InteractiveFly: GeneBrief

brain tumor: Biological Overview | References


Gene name - brain tumor

Synonyms -

Cytological map position - 37C3--5

Function - post-transcriptional regulation

Keywords - segmentation, cell proliferation, growth, brain

Symbol - brat

FlyBase ID: FBgn0010300

Genetic map position -

Classification - C3HC4 type RING finger, NHL domain proteins

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Laver, J. D., et al. (2015). Brain tumor is a sequence-specific RNA-binding protein that directs maternal mRNA clearance during the Drosophila maternal-to-zygotic transition. Genome Biol 16: 94. PubMed ID: 25962635
Summary:
Brain tumor (BRAT) is a Drosophila member of the protein family. This family is conserved among metazoan and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. These results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.

Newton, F. G., Harris, R. E., Sutcliffe, C. and Ashe, H. L. (2015). Coordinate post-transcriptional repression of Dpp-dependent transcription factors attenuates signal range during development. Development 142(19):3362-73. PubMed ID: 26293305
Summary:
Precise control of the range of signalling molecule action is critical for correct cell fate patterning during development. For example, Drosophila ovarian germline stem cells (GSCs) are maintained by exquisitely short-range BMP signalling from the niche. In the absence of BMP signalling, one GSC daughter differentiates into a cystoblast (CB) and this fate is stabilised by Brain Tumour (Brat) and Pumilio (Pum)-mediated post-transcriptional repression of mRNAs, including that encoding the Dpp transducer, Mad. However, the identity of other repressed mRNAs and the mechanism of post-transcriptional repression are currently unknown. This study identified the Medea and schnurri mRNAs, which encode transcriptional regulators required for activation and/or repression of Dpp target genes, as additional Pum-Brat targets suggesting that tripartite repression of the transducers is deployed to desensitise the CB to Dpp. In addition, this study shows that repression by Pum-Brat requires recruitment of the CCR4 and Pop2 deadenylases, with knockdown of deadenylases in vivo giving rise to ectopic GSCs. Consistent with this, Pum-Brat repression leads to poly(A) tail shortening and mRNA degradation in tissue culture cells and a reduced number of Mad and shn transcripts in the CB relative to the GSC based on single molecule mRNA quantitation. Finally, the generality of the mechanism was shown by demonstrating that Brat also attenuates pMad and Dpp signalling range in the early embryo. Together these data serve as a platform for understanding how post-transcriptional repression restricts interpretation of BMPs and other cell signals in order to allow robust cell fate patterning during development.

Loedige, I., Jakob, L., Treiber, T., Ray, D., Stotz, M., Treiber, N., Hennig, J., Cook, K. B., Morris, Q., Hughes, T. R., Engelmann, J. C., Krahn, M. P. and Meister, G. (2015). The crystal structure of the NHL domain in complex with RNA reveals the molecular basis of Drosophila Brain-tumor-mediated gene regulation. Cell Rep 13: 1206-1220. PubMed ID: 26527002
Summary:
TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, this study identified Brat-bound mRNAs in Drosophila embryos and defined consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, the Brat-NHL domain in complex was crystallize with RNA, and a high-resolution protein-RNA structure of this fold is presented.

Mukherjee, S., Tucker-Burden, C., Zhang, C., Moberg, K., Read, R., Hadjipanayis, C. and Brat, D.J. (2016). Drosophila Brat and human ortholog TRIM3 maintain stem cell equilibrium and suppress brain tumorigenesis by attenuating Notch nuclear transport. Cancer Res [Epub ahead of print]. PubMed ID: 