians (Reim, 2014).

The data show that Als regulates Armadillo protein levels. Based on epistasis experiments, Als acts downstream of Shaggy/GSK3β and upstream of the SCF/Slimb/βTRCP E3 Ub ligase, which is known to ubiquitinate Arm, a prerequisite for proteasomal degradation. Consistent with this, the degradation-resistant form of Arm could completely bypass the requirement for als, in contrast to the wild-type form of Arm. This suggests that proteasomal degradation acts downstream of als; however, this cannot be taken as an unambiguous proof. Importantly, depletion of ubiquitin and overexpression of CSN6, a negative regulator of SCF/Slimb/βTRCP E3 Ub ligase, could ameliorate the als phenotype (as well as phenotypes based on the overexpression of Axin or Shaggy, which overactivate the destruction complex, thus resulting in enhanced Arm degradation). In contrast, altering these factors did not ameliorate the Lgs phenotype, which is caused by interfering more downstream in the Wg pathway. These findings suggest that als works upstream of proteasomal degradation. A further informative experiment was monitoring Wg pathway components with respect to protein levels: Arr, Fz, Axin, APC, Sgg, and Arm. The only change in the absence of Als function was Arm: its levels were strongly reduced upon als depletion. The effects on Arm levels could be due either to a direct effect on Arm or to an indirect effect on a negative component. Importantly, the rate-limiting factor Axin as well as other key negative components of the Arm/β-Catenin destruction complex were unaltered uponals depletion (Reim, 2014).

Some further mechanistic insight was obtained with the finding that Ter94 interacts in vitro and in vivo with Als. Interestingly, Als-Ter94 was found to localize at the cell cortex, as was similarly observed for the Arm/β-Catenin destruction complex. The studies are consistent with earlier work that showed that the human ortholog of Ter94, p97, interacts with UBXN6 (Madsen, 2008). Ter94/p97/Cdc48 is a conserved and highly abundant AAA ATPase that was found to associate with SCF/Slimb/βTRCP E3 Ub ligases or proteasomal shuttle factors to mediate UPS-mediated protein degradation. Specifications of the diverse activities of Ter94/p97 and the fate of its substrates are mainly exerted by UBX domain protein co-factors, which eventually either promote or hinder p97's function in protein turnover; an example of the latter involves the dissociation of the SCF/Slimb/βTRCP E3 Ub ligase complex, eventually leading to its inactivation. Interestingly, it was recently reported that inactivation of the E3 ligase complex upon Wnt signaling is achieved by its dissociation from the destruction complex. Based on the current experiments and what is known about Ter94/p97, a possible mechanism is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, thereby rescuing Arm levels. No increased protein levels were observed of Slimb, Axin, Shaggy, or APC in this analyses; thus, the results favor a model in which Als antagonizes Ter94 to hinder the transfer of Arm to the proteasome by interfering with the SCF/Slimb/βTRCP E3 Ub ligase function or its assembly. Importantly, no interaction was found between Arm and Als. This is consistent with the finding that UBX domain family members lacking an UBA domain, such as UBXN6/Als, do not directly interact with substrate proteins (Beskow, 2009), but are necessary for the activity or fate of the Ter94/p97 (Reim, 2014).

Interestingly, Zhang (2013) found that ter94 depletion affected the partial proteolysis of Ci. However, that study observed neither any typical consequence of disturbed Hh signaling per se (i.e., no alteration of Hh target gene expression in genes such as ptc) nor any phenotypical consequence upon overexpression of a dominant negative form of Ter94 (i.e., aberrant wing patterning and growth typical for Hh signaling). This is consistent with the current data that neither Ci target expression nor Hh signaling was affected upon als or ter94 depletion (Reim, 2014).

p97/Ter94 is known as a highly pleiotropic AAA ATPase associated with many cellular functions. Further, p97/Ter94 acts in multifaceted and large protein-protein complexes, and it is its regulatory co-factors, including UBX domain proteins, that render p97/Ter94 specific for a certain task in a particular cellular context. For example, p47/Shp1 is a co-factor of p97/Ter94 that blocks other co-factors from Ter94 binding (Kondo, 1997; Bruderer, 2004). Interestingly, in Kc-167 cell mass spectroscopy experiments, this study found p47 in Ter94/Als protein complexes, but only in the absence of Wg stimulation. On the other hand, als transcript and Als protein levels were elevated upon Wg signaling. These findings suggest a dynamic regulation of the Ter94 complex upon signaling inputs. The identification and functional analysis of all key components of the Als-Ter94 complex will be needed to obtain a refined insight into Als-Ter94's molecular mechanism (Reim, 2014).

Critically, this work spotlights an underappreciated facet in the control of the output of the entire canonical Wg/Wnt pathway -- how Arm/β-Catenin is handed over to the proteasome -- and the potential for regulating this step; this works also indicates that this step, in contrast to the conventional wisdom, is tunable. The identification and characterization of the UBX protein Als as a positive regulator of Wg/Wnt signaling contributes to this layer of pathway control (Reim, 2014).

The Spartan ortholog Maternal haploid is required for paternal chromosome integrity in the Drosophila zygote

The animal sperm nucleus is characterized by an extremely compacted organization of its DNA after the global replacement of histones with sperm-specific nuclear basic proteins, such as protamines. In the absence of DNA repair activity in the mature gamete, the integrity of the paternal genome is potentially challenged by the unique topological constraints exerted on sperm DNA. In addition, the maintenance of paternal DNA integrity during the rapid remodeling of sperm chromatin at fertilization has long been regarded as a maternal trait. However, little is known about the nature of the egg proteins involved in this essential aspect of zygote formation. Previous work has characterized the unique phenotype of the classical Drosophila maternal effect mutant maternal haploid (mh), which specifically affects the integration of paternal chromosomes in the zygote. This study shows that MH is the fly ortholog of the recently identified human DVC1/Spartan (Davis, 2012; Mosbech, 2012), a conserved regulator of DNA damage tolerance. Like Spartan, MH protein is involved in the resistance to UV radiation and recruits the p97/TER94 segregase to stalled DNA replication forks in somatic cells. In the zygote, mh phenotype was found to be consistent with perturbed or incomplete paternal DNA replication. Remarkably, however, the specific accumulation of MH in the male pronucleus before the first S phase suggests that this maternal protein is required to maintain paternal DNA integrity during nuclear decondensation or to set the paternal chromatin landscape in preparation of the first zygotic cycle (Delabaere, 2014).

The original mh1 allele [or fs(1)1182] was isolated in an ethyl methanesulfonate mutagenesis screen for X-linked female sterile mutations]. Complementation analyses with newly available deficiencies allowed reduction of the mh genetic interval to a 33 kb region (13C3-13C5) with 12 predicted genes. As mh is a strict maternal effect mutation, focus was placed on CG9203, a gene mainly expressed in adult ovary. A P element (P{SUPor-P}CG9203KG05829) inserted in CG9203 was mobilized to generate deletion alleles by imprecise excision. A female sterile mutation was isolated, mh², which did not complement the maternal effect embryonic lethal phenotype of mh1. Molecular analysis of the mh² allele revealed the presence of a 903 bp deletion in CG9203. The deletion generated a premature STOP codon at the end of the second exon, leaving only 65 residues of the 724 aa predicted wild-type protein. The identity of mh and CG9203 was finally confirmed by the rescue of mh² female sterility with a transgenic bacterial artificial chromosome (BAC) fragment containing the entire CG9203 gene and with a transgene expressing Maternal Haploid (MH) tagged in its N terminus with the V5 peptide (V5::MH) (Delabaere, 2014).

Homozygous mh² females produced eggs in normal quantities (hereafter mh² eggs), but that failed to hatch. Hemizygous mh²/Y males were viable and fertile. Fertilization, pronuclear decondensation and apposition seemed to occur normally in mh² eggs. However, in metaphase of the first zygotic mitosis, paternal chromosomes appeared improperly condensed and systematically formed a chromatin bridge in anaphase in a way identical to what has been described for mh1 eggs. As for mh1, most mh² embryos arrested their development after a few catastrophic mitoses, but about 20% escaped this early arrest and developed as nonviable gynogenetic haploid embryos after the loss of paternal chromosomes at the first mitosis (Delabaere, 2014).

Interestingly, sequencing of the CG9203 coding region in mh1 revealed the presence of five nonsynonymous substitutions (M151K, A368D, K372N, E381K, and K441T) compared to the reference genome sequence. The high evolutionary conservation of methionine 151 suggests that the M151K mutation is responsible for the mh1 phenotype, while the other changes could be polymorphisms (Delabaere, 2014).

Since maternal chromosomes in mh mutant eggs condense and divide normally, the mh phenotype cannot be explained by a general defect in mitotic chromosome condensation. Several studies have established that perturbing DNA replication can lead to abnormal mitotic chromosome condensation and a chromatin bridge in mitosis. To determine if the male pronucleus in mh eggs is capable of replicating its DNA, attempts were made to incorporate the thymidine analog EdU (5-ethynyl-2′-deoxyuridine) during the first zygotic S phase. Feeding adult females with EdU allowed observation, on very rare occasions, of its incorporation in DNA during the first zygotic S phase. In all four cases of mh cycle 1 embryos that had been exposed to the reagent, EdU had been clearly incorporated in both parental sets of chromosomes. Although the rarity of these cases and the relative faintness of the staining did not allow ureliable appreciation of the level of EdU incorporation, this experiment nevertheless confirmed that paternal chromosomes at least initiate replication in mh eggs. Attempts were made to directly evaluate the phenotypic consequences of impeding DNA synthesis during the first zygotic replication. The expression of the large catalytic subunit of DNA polymerase epsilon complex (DNApol-ε) was knocked down by expressing a small hairpin RNA in the female germline. Strikingly, DNApol-ε knocked-down females produced embryos where both parental sets of chromosomes failed to condense normally in metaphase and systematically formed a chromatin bridge in anaphase, in a way similar to paternal chromosomes in mh mutant eggs. Interestingly, in both cases, the dynamics of proliferating cell nuclear antigen (PCNA) in pronuclei appeared normal, again suggesting that DNA replication was not entirely disrupted. Taken together, these observations indicate that the mh phenotype is indeed compatible with a paternal DNA replication problem (Delabaere, 2014).

The distribution of MH protein was investigated at fertilization. In Drosophila, eggs are fertilized shortly before deposition and male pronucleus formation occurs rapidly as female meiosis resumes. A polyclonal antibody directed against two MH peptides was generated. Remarkably, in wild-type eggs, the anti-MH antiserum specifically stained the decondensing male nucleus but not female chromosomes in meiosis II. No staining was detected in the male nucleus in mh² eggs, thus demonstrating the specificity of the antibody and confirming that mh² is a null allele. In contrast, the mh1 mutation did not affect the localization of the protein. Finally, in eggs from mh²; V5:mh females, it was confirmed that V5::MH also specifically localized in the male nucleus using anti-V5 antibodies. Importantly, the localization of MH or V5::MH in the male nucleus appeared very transient. MH was most abundant in the male nucleus of eggs in metaphase of female meiosis II but then rapidly vanished. As metaphase of female meiosis II is the earliest stage that can be practically observed, it is not known if MH localizes earlier in the male nucleus, notably when protamine-like proteins are replaced with histones. Importantly, MH was no longer detected in the male nucleus at the onset of the first zygotic S phase, just before pronuclear apposition. Thus, although the improper condensation of paternal chromosomes in mh mutant zygotes is reminiscent of perturbed DNA replication, the results clearly show that MH is required in the male nucleus well before the first S phase, indicating that the observed phenotype is an indirect consequence of an earlier defect (Delabaere, 2014).

Blast analyses revealed that MH is highly conserved among metazoans and, recently, its human ortholog Spartan/C1orf124/DVC1 was characterized by several groups. Like Spartan, MH is characterized by the presence of a large and remarkably conserved SprT domain in its N terminus. The domain spans 185 residues (111-296) including a stretch of 28 residues that are identical in human Spartan and that contain a predicted HExxH catalytic motif found in zinc-dependent metalloproteases. Searching protein databases identified only two human proteins with an SprT domain, Spartan and ACRC (Acidic-repeat containing protein), and four in Drosophila melanogaster, including MH. Thus, SprT-containing proteins seem relatively rare in animal proteomes (Delabaere, 2014).

Another key feature of Spartan/MH orthologs is the presence of at least one ubiquitin binding zinc finger (UBZ) of the CCHC-type in its C terminus. The association of the SprT domain with one or two UBZ domains is unique to Spartan orthologs, and on the basis of this criterion, a single Spartan/MH representative was found in most animal species (Delabaere, 2014).

To investigate the functional contribution of these protein domains at fertilization, a series of transgenes was generated expressing various mutated versions of V5::MH under the control of its endogenous upstream sequences. All transgenes were inserted in the same attP landing platform in order to achieve similar expression levels. A control transgene expressing wild-type V5::MH fully rescued the fertility of mh² females. In the ovaries of these females, V5::MH accumulated in nurse cell nuclei and in the germinal vesicle (the oocyte nucleus) from stage 10 egg chambers onward, thus reflecting the main germline expression of mh in adult females. Two independent transgenes were generated with point mutations designed to inactivate the predicted HEMIH peptidase motif of the SprT domain. Changing both histidine residues of the motif or the glutamic acid completely annihilated the ability of the transgenes to rescue mh². Unexpectedly however, these mutated versions of V5::MH could not be detected in ovaries despite a level of mRNA expression equivalent to the endogenous gene, suggesting that an intact peptidase motif is required for protein stability. Unfortunately, failure to detect MH or V5::MH in a western blot assay using anti-MH or anti-V5 antibodies, respectively, prevented a direct testing of this possibility. The essential role of the SprT domain of MH is also supported by the presence of the M151K mutation in the original mh1 allele. This residue lies upstream the HEMIH catalytic motif within the SprT domain and is highly conserved in Spartan/MH orthologs. These results indicate that the SprT domain and its putative proteolytic activity are essential for the function of MH. However, the role of this metalloprotease domain in the decondensing male nucleus remains elusive, as it is for its human ortholog in somatic cells (Delabaere, 2014).

In another series of transgenes, the predicted UBZ motifs of MH were mutated or deleted. Removing only the second UBZ (V5:mhC699∗ transgene) did not abolish the fertility of rescued females. However, inactivation of the first UBZ (V5:mhC588F) or deletion of both motifs (V5:mhΔUBZ) destabilized the protein and prevented mh² rescue. Besides the SprT and UBZ domains, human Spartan contains a PCNA-interacting protein (PIP) box and a SHP motif required for the interaction with the VCP/p97 segregase. Both motifs seem to be conserved in MH although they do not perfectly match the consensus. However, deletion of the PIP (V5:mhΔPIP) or SHP (V5:mhΔSHP) reduced but did not abolish the fertility of rescued females, demonstrating that these motifs are not essential for the pronuclear function of MH (Delabaere, 2014).

A recent series of papers collectively established that Spartan/DVC1 is a regulator of translesion synthesis (TLS) and is involved in the response to UV-induced DNA damage in human cells TLS is a mechanism of DNA damage tolerance in which the replacement of replicative polymerases with specialized TLS polymerases allows stalled replication forks to bypass certain types of DNA lesions. The finding that mh encoded a putative conserved regulator of TLS was unexpected when considering its highly specialized and replication-independent function in the male pronucleus. To explore the possibility that MH could play an additional role in TLS in somatic cells, the sensitivity of mh² mutant larvae to UV radiation was tested. Strikingly, mh² third instar larvae displayed the same sensitivity to UV light than larvae deficient for the TLS polymerase DNA pol-η. Importantly, the UV resistance of mh² larvae was fully restored by two copies of the V5:mh transgene, thus confirming the specific implication of MH in this resistance (Delabaere, 2014).

In human cells, Spartan is almost systematically found associated with the replication factor PCNA, which is also a key regulator of the polymerase switch at the site of DNA lesions during TLS. Spartan and PCNA hence colocalize in multiple nuclear foci that correspond to stalled DNA replication forks. To test the intrinsic ability of MH to associate with PCNA in somatic cells, this study took advantage of salivary gland polytene chromosomes that naturally accumulate PCNA at stalled replication forks. In wild-type third instar larvae, PCNA was observed accumulated in randomly distributed foci that usually did not span the entire width of polytene chromosomes. As mh is not expressed in salivary glands (Flybase), transgenic lines were generated expressing V5::MH under the control of the inducible UAS/Gal4 system. Strikingly, induction of the UAS-V5:mh transgene in salivary gland led to a near perfect colocalization of V5::MH with PCNA foci. V5::MH foci were indistinguishably detected using anti-MH or anti-V5 antibodies. It was thus concluded that MH is indeed capable of accumulating with PCNA at stalled replication forks, thus sharing this property with its human ortholog. The precise role of Spartan at DNA replication blocks is, however, not entirely clear. Several studies have proposed that Spartan regulates TLS by enhancing Rad18-dependent PCNA ubiquitination or by stabilizing ubiquitinated PCNA and RAD18 at DNA damage sites. The apparent absence of a RAD18 homolog in Drosophila prevented a test this possibility. Two other groups have alternatively proposed that Spartan could be involved in the recruitment of the p97/VCP segregase to replication blocks to limit the extent of TLS by extracting TLS polymerases. The molecular chaperone p97/VCP is a conserved AAA-ATPase (ATPase associated with diverse cellular activities) represented in Drosophila by the ubiquitously expressed TER94 protein (Delabaere, 2014).

Immunofluorescence of salivary glands with anti-p97/VCP antibody (that recognizes TER94) barely stained squashed polytene chromosomes. Surprisingly however, the accumulation of TER94 was observed at MH foci when V5::MH was expressed in salivary glands. These results demonstrate that MH is indeed capable of recruiting TER94 at stalled forks, thus strongly supporting the same property for Spartan/DVC1 in human cells. However, despite the functional conservation of MH and Spartan in somatic cells, neither PCNA nor TER94 were detected in the decondensing male nucleus, thus reinforcing the idea that MH functions differently in the egg and in larval somatic cells. In addition, the fertility of DNApol-η12 mutant females argues against a specific implication of TLS per se in the male pronucleus (Delabaere, 2014).

Finally, during the course of this study, it was discovered that the DNA damage marker γgamma;H2A.Z (histone H2A.Z phosphorylated at serine 137) specifically accumulated as nuclear foci in the decondensing male nucleus, in a way similar to the asymmetric distribution of γH2A.X foci in the male pronucleus of mouse zygotes. However, the dynamic distribution of these paternal γH2A.Z foci did not seem altered in mh mutant eggs, suggesting that MH function is at least not directly related to the presence of these marks, although this does not rule out that these putative paternal DNA damage could persist in mutant eggs (Delabaere, 2014).

Although MH shares with Spartan a somatic role in UV resistance, the essential replication-independent function of this metalloprotease in the male pronucleus is not consistent with canonical TLS. A role of MH is instead favored specifically related to the unique chromatin or topological features of sperm DNA. Although mh does not perturb global paternal chromatin assembly triggered by the eviction of sperm protamines, it is possible that yet unknown sperm nuclear proteins require MH for their timely proteolysis at fertilization, in preparation of the first S phase. Alternatively, the recently formulated hypothesis that Spartan could repair DNA-protein crosslinks, such as those involving topoisomerases, is particularly stimulating as these enzymes are involved in sperm DNA topological rearrangements at the histone-to-protamine transition. Finally, as Drosophila and humans share a protamine-based organization of sperm chromatin, future work will have to determine if Spartan/DVC1 is similarly required in mammals to preserve the integrity of paternal chromosomes at the onset of embryogenesis (Delabaere, 2014).

Ecdysone-induced receptor tyrosine phosphatase PTP52F regulates Drosophila midgut histolysis by enhancement of autophagy and apoptosis

The rapid removal of larval midgut is a critical developmental process directed by molting hormone ecdysone during Drosophila metamorphosis. To date, it remains unclear how the stepwise events can link the onset of ecdysone signaling to the destruction of larval midgut. This study investigated whether ecdysone-induced expression of receptor protein tyrosine phosphatase PTP52F regulates this process. The mutation of the Ptp52F gene caused significant delay in larval midgut degradation. Transitional endoplasmic reticulum ATPase (TER94), a regulator of ubiquitin proteasome system, was identified as a substrate and downstream effector of PTP52F in the ecdysone signaling. The inducible expression of PTP52F at the puparium formation stage resulted in dephosphorylation of TER94 on its Y800 residue, ensuring the rapid degradation of ubiquitylated proteins. One of the proteins targeted by dephosphorylated TER94 was found to be Drosophila inhibitor of apoptosis 1 (DIAP1), which was rapidly proteolyzed in cells with significant expression of PTP52F. Importantly, the reduced level of DIAP1 in response to inducible PTP52F was essential not only for the onset of apoptosis but also for the initiation of autophagy. This study demonstrates a novel function of PTP52F in regulating ecdysone-directed metamorphosis via enhancement of autophagic and apoptotic cell death in doomed Drosophila midguts (Santhanam, 2014).

This study shows that ecdysone-induced expression of PTP52F and the subsequent tyrosine dephosphorylation of TER94 coordinate to construct upstream signaling determinants for a precise time-dependent degradation of larval midgut. The transient expression of Ptp52F gene at the PF stage is regulated by the functional EcR. Immediately after the level of endogenous PTP52F protein is detectable in larval midgut, TER94 becomes dephosphorylated on its Y800 residue. This modification may be critical to the rapid degradation of ubiquitylated proteins through a TER94-dependent regulation of ubiquitin proteasome system (UPS). Although the exact mechanism remains elusive, recent studies have suggested that only the tyrosine-dephosphorylated form and not the tyrosine-phosphorylated form of VCP interacts with cofactors for processing ubiquitylated substrates of UPS. Because VCP and TER94 share some evolutionarily conserved features, it is proposed that the same phosphorylation- and dephosphorylation-dependent mechanism may be adopted by TER94. Ubiquitylated DIAP1, a potential substrate of UPS, was found to be targeted by the Y800 dephosphorylated form of TER94. DIAP1 was rapidly degraded in cells in which levels of PTP52F were increased, as illustrated by in vivo observations in Drosophila midgut during metamorphosis. Consequently, the proteolysis of DIAP1 in response to inducible expression of PTP52F terminates the inhibitory effect on autophagy, allowing the initiation of autophagic cell death accompanied by apoptotic cell death for the destruction of the larval midgut tissues. Since the regulatory role of all Drosophila homologs of caspases have been ruled out in the process of larval midgut histolysis, it is likely that DIAP1 degradation-induced autophagic signaling may activate a yet-unknown pathway leading to the onset of apoptotic cell death in dying midgut. Additional experiments are needed to identify downstream effectors of PTP52F that modulate the cross talk between autophagy and apoptosis in the context of midgut maturation (Santhanam, 2014).

Identification of TER94 as a substrate dephosphorylated by PTP52F in larval midgut is interesting and important. From the time of their original cloning and identification, Drosophila TER94 and its vertebrate ortholog VCP have been characterized as key mediators involved in ER-associated degradation (ERAD), a major quality control process in the protein secretary pathway. Additional investigations have demonstrated degradation of proteins with no obvious relationship to ERAD by a VCP-mediated process, suggesting that TER94 and VCP may perform general functions in the proteolysis of ubiquitylated proteins. However, it remains unknown how this process is regulated under physiological conditions. The current study presents evidence that TER94-dependent degradation of ubiquitylated proteins is enhanced by PTP52F-mediated dephosphorylation of the penultimate Y800 residue. It has been suggested that the penultimate tyrosine (Y805 in VCP and Y800 in TER94) must be in a dephosphorylated form in order to interact with substrate-processing cofactors, such as the peptides N-glycanase (PNGase) and Ufd3, during UPS-mediated proteolysis. In addition, tyrosine phosphorylation levels of VCP/TER94 determine how fast ubiquitylated proteins are degraded by the USP pathway. Clearly, the finding that PTP52F is responsible for dephosphorylation of the penultimate tyrosine residue is critical for uncovering the functional role of VCP/TER94 in the regulation of protein degradation under physiologically relevant conditions (Santhanam, 2014).

This study has demonstrated that the timely degradation of DIAP1 in doomed larval midgut of developing flies is regulated by ecdysone-induced PTP52F. DIAP1 was identified to ubiquitylate proapoptotic proteins in living cells, thereby suppressing cell death signaling. Interestingly, DIAP1 can be ubiquitylated for degradation itself. The proteolytic process of ubiquitylated DIAP1 remained unclear until a recent report suggesting that TER94-mediated UPS pathway is involved in this process. This study has further shown that it is the dephosphorylated form of TER94 that is responsible for rapid DIAP1 degradation. In addition, although a previous study suggested that DIAP1 might suppress Atg1-mediated PCD, it was not known whether degradation of ubiquitylated DIAP1 could promote autophagy in vivo. This study has explored the underlying mechanism through which autophagic cell death is initiated by degradation of DIAP1. The data show that the constitutively tyrosine-phosphorylated form of TER94 acts as a gatekeeper ensuring the death signaling downstream of DIAP1 in'switch-off' mode. Developmental stage-dependent dephosphorylation of TER94 by inducible expression of PTP52F converts the autophagic death signaling into 'switch-on' mode through degradation of DIAP1. These findings thus explain, at least in part, how the massive destruction of larval midgut is precisely controlled by autophagic cell death. In conclusion, this study shows a novel function of PTP52F involved in the onset of autophagy and apoptosis essential for the removal of obsolete midgut tissues. Reversible tyrosine phosphorylation signaling controlled by PTP52F plays an indispensable role in the process of cell death-directed midgut maturation. Therefore, these findings open a new avenue for understanding the previously unexplored function of R-PTPs linked to regulation of autophagic and apoptotic cell death (Santhanam, 2014).

Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS

In humans, mutations in the fused in sarcoma (FUS) gene have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Cabeza (Caz) is the Drosophila ortholog of human FUS. Previous studies have established Drosophila models of ALS harboring Caz-knockdown. These flies develop locomotive deficits and anatomical defects in motoneurons (MNs) at neuromuscular junctions; these phenotypes indicate that loss of physiological FUS functions in the nucleus can cause MN degeneration similar to that seen in FUS-related ALS. This study aimed to explore molecules that affect these ALS-like phenotypes of Drosophila models with eye-specific and neuron-specific Caz-knockdown. Several previously reported ALS-related genes were examined, and genetic links were found between Caz and ter94, the Drosophila ortholog of human Valosin-containing protein (VCP). Genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown strongly enhanced the rough-eye phenotype and the MN-degeneration phenotype caused by Caz-knockdown. Conversely, the overexpression of wild-type ter94 in the background of Caz-knockdown remarkably suppressed those phenotypes. These data demonstrated that expression levels of Drosophila VCP ortholog dramatically modified the phenotypes caused by Caz-knockdown in either direction, exacerbation or remission. These results indicate that therapeutic agents that up-regulate the function of human VCP could modify the pathogenic processes that lead to the degeneration of MNs in ALS (Azuma, 2014).

VCP is a member of the AAA (ATPase associated with a variety of cellular activities) family of proteins, which are implicated in a large variety of biological functions including the regulation of ubiquitin-dependent protein degradation, control of membrane fusion and of dynamics of subcellular components, vesicle-mediated transport and nucleocytoplasmic shuttling. Association of VCP mutations with human disease was first identified in patients with IBMPFD (inclusion body myopathy with early-onset Paget disease and frontotemporal dementia) and more recently in those with ALS. There is a single ortholog of human VCP in Drosophila, named ter94, which is predicted to share ~83% amino acid sequence identity with human VCP (Azuma, 2014).

This study found that genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown severely enhanced the Caz-knockdown phenotypes in flies; it severely exacerbated locomotive disabilities and the degeneration of MNs induced by neuron-specific Caz-knockdown. Conversely, the overexpression of ter94 rescued those phenotypes (Azuma, 2014).

These results demonstrate, for the first time, a genetic link between Caz and ter94, the Drosophila orthologs of FUS and VCP, respectively. Although it would be necessary to confirm whether that is Drosophila-specific or not, the results suggest genetic interaction between FUS and VCP in human. Genetic interaction between TDP-43 and VCP in Drosophila was demonstrated previously; IBMPFD-causing mutations in ter94 lead to redistribution of TDP-43, from the nucleus to the cytoplasm, and redistribution of TDP-43 is sufficient to induce morphologically aberrant rough eyes (Ritson, 2010). This previous report suggests that VCP can balance the amount of TDP-43, which is a constituent of larger heteronuclear ribonucleoprotein (hnRNP) complexes, between nucleus and cytoplasm by acting as a nucleocytoplasmic shuttling molecule. In this schema, VCP functions to remove TDP-43 from RNP complexes, import TDP-43 into nuclei and degrade TDP-43 via autophagy. VCP might have similar functions with respect to FUS because FUS and TDP-43 have significant structural and functional similarities and are implicated in similar molecular processes. For example, TDP-43 and FUS act in the context of larger hnRNP complexes. FUS also continuously moves between the nucleus and the cytoplasm; therefore, FUS not only regulates gene expression in the nucleus, but also has important functions in the cytoplasm. This study showed that the decreased level of Caz in the nucleus and the resultant motor disturbance induced by neuron-specific Caz-knockdown could be rescued by overexpressed wild-type ter94 despite lacking any change of Caz protein in the CNS. If VCP has a shuttling function, wild-type ter94 overexpression could translocate Caz from cytoplasm to nucleus because nuclear importing function of ter94 would be dominantly induced in the situation with the deficiency of Caz in the nucleus. Conversely, the loss-of-function allele of ter94 (ter94^k15502) exacerbated the depletion of Caz from the nucleus probably because ter94-mediated nuclear import of Caz was compromised (Azuma, 2014).

It has been demonstrated that a polyQ tract can interact with VCP in Drosophila; specifically, either the strongest (ter94^k15502) or strong (ter94⁰³⁷⁷⁵) loss-of-function allele of ter94 suppressed the eye degeneration induced by an expanded polyQ tract, whereas the overexpression of wild-type ter94 in the background of Caz-knockdown enhanced this phenotype. Additionally, a chromosomal deletion of 46C3-46E02, the genomic region that contains ter94, acted as a dominant suppressor of the polyQ-induced phenotype. The present study and these previous reports together indicate that gain and loss of ter94 function rescued and exacerbated Caz-knockdown phenotypes, respectively, and that they had the converse effects on polyQ-induced phenotypes. These converse effects could be explained by the difference in disease pathogenesis; in polyQ-induced disease models, polyQ-containing pathogenic aggregates exist in nuclei of affected neurons; in contrast, Caz expression in nuclei is deficient in Caz-knockdown disease models. Overexpression of wild-type ter94, which functions in nuclear import of polyQ or Caz, would exacerbate nuclear polyQ aggregation, but could alleviate the nuclear deficiency of Caz protein (Azuma, 2014).

Ter94/VCP is a novel component involved in BMP signaling

Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor (TGF)-beta family, transduce their signal through multiple components downstream of their receptors. Even though the components involved in the BMP signaling pathway have been intensely studied, many molecules mediating BMP signaling remain to be addressed. To identify novel components that participate in BMP signaling, RNA interference (RNAi)-based screening was established by detecting phosphorylated Mad (pMad) in Drosophila S2 cells. Ter94, a member of the family of AAA ATPases, was identified as a novel mediator of BMP signaling, which is required for the phosphorylation of Mad in Drosophila S2 cells. Moreover, the mammalian orthlog of Ter94 valosin-containing protein (VCP) plays a critical role in the BMP-Smad1/5/8 signaling pathway in mammalian cells. Genetic evidence suggests that Ter94 is involved in the dorsal-ventral patterning of the Drosophila early embryo through regulating Decapentaplegic (Dpp)/BMP signals. Taken together, these data suggest that Ter94/VCP appears to be an evolutionarily conserved component that regulates BMP-Smad1/5/8 signaling (Zeng, 2014: PubMed).

Wnt-Dependent inactivation of the Groucho/TLE co-repressor by the HECT E3 ubiquitin ligase Hyd/UBR5

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF; see Pangolin) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin (see Armadillo) binds to TCF to overcome this repression, but how it achieves this is unknown. This study discovered that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. Groucho/TLE was identified as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription (Flack, 2017).

An essential step enabling Wnt-dependent transcription is the conversion of the Wnt enhanceosome from silent to active. This involves the binding of the Wnt effector β-catenin to TCF, which releases the transcriptional silence imposed on the linked genes by TCF-bound Groucho/TLE. This study has discovered a crucial role of Hyd/UBR5 in this process, and the evidence suggests that β-catenin directs the activity of this HECT ubiquitin ligase toward Groucho/TLE, to block its repressive activity. The evidence also implicates VCP/p97 in this UBR5-dependent inactivation of Groucho/TLE during Wnt signaling (Flack, 2017).

By generating UBR5 null mutant cell lines, it was possible to resolve previous inconsistencies regarding the effects of UBR5 depletion on Wnt/β-catenin responses in human cell lines. UBR5 KO cell lines consistently showed reduced Wnt responses, but no changes in β-catenin levels. This parallels the results from hyd mutant clones in flies, providing unequivocal evidence for Hyd/UBR5 as a positive regulator of Wnt signaling in fly and human cells (Flack, 2017).

Three strands of evidence implicate Groucho/TLE as a physiologically relevant substrate of Hyd/UBR5 during Wnt signaling. First, epistasis analysis revealed that Hyd/UBR5 acts below Armadillo/β-catenin, and thus likely targets a substrate in the nucleus, consistent with its nuclear localization. Second, the activity of UBR5 in ubiquitylating Groucho/TLE is triggered by Wnt/β-catenin signaling. Third, in Drosophila wing discs, hyd is largely dispensable in the absence of Groucho (as revealed by hyd gro double mutant clones), which provides powerful evidence that Hyd acts by antagonizing Groucho (Flack, 2017).

Two possible mechanisms by which β-catenin might activate UBR5 toward TLE3 during Wnt signaling are considered. Either, β-catenin might disinhibit UBR5 if this enzyme were normally autoinhibited, like the NEDD4 family HECT ligases. Indeed, one of these ligases (WWP2) is disinhibited by Dishevelled, which, upon polymerization, engages in multivalent interactions with WWP2 to release its cognate binding sites from autoinhibitory contacts. However, the strong activity of UBR5 toward PAIP2 in the absence of Wnt signaling argues against this mechanism. An alternative mechanism is favored, namely that β-catenin apposes enzyme and substrate, e.g., via triggering a conformational change of the Wnt enhanceosome that results in proximity between UBR5 and Groucho/TLE. Support for this mechanism comes from previous proximity labeling experiments that revealed a β-catenin-dependent rearrangement of some of the components within the Wnt enhanceosome (van Tienen, 2017), and from coIP assays showing that β-catenin promotes the association between UBR5 and TLE3 (Flack, 2017).

How does UBR5-dependent ubiquitylation of Groucho/TLE inactivate its co-repressor function? The most obvious mechanism involves proteasomal turnover of Ub-TLE, given the specificity of UBR5 in generating K48-linked Ub chains, which are efficient proteasomal targeting signals. In support of this, the levels of UBR5-dependent Ub-TLE3 are elevated after proteasome inhibition. However, negative results from the cycloheximide chase experiments argue against rapid proteosomal degradation being the primary mechanism underlying the UBR5-dependent inactivation of Groucho/TLE (Flack, 2017).

It was also considered that the ubiquitylation of the WD40 domain might interfere with its binding to cognate ligands, and thus weaken the association of Groucho/TLE with the Wnt enhanceosome. However, this does not seem to be the case since Ub-TLE3 appears to bind to its ligands as efficiently as unmodified TLE, including a K-only mutant which can only be ubiquitylated at K720, a WD40 pore residue that is crucial for ligand binding and co-repression. Evidently, the extended C terminus through which ubiquitin is attached to K720 is flexible enough to allow simultaneous ligand binding. However, for technical reasons, it was not possible to test the binding of Ub-TLE to the key ligand through which Groucho/TLE exerts its repressive function -- namely the nucleosomes to which Groucho/TLE binds via both its structured domains, to promote chromatin compaction. Nevertheless, it is plausible that the attachment of multiple ubiquitin chains to the WD40 domain would loosen up the binding of Groucho/TLE to nucleosomes, and thus attenuate its ability to compact chromatin (Flack, 2017).

Evidence based on dominant-negative VCP/p97 and two distinct VCP/p97 inhibitors implicates this ATPase in the Wnt-dependent inactivation of Ub-TLE. Intriguingly, a recent proteomic screen for NMS-873-induced VCP/p97-associated proteins identified TLE1 and TLE3 as the only Wnt signaling components, along with VCP/p97 adaptors and other putative substrates, consistent with the notion of Groucho/TLE is a substrate of this ATPase. VCP/p97 regulates the folding of ubiquitylated proteins, to promote their segregation from large structures, such as endomembranes, and also from large protein complexes, including DNA repair and chromatin complexes. It is therefore conceivable that VCP/p97 unfolds Groucho/TLE upon its ubiquitylation, especially if this modification loosened the interaction of Groucho/TLE with nucleosomes. Whatever the case, unfolding of the Groucho/TLE tetramer by VCP/p97 is likely to destabilize it, which would disable its repressive function. This is consistent with a recent proposal that the relief of Groucho-dependent repression is based on kinetic destabilization of the Groucho complex (Chambers, 2017), which may be facilitated by its ubiquitylation and unfolding by VCP/p97 (Flack, 2017).

One other E3 ligase has been shown to ubiquitylate TLE3, namely the RING ligase XIAP, which constitutively monoubiquitylates the Q domain of TLE3, apparently stimulating Wnt-dependent transcription by blocking its binding to TCF4. This contrasts with the Wnt-induced activity of UBR5 toward TLE3 revealed by this study. Evidently, the two ligases act distinctly, and also independently, given that the UBR5-dependent polyubiquitylation of TLE3 is normal in XIAP KO cells. However, it is also noted that the reduction of Wnt-dependent transcription in the XIAP KO cells was modest at best, compared to the substantial reduction in UBR5 KO cells. Either XIAP plays a lesser role in promoting transcriptional Wnt responses or a compensating E3 ligase was upregulated during the process of establishing XIAP KO cells. It is noted that the XIAP KO mice are viable, and without any overt mutant phenotypes, and that the Drosophila XIAP mutants do not show wg-like phenotypes, in contrast to the hyd mutant clones that phenocopy strong wg-like mutant phenotypes. All in all, it appears that UBR5 has a more profound role than XIAP in enabling transcriptional Wnt responses (Flack, 2017).

Inactivation of Groucho/TLE by UBR5 and VCP/p97 could also underlie other signaling-dependent gene switches that involve Groucho/TLE-dependent repression, e.g., Notch signaling, which depends on binding of Groucho/TLE to HES repressors. Indeed, recent genetic screens in C. elegans have identified the UBR5 ortholog sog-1 as a negative regulator of Notch signaling during nematode development. Although it is conceivable that hyd also affects Notch responses in flies, this study found that the derepression of the Notch target gene wg in hyd mutant wing disc clones is not sensitive to blockade by dominant-negative Mastermind, which argues against a role of Hyd in Notch-dependent transcription in this tissue. It is also noted that Ubr5 has been linked to defective Hedgehog signaling in mice, following an earlier lead of Groucho as a putative Hyd target in the context of Hedgehog signaling, although these links between Hyd/Ubr5 and Hedgehog signaling appear to be indirect (Flack, 2017).

However, UBR5 clearly also modifies substrates other than Groucho/TLE, including proteins with PAM2 motifs that are recognized by its MLLE domain, e.g., PAIP2 involved in translational control. Furthermore, via its UBR domain, UBR5 may recognize substrates of the N-end rule pathway, though few of these have been identified to date. Given the nuclear location of UBR5, it seems highly likely that most of its physiologically relevant substrates are nuclear proteins, e.g., the RING E3 ligase RNF168, which is ubiquitylated and destabilized by UBR5 during the DNA damage response (Flack, 2017).

UBR5 has been heavily implicated in cancer, although it is somewhat unclear whether it promotes or antagonizes tumor progression, which may depend on context. However, UBR5 amplification is the predominant genetic alteration in many types of cancers (far more prevalent than loss-of-function UBR5 mutations), and amplified UBR5 correlates with poor outcomes in breast cancer. This implies a tumor-promoting role of UBR5, consistent with its role in relieving Groucho/TLE-dependent repression of Wnt responses. It will be interesting to test whether UBR5 loss-of-function inhibits β-catenin-dependent tumorigenesis, e.g., in the intestine. This might be expected, given the results from the colorectal cancer cell line HCT116 whose β-catenin-dependent transcription is attenuated by UBR5 KO and whose proliferation is slowed down by VCP/p97 inhibition. If this were to apply generally to other colorectal cancer lines, this would indicate the potential of UBR5 and VCP/p97 as new enzymatic targets for therapeutic intervention in colorectal and other β-catenin-dependent cancers. It could widen the application of CB-5083, an orally bioavailable VCP/p97 inhibitor currently in phase 1 clinical trials (Flack, 2017).

Lipid-mediated phase separation of AGO proteins on the ER controls nascent-peptide ubiquitination

AGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. This study unexpectedly discovered that fly and human AGO proteins (see Drosophila Ago1), which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P(2). The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, this study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation (Gao, 2022).

AGO proteins play principal roles in regulating small-RNA-mediated gene silencing. Interestingly, AGO proteins are present in cytoplasmic RNA granules and associate with membrane-bound organelles (e.g., ER). However, the functional importance of membrane-associated AGO proteins has long been underestimated. This study focused on the membrane function of AGO proteins. Investigation of the dmAGO1 revealed a mechanism where ER-localized dmAGO1 forms a complex with the E3 ubiquitin ligase Ltn1 to catalyze the ubiquitination of nascent peptides and then coordinates with the VCP-Ufd1-Npl4 complex to process unwanted new protein products, which are ultimately degraded by the proteasome. Thus, in addition to facilitating miRNA-guided repression of RNA translation, dmAGO1 also acts in concert with the ribosome quality control machinery to ensure efficient repression of gene products. Given that AGO proteins play evolutionarily conserved roles in gene expression, this study provides a critical starting point toward mechanistic understandings of post-transcription-translation coupling controlled by AGO proteins (Gao, 2022).

AGO proteins associate with cellular membranes (e.g., ER membrane). However, the functional role of AGO proteins' association with the ER membrane and how this association is regulated have remained unresolved. Prior structural studies demonstrated that AGO proteins have six well-defined domains. The PAZ domain and the MID-PIWI domains function to anchor the 3′ and 5′ ends of the small RNA guide, respectively. This study showed that lipid binding might be a fundamental function of the N domain of dmAGO1 and hsAGO2. Sequence analysis revealed that these AGO proteins do not have any transmembrane domain. However, comparison assays between the N domain of AGOs and the PH domain from phospholipase C-δ1 suggested that the two domains display a striking similarity in topological patterns. Moreover, in vitro protein-lipid binding assays showed that AGOs selectively interacted with PI(4.5)P2. Finally, this study showed that vcp knockdown caused a significant increase in PI(4,5)P2 levels in ER membranes. The increased levels of PI(4,5)P2 serve as docking modules for AGO proteins to access the ER membrane and execute their function. Consistently, expression of BiP-Flag-AGOs-KDEL enhanced the gene silencing activity when compared with wild-type AGOs. It is worth noting that the 4-residue (KDEL) extension at the C terminal of AGOs (dmAGO1 or hsAGO2) did not affect the slicer activities to cleave the targets. Collectively, these findings revealed that lipid-mediated condensation of AGOs provides a primary mechanism for function of AGOs on the ER membrane, where AGOs repress target mRNA translation and control nascent-peptide ubiquitination (Gao, 2022).

Human AGO2 plays a compelling role in tumorigenesis and cancer aggressiveness; however, the mechanisms underlying the action of hsAGO2 in cancer remain elusive. Given that lipid binding is important for AGOs' function on cellular membrane and that the LBM contains two cancer-related mutations (based on COSMIC database), it would be interesting to study whether the lipid-mediated membrane function of hsAGO2 is related to cancer in future (Gao, 2022).

It is well documented that AGOs form a complex with miRNAs and utilize miRNAs to bind the target mRNAs for post-transcriptional repression. This study uncovered a mechanism by which AGOs form a complex with Ltn1 on the ER, where AGOs facilitate Ltn1 to catalyze nascent peptides for ubiquitination, thus ensuring efficient gene silencing. Although this study identified more than one hundred high-confident targets for dmAGO1/Ltn1, they were not analyze in depth in this study. The identified targets are of potential interest because they are involved in a variety of biological processes. For example, a significant portion of targets of dmAGO1/Ltn1 is involved in the secretion pathway, which is consistent with the previous findings. Interestingly, this study also found that a number of targets of dmAGO1/Ltn1 are mitochondrial proteins; this finding raises an intriguing possibility that dmAGO1/Ltn1-mediated PQC might contribute to the homeostasis and function of mitochondria. It would be important to solve this issue in the future studies (Gao, 2022).

A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. This study shows that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. These results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97(Ufd1/Npl4) complex. These data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome (Beskow, 2009).

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components, proteins were isolated that associate in early embryos with Vasa (Vas) and Tudor (Tud), two known polar granule associated molecules. Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) were identified as components of both Vas and Tud complexes and their localization to polar granules was confirmed by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. The recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly (Thomson, 2008).

Little is understood of the molecular events that link the assembly of germ plasm to the formation of germ cells. There is a strong correlation between polar granule formation and germ cell formation, yet their functional relationship is still unclear. In an attempt to understand polar granule formation and function this study set out to isolate polar granule components with a biochemical approach; proteins common to both Tud and Vas complexes were isolated. These complexes were isolated by cross-linking proteins from early embryonic extracts followed by anti-Tud or anti-Vas immunoprecipitation; proteins found in both complexes were then immunolocalized using EM. Using this method it was confirmed that Aub is a polar granule component and three new polar granule components were identified: ME31B, TER94 and eIF4A. Through genetic interaction analysis in transheterozygous embryos it was shown that decreasing the levels of Vas or Tud along with either Aub, ME31B, Ter94 or eIF4A reduces germ cell number. This approach both identified novel polar granule components and implicated novel processes in germ cell formation (Thomson, 2008).

The presence of Aub, ME31B and eIF4A in polar granules supports the hypothesis that polar granules and P bodies are structurally, and perhaps functionally, related. Recovery of CUP, an ME31B-interacting protein, in the Tud complex further supports this. Similar parallels have recently been found for the mouse chromatoid body, an electron dense structure in the male germ line with similarities to Drosophila polar granules and nurse cell nuage. RNAs in P bodies are stored in a translationally quiescent state and can later be either degraded or translationally activated in response to physiological cues. Translational repression in P bodies occurs at the level of mRNA recruitment to the ribosome, and through miRNA silencing pathways. The polar granule components that were identified suggest their involvement in both types of post-transcriptional regulation. Aub has been implicated in processing of germ line specific piRNAs. Findings that Aub associates with polar granules implicates piRNAs in germ cell formation, as has a previous study. In contrast, Vas and eIF4A have been closely linked with translational regulation and are not known to participate in miRNA silencing pathways (Thomson, 2008).

The RNA-rich nature of early polar granules supports the idea that specific germ line-specific mRNAs are stored in polar granules in a translationally repressed state. Subsequently, these RNAs are translated and their function may be required for germ cell formation and further development. How could general translational repression mediated by polar granules be overcome? Conceivably Vas could be a key factor. Vas, a highly conserved polar granule component with homologues in other species involved in germ line formation, binds directly to eIF5B. Disrupting the eIF5B-Vas interaction abrogates germ cell formation, presumably due to the loss of the ability of Vas to initiate translation of yet unidentified mRNAs. Thus, Vas may act as a germ line specific mRNA translation derepression factor. Other tissue specific factors could adapt a P body to a specific function or cell line. Identification of mRNAs that localize in polar granules and are dependent on Vas for their translation will no doubt provide more insight into this mechanism (Thomson, 2008).

Ultrastructural analysis of proteins found in the Vas and Tud containing complexes revealed that polar granules were often in close proximity or in contact with ER. Supporting such a link, Ter94 and ME31B were present in both Tud and Vas complexes, and are enriched in polar granules. Further work is required to elucidate what proportion of polar granules associate with ER, and whether this association is stage dependent. The presence of Ter94, an ER exit site marker, with Vas, ME31B, Aub and eIF4A in the same structure suggests that ER exit sites directly associate with the translational machinery with both activating and repressing factors. Polar granules may form at ER exit sites, which could provide a mechanism for the localization and assembly of mRNPs required for the translational regulation of their constituent mRNAs. There is evidence that P bodies associate with ER exit sites. In the Drosophila ovary, Trailer Hitch (TRAL) associates with ER exit sites and associates with P body components such as ME31B and CUP. The C. elegans homologue of TRAL, Car-1, associates with DCAP-1, a P-body marker, and car-1 mutations affect ER assembly. A single TRAL peptide was recovered in one immunoprecipitation with Tud, perhaps lending additional support to an association between polar granules and ER exit sites (Thomson, 2008).

Repeated attempts to biochemically isolate polar granules were made over 30 years ago, before the advent of modern analytical techniques that allow the identification of very small amounts of protein. From this work a major polar granule component of approximately 95 kDa was identified. The nature of this protein was not determined although Ter94 has approximately the same molecular mass, as does PIWI, a 97-kDa likely polar granule component that has eluded the currently used screens. PIWI associates with Vas, a polar granule component, as well as with components of the miRNA machinery. PIWI RNA and protein are enriched in germ plasm and piwi mutants have defects in germ cell formation. The screen also did not identify Osk, which was shown by a yeast two-hybrid screen to bind directly to Vas. This may be because these proteins were not present in high enough abundance for detection. Alternatively, since the reactive ends of the cross-linkers that were used specifically cross-link cysteine residues, they would not stabilize a particular protein-protein interaction unless a pair of cysteine residues is within the range of the cross-linker. The work demonstrates that a molecular approach can be a powerful complement to genetics, and that purification schemes based on two independent reagents can reduce signal-to-noise problems that are inherent in co-immunoprecipitation experiments. Molecular approaches such as this one also have the capacity to identify proteins involved in a developmental process that are encoded by genes with multiple functions, or required for cellular viability, that will therefore elude phenotype-based genetic screens (Thomson, 2008).

The Drosophila eve insulator Homie promotes eve expression and protects the adjacent gene from repression by polycomb spreading

The Drosophila even skipped (eve)gene has a Polycomb-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. This study show that this insulator has three major functions. It blocks the spreading of the eve Pc-silenced region, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When the insulator Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is 'replaced' by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc-silenced domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. These data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin (Fujioka, 2013).

A chromatin insulator mediates transgene homing and very long-range enhancer-promoter communication

Insulator sequences help to organize the genome into discrete functional regions by preventing inappropriate cross-regulation. This is thought to be mediated in part through associations with other insulators located elsewhere in the genome. Enhancers that normally drive Drosophila even skipped (eve) expression are located closer to the TER94 transcription start site than to that of eve. It was discovered that the region between these genes has enhancer-blocking activity, and that this insulator region also mediates homing of P-element transgenes to the eve-TER94 genomic neighborhood. Localization of these activities to within 0.6 kb failed to separate them. Importantly, homed transgenic promoters respond to endogenous eve enhancers from great distances, and this long-range communication depends on the homing/insulator region, which has been called Homie. The eve promoter contributes to long-distance communication. However, even the basal hsp70 promoter can communicate with eve enhancers across distances of several megabases, when the communication is mediated by Homie. These studies show that, while Homie blocks enhancer-promoter communication at short range, it facilitates long-range communication between distant genomic regions, possibly by organizing a large chromosomal loop between endogenous and transgenic Homies (Fujioka, 2009).

Some of the eve enhancers are close to the TER94 promoter, yet they do not activate TER94. Although TER94 is expressed nearly ubiquitously in embryos, it is expressed only at a low level in the mesoderm and anal plate, where eve expression is high in a subset of cells, making it unlikely that eve enhancers acting on TER94 would be masked by this expression. Therefore, something isolates TER94 from eve enhancers (and probably vice versa). Indeed, the region between the 3'-most eve regulatory element, a PRE, and the TER94 transcription start site has the properties of an enhancer-blocking insulator. It exhibits directional enhancer blocking in transgenes carrying eve enhancers in combination with either the eve promoter region or heterologous promoters, as well as between heterologous enhancers and promoters (Fujioka, 2009).

This insulator region was dissected in the context of transgenes carrying two different enhancers between divergently transcribed reporter genes. Some deletion mutants were still able to block the AR enhancer from activating the mini-white reporter, while allowing the eve mesodermal enhancer to activate the eve-promoter-lacZ reporter across the mutant insulator. This might result from a relatively weak interaction between the eve AR enhancer and the heterologous mini-white promoter, which suggests a degree of specificity of eve enhancers for their cognate promoter. This mechanism also contributes to long-range E-P communication mediated by the insulator. Furthermore, the recently discovered presence of an insulator at the 3' end of mini-white might contribute to stronger enhancer blocking in this direction (Fujioka, 2009).

First enhancer-blocking activity was narrowed down to an 800 bp sequence that spans the 5' end of TER94. Further dissection showed that the start site of TER94 is not required. This makes it unlikely that transcriptional interference makes a strong contribution to the results, although it could be significant in some cases, such as for δF, which retains the TER94 start site. Notably, region F, extending from ~150 to 45 bp upstream of this start site, seems particularly important for enhancer blocking. A similar situation pertains to the well-studied insulators scs and scs'. Perhaps some promoter regions induce a chromatin configuration that blocks the progression of activating complexes or chromatin modifications, through which enhancers communicate with target promoters (Fujioka, 2009).

The region between eve and TER94 also induces transgene homing. About 7% of transgenes carrying this region (27 out of 380 lines tested) inserted within 180 kb of eve. Among 27 homed lines, eight inserted within 1.5 kb of the endogenous insulator, suggesting that homing involves direct tethering, possibly through a homophilic protein complex formed on the element in the germline, where transgenic insertion occurs. The responsible element has been called Homie, for homing insulator at eve (Fujioka, 2009).

Although it is more difficult to dissect the region required for homing than it is to dissect the region required for enhancer blocking (due to the number of transgenic insertions required to validate a negative result), there is a clear correlation between these activities. Of the 210 transgenes tested for homing that carry all or part of the 800 bp R100 insulator, nine of them (4.3%) were homed, even though the 'homed' region is less than 0.4% of the genome. Protein-protein interactions among insulators, when they occur in the germline, might lead to transgene homing (Fujioka, 2009).

In previous studies of the eve 3' region, hundreds of lines were produced that carried the eve PRE, yet homing was not observed. Therefore, the eve PRE is not sufficient for homing. Furthermore, as the minimal homing element does not contain the PRE, this PRE is not required for either homing activity or long-range E-P communication. However, the engrailed homing region has PRE activity, indicating that some PREs may engage in homotypic interactions that facilitate homing. Consistent with this, long-range interactions among PREs were seen in the BX-C. Furthermore, the engrailed PRE may also facilitate long-distance E-P communication (Fujioka, 2009).

The eve-promoter-lacZ reporter in a homed transgene is usually expressed in a full eve pattern, showing communication with all of the endogenous eve enhancers from as far away as 180 kb, and across a number of other genes. Beyond the homing target region, there is a tendency for Homie-carrying transgenes to insert on chromosome 2R, particularly centromere proximal from eve. These insertions have not been referred to as 'homed', mainly to distinguish them from transgenes that pick up a full eve pattern of expression. However, they usually (9 out of 12) pick up a partial eve pattern. Intriguingly, Homie-carrying transgenes inserted as far as 3300 kb away, are capable of interacting with the endogenous eve AR and mesodermal enhancers. Previous indications of long-range E-P interactions mediated by transgenic insulators have come from the genetic and phenotypic analysis of transvection and related regulatory interactions (Fujioka, 2009).

The requirement for Homie in long-range E-P communication was directly tested using PhiC31-RMCE to compare transgenes with and without this region at the same chromosomal insertion site. Removal of Homie resulted in complete loss of the eve pattern. The same results were obtained at two different landing sites, at opposite ends of the homing region. Communication of distant 'shadow' enhancers with promoters across several intervening genes has recently been proposed, based upon bioinformatics-based identification of functionally conserved enhancer regions with no other apparent target promoters. The results suggest that for such distant enhancers to communicate effectively, they may need promoter-targeting and/or promoter-tethering sequences, and that some of these sequences might also act as insulators, generating a chromosomal architecture that facilitates functionally important interactions while preventing deleterious ones (Fujioka, 2009).

How does Homie mediate such long-range E-P communication? Both preferential insertion and the ability to pick up a partial eve pattern from long range could be explained by a homologous tethering mechanism, if it is assumed that this region of 2R is in relative proximity to the eve locus within a chromosome territory, both in the germline and in the developing AR and mesoderm. Homologous tethering might stabilize a functional E-P interaction, which in turn might facilitate transcription initiation through a combination of mechanisms, including targeting to regions of active transcription within the nucleus (Fujioka, 2009).

{Phi}C31 recombinase-mediated cassette exchange was used to test the role of promoter specificity in long-range communication. Exchanging a basal hsp70 promoter for the eve promoter caused a complete loss of communication with some endogenous eve enhancers but not others. The communication that remained was with the AR and mesodermal enhancers, the same ones that often communicate with either the eve or hsp70 promoters in transgenes inserted up to 3300 kb away. The ability of these enhancers to communicate at a much longer range than others might indicate relatively stable E-P interactions that can survive entropic forces tending to randomize their positions in the nucleus. Alternatively, the interactions of these enhancers might be specifically facilitated by Homie (Fujioka, 2009).

Another indication of the effects of promoter specificity in long-range E-P communication is that when the eve promoter was replaced by that of hsp70, β-gal reporter expression in the CNS changed from an eve-like pattern to one similar to that of TER94. Although it is possible that this TER94-like expression is driven by enhancers located near the insertion site, it is clear that which enhancers are targeted by the transgenic promoter depends in part on promoter specificity. Similar influences have recently been found on E-P communication at the engrailed locus (Fujioka, 2009).

How can Homie act as an insulator and also mediate long-range communication? The key may lie in the details of the resulting chromosomal architecture. Precedence for this idea comes from the phenomenon of insulator bypass, in which the enhancer-blocking activity of a single insulator can be negated by placing a second insulator between the enhancer and promoter. This phenomenon is consistent with data from those homed insertions that lie just downstream of endogenous Homie. In these cases, both the transgenic and endogenous Homies are interposed between the lacZ reporter and the endogenous enhancers that drive its expression. The data also show that the apparent bypass of endogenous Homie does not require that transgenic Homie lies between the interacting enhancer and promoter. In one case, the transgenic promoter lies between the two Homies, with the interacting enhancers on the outside. It is proposed that Homie has directionality, so that the two copies of Homie line up in parallel with each other within a wall-like structure. In the cases where both Homies are between the interacting enhancer and promoter, the Homies are inverted in orientation, whereas in the other case they are in the same orientation. In both cases, their lining up in parallel would tend to place the interacting enhancer and promoter on the same side of this wall-like structure, facilitating their communication. By contrast, a single copy of Homie would tend to block communication between sequences on either side, by placing them on opposite sides of the structure. Similar effects of insulator directionality have been seen for the Fab-8 and Mcp insulators (Fujioka, 2009).

In most homed lines, mini-white expression is not seen in an eve pattern. This might be due to the mini-white promoter being relatively weak and/or less compatible with eve enhancers than is the eve promoter, or even the hsp70 promoter, which also often picked up AR or mesodermal enhancer activity from great distances (facilitated by Homie). Intriguingly, however, although in most of the transgenes carrying Homie its 5' end was oriented toward the lacZ reporter, in one line (inserted at +46 kb), this orientation was reversed, and in that line mini-white was expressed in the eve pattern. Thus, it is possible that Homie directionality, through the mechanism described above for insulator bypass, might play a role in determining whether or not a weak E-P interaction is facilitated (Fujioka, 2009).

There are two likely possibilities for how Homie functions in the regulation of eve and TER94. The first is that it simply prevents eve enhancers from activating TER94, and also prevents eve from being expressed broadly in the CNS like TER94, which would probably cause mis-specification of neurons. Another, not mutually exclusive, possibility is that Homie works in conjunction with the nearby PRE to orchestrate functionally appropriate chromosomal architectures during development. Known insulators in the BX-C are each situated near a PRE, and these PRE-insulator regions interact with promoters in several contexts. The data suggest a similar interaction with the eve promoter region, based on the fact that three of the homed lines are inserted within the eve promoter region. Such an interaction might help enhancers from the 3' end of the eve locus communicate with the eve promoter, while also preventing inappropriate interaction with TER94 enhancers. One motivation for such a model is that in mutants for the PcG gene polyhomeotic, eve is ectopically expressed throughout the CNS, which is reminiscent of normal TER94 expression. Thus a loss of PcG repression, acting through the PRE, might disrupt the normal insulator function that prevents inappropriate activation of eve. This suggests that the functions of the PRE and Homie are coordinated during development, allowing the PRE to maintain either an activated or repressed state of eve in different cells, while maintaining the functional isolation of eve from TER94 (Fujioka, 2009).

Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber

A genetic screen was carried out in Drosophila to identify mutations that disrupt the localization of Oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components that are required during the mitotic divisions will also be required for Oskar mRNA localization during oogenesis, the following genetic screen was designed. A screen was carried out for P-element insertions in genes that slow down the blastoderm mitotic divisions. A secondary genetic screen was used to generate female germ-line clones of these potential cell division cycle genes and to identify those that cause the mislocalization of Oskar mRNA. Mutations were identified in ter94 that disrupt the localization of Oskar mRNA to the posterior pole of the oocyte. Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins that are involved in homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. A tested was carried out to see whether other membrane biosynthesis genes are required for localizing Oskar mRNA during oogenesis. Ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in Oskar mRNA localization during oogenesis (Ruden, 2000).

In order to identify new genes required for OSK mRNA localization, OSK localization defects in egg chambers were sought in mutants for cell division cycle (CDC) genes that had been isolated in a 'mitotic delay-dependent survival' (MDDS) genetic screen. The rationale for this is that many cytoskeletal proteins required for mitotic divisions may also be required for mRNA localization. The advantage of studying the function of CDC genes during oogenesis, in which all of the mitotic divisions occur in region 1 of the germarium, is that later in oogenesis one can analyze the biological functions of the CDC genes independent of their mitotic functions. For example, Klp38B, a chromatin-binding kinesin-like-protein isolated in the MDDS genetic screen, is required not only for chromosome segregation during the meiotic and mitotic divisions, but also for the proper development of the oocyte, possibly by localizing mRNA or protein in the oocyte (Ruden, 2000 and references therein).

Based on the phenotypes of syx-1a, ter94, rop and syt mutant egg chambers, a three-step genetic pathway is proposed for the role of membrane fusion proteins on OSK mRNA localization during oogenesis. (1) Syx-1a is required in stage 1 egg chambers to get OSK mRNA to the oocyte. Syx was originally identified as a Drosophila homolog of a human tSNARE that is required for synaptic vesicle fusion in neurons. Interestingly, Syx5 in humans has recently been shown to be required for TERA-mediated (the human Ter94 ortholog) assembly of Golgi cisternae from mitotic Golgi fragments in vitro (Rabouille, 1998). (2) Ter94 is required to localize OSK mRNA within the oocyte. It is speculated that OSK mRNA might be transported in membranous particles because both the endoplasmic reticulum and OSK mRNA form particulate complexes in ter94-mutant egg chambers. (3) The final step in OSK mRNA localization is anchoring the mRNA to the posterior pole of the oocyte. It is proposed that Rop and Syt are required for this process because rop and syt mutant egg chambers have poorly formed cytoplasmic membranous structure in the oocytes, and, possibly as a result, OSK mRNA fails to remain localized at the posterior pole. Rop is a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins and is a negative regulator of neurotransmitter release in vivo (Schulze, 1994). Syt controls and modulates synaptic vesicle fusion in a Ca2+ dependent manner (Littleton, 1993). It is concluded that many synaptic vesicle fusion proteins also function during other cellular processes such as OSK mRNA localization during oogenesis (Ruden, 2000).

Identification of TER94, an AAA ATPase protein, as a Bam-dependent component of the Drosophila fusome

The Drosophila fusome is a germ cell-specific organelle assembled from membrane skeletal proteins and membranous vesicles. Mutational studies that have examined inactivating alleles of fusome proteins indicate that the organelle plays central roles in germ cell differentiation. Although mutations in genes encoding skeletal fusome components prevent proper cyst formation, mutations in the bag-of-marbles gene disrupt the assembly of membranous cisternae within the fusome and block cystoblast differentiation altogether. To understand the relationship between fusome cisternae and cystoblast differentiation, attempts have been made to identify other proteins in this network of fusome tubules. Evidence is presented that the fly homolog of the transitional endoplasmic reticulum ATPase (TER94) is one such protein. The presence of TER94 suggests that the fusome cisternae grow by vesicle fusion and are a germ cell modification of endoplasmic reticulum. Fusome association of TER94 is Bam-dependent, suggesting that cystoblast differentiation may be linked to fusome reticulum biogenesis (Leon, 1999).

Antisera raised against a TER94 internal peptide reacts with bands of 94,000 Da in wild-type ovarian extracts and 57,000 Da in Escherichia coli cells expressing a fragment of TER94 as a GST-fusion protein. Both Cdc48p and vertebrate TERs oligomerize to form homohexameric complexes. When ovarian extracts were analyzed on native sucrose gradients, the peak of TER94 from flies sedimented was Mr ~500,000, which is close to the expected size (Mr ~530,000) for a homohexameric complex (Leon, 1999).

TER94 protein is present in both ovarian germ cells and somatic cells. TER94 is largely cytoplasmic in follicle and germ cells. Significantly, germ cells often contain one or several especially intense fluorescent signals, suggesting that TER94 is distributed unevenly in the cytoplasm. In cystocytes in germarial Region 1, these are usually somewhat diffuse bright regions, whereas in more mature cystocytes the bright spots are more sharply defined (Leon, 1999).

The number and positions of the TER-enriched regions suggest that they might correspond to fusomes. Stem cell fusomes in germ cells nearest the anterior tip appear as a single dot of intense staining, whereas those in a more posterior position (i.e. more mature cysts) contain elongated, branched fusomes. Precise colocalization of TER94 and Hu-li tao shao is strongest in Region 1 germ cells and declines in regions containing mature cysts. Because a fraction of TER is nuclear in yeast and mammals, Drosophila nuclei were examined closely. Most germ cell nuclei are faintly TER94 positive. Many examples of strong nuclear and perinuclear staining have been found in nonovarian somatic cells in larvae and adults (Leon, 1999).

Fusomes are the primary site of ER-like cisternae in young germ cells. If TER94 enrichment in fusomes represents accumulation at the fusome reticulum, TER94 distribution might be altered when the reticulum is not properly assembled. Bam is a fusome-associated protein and bam mutant fusomes are deficient in cisternae. The distribution of TER94 protein was examined in bam germ cells; it is distributed uniformly without signs of enrichment at the site of fusomes as is observed in wild-type germaria. Indeed, when the bam stem cell fusomes are visualized with Hts antibodies, it is clear that TER94 is no more abundant within or near stem cell fusomes than in any other cytoplasmic regions. Consistent with this conclusion, the merged images of TER94 and Hts distributions do not show immunofluorescent overlap, indicating that bam fusomes do not accumulate detectable TER94 (Leon, 1999).

TER94 is also enriched at a few sites that do not correspond to fusomes. It is speculated that these may be sites of Golgi bodies or transport vesicles, although unambiguous identification requires additional reagents as markers. These extrafusome sites of TER94 enrichment are also abolished in bam mutant cells (Leon, 1999).

The observation that TER94 fusome association is linked to Bam function can be explained by either a direct or indirect Bam dependent mechanism. Although loss of bam function might block fusome reticulum assembly before TER94 arrival, it is also possible that Bam recruits TER94 to the reticulum as part of the assembly process. This hypothesis has been difficult to test because Bam is a low-abundance protein in ovaries, and in vitro assays for Bam and TER94 interaction have produced inconsistent results. The interaction of Bam and TER94 as two-hybrid partners supports the hypothesis of in vivo interaction. Finding the Drosophila homolog of the S. cerevisiae protein Ufd3p as a second Bam interacting protein strengthens the significance of the Bam-TER94 interaction. Ufd3p and the yeast TER (i.e., Cdc48p) interact with one another directly. Ufd3p is required for efficient organelle vesicle fusion (Leon, 1999 and references therein).

TER94, a Drosophila homolog of the membrane fusion protein CDC48/p97, is accumulated in nonproliferating cells: in the reproductive organs and in the brain of the imago

A Drosophila homolog of the membrane fusion protein CDC48/p97 has been cloned. The open reading frame of the Drosophila homolog encodes an 801 amino acid long protein (TER94), which shows high similarity to the known CDC48/p97 sequences. The chromosomal position of TER94 is 46 C/D. TER94 is expressed in embryo, in pupae and in the adult, but is suppressed in larva. In the adults, the immunoreactivity is exclusively present in the head and in the gonads of both sexes. In the head the most striking staining is observed in the entire neuropil of the mushroom body and in the antennal glomeruli. Besides TER94, sex-specific forms are also detected in adult gonads: p47 in the ovaries and p98 in the testis. TER94/p47 staining is observed in the nurse cells and often in the oocytes, while TER94/p98 staining is present in the sperm bundles. On the basis of the TER94 distribution it is suggested that TER94 functions in the protein transport utilizing endoplasmic reticulum and Golgi derived vesicles (Pinter, 1998)

Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin

During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double-membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood. In vitro, nucleus formation requires p97, a hexameric ATPase implicated in membrane fusion and ubiquitin-dependent processes (Drosophila homolog: TER94). However, the role and relevance of p97 in nucleus formation have remained controversial. This study shows that p97 stimulates nucleus reformation by inactivating the chromatin-associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin-dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation (Ramadan, 2007).

Search PubMed for articles about Drosophila Ter-94

Azuma, Y., Tokuda, T., Shimamura, M., Kyotani, A., Sasayama, H., Yoshida, T., Mizuta, I., Mizuno, T., Nakagawa, M., Fujikake, N., Ueyama, M., Nagai, Y. and Yamaguchi, M. (2014). Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS. Hum Mol Genet. PubMed ID: 24497576

Beskow, A., Grimberg, K. B., Bott, L. C., Salomons, F. A., Dantuma, N. P. and Young, P. (2009). A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation. J Mol Biol 394: 732-746. PubMed ID: 19782090

Bruderer, R. M., Brasseur, C. and Meyer, H. H. (2004). The AAA ATPase p97/VCP interacts with its alternative co-factors, Ufd1-Npl4 and p47, through a common bipartite binding mechanism. J Biol Chem 279: 49609-49616. PubMed ID: 15371428

Chambers, M., Turki-Judeh, W., Kim, M. W., Chen, K., Gallaher, S. D. and Courey, A. J. (2017). Mechanisms of Groucho-mediated repression revealed by genome-wide analysis of Groucho binding and activity. BMC Genomics 18(1): 215. PubMed ID: 28245789

Davis, E. J., Lachaud, C., Appleton, P., Macartney, T. J., Nathke, I. and Rouse, J. (2012). DVC1 (C1orf124) recruits the p97 protein segregase to sites of DNA damage. Nat Struct Mol Biol 19: 1093-1100. PubMed ID: 23042607

Delabaere, L., Orsi, G. A., Sapey-Triomphe, L., Horard, B., Couble, P. and Loppin, B. (2014). The spartan ortholog maternal haploid is required for paternal chromosome integrity in the Drosophila zygote. Curr Biol 24: 2281-2287. PubMed ID: 25242033

Flack, J. E., Mieszczanek, J., Novcic, N. and Bienz, M. (2017). Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5. Mol Cell 67(2): 181-193.e185. PubMed ID: 28689657

Fujioka, M., Wu, X. and Jaynes, J. B. (2009). A chromatin insulator mediates transgene homing and very long-range enhancer-promoter communication. Development 136(18): 3077-87. PubMed Citation: 19675129

Fujioka, M., Sun, G. and Jaynes, J. B. (2013). The Drosophila eve Insulator Homie Promotes eve Expression and Protects the Adjacent Gene from Repression by Polycomb Spreading. PLoS Genet 9: e1003883. PubMed ID: 24204298

Gao, Y., Zhu, Y., Wang, H., Cheng, Y., Zhao, D., Sun, Q. and Chen, D. (2022). Lipid-mediated phase separation of AGO proteins on the ER controls nascent-peptide ubiquitination. Mol Cell 82(7): 1313-1328. PubMed ID: 35325613

Kondo, H., Rabouille, C., Newman, R., Levine, T. P., Pappin, D., Freemont, P. and Warren, G. (1997). p47 is a cofactor for p97-mediated membrane fusion. Nature 388: 75-78. PubMed ID: 9214505

Leon, A. and McKearin, D. (1999). Identification of TER94, an AAA ATPase protein, as a Bam-dependent component of the Drosophila fusome. Mol. Biol. Cell 10(11): 3825-34. 10564274

Madsen, L., Andersen, K. M., Prag, S., Moos, T., Semple, C. A., Seeger, M. and Hartmann-Petersen, R. (2008). Ubxd1 is a novel co-factor of the human p97 ATPase. Int J Biochem Cell Biol 40: 2927-2942. PubMed ID: 18656546

Mosbech, A., Gibbs-Seymour, I., Kagias, K., Thorslund, T., Beli, P., Povlsen, L., Nielsen, S. V., Smedegaard, S., Sedgwick, G., Lukas, C., Hartmann-Petersen, R., Lukas, J., Choudhary, C., Pocock, R., Bekker-Jensen, S. and Mailand, N. (2012). DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks. Nat Struct Mol Biol 19: 1084-1092. PubMed ID: 23042605

Pinter, M., et al. (1998). TER94, a Drosophila homolog of the membrane fusion protein CDC48/p97, is accumulated in nonproliferating cells: in the reproductive organs and in the brain of the imago. Insect Biochem. Mol. Biol. 28(2): 91-8. PubMed ID: 9639875

Ramadan, K., et al. (2007). Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin. Nature 450(7173): 1258-62. Medline abstract: 18097415

Reim, G., Hruzova, M., Goetze, S. and Basler, K. (2014). Protection of Armadillo/beta-Catenin by Armless, a novel positive regulator of Wingless signaling. PLoS Biol 12: e1001988. PubMed ID: 25369031

Ruden, D. M., et al. (2000). Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber. Dev. Biol. 218: 314-325. PubMed Citation: 10656772

Rumpf, S., Bagley, J. A., Thompson-Peer, K. L., Zhu, S., Gorczyca, D., Beckstead, R. B., Jan, L. Y. and Jan, Y. N. (2014), Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism. Proc Natl Acad Sci U S A 111(20):7331-6. PubMed ID: 24799714

Santhanam, A., Peng, W. H., Yu, Y. T., Sang, T. K., Chen, G. C. and Meng, T. C. (2014). Ecdysone-induced receptor tyrosine phosphatase PTP52F regulates Drosophila midgut histolysis by enhancement of autophagy and apoptosis. Mol Cell Biol 34: 1594-1606. PubMed ID: 24550005

Thomson, T., Liu, N., Arkov, A., Lehmann, R. and Lasko, P. (2008). Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins. Mech. Dev. 125(9-10): 865-73. PubMed Citation: 18590813

van Tienen, L. M., Mieszczanek, J., Fiedler, M., Rutherford, T. J. and Bienz, M. (2017). Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9. Elife 6. PubMed ID: 28296634

Zeng, Z., de Gorter, D. J., Kowalski, M., Ten Dijke, P. and Shimmi, O. (2014). Ter94/VCP is a novel component involved in BMP signaling. PLoS One 9: e114475. PubMed ID: 25469707

Zhang, Z., Lv, X., Yin, W. C., Zhang, X., Feng, J., Wu, W., Hui, C. C., Zhang, L. and Zhao, Y. (2013). Ter94 ATPase complex targets k11-linked ubiquitinated Ci to proteasomes for partial degradation. Dev Cell 25: 636-644. PubMed ID: 23747190

date revised: 25 September 2023

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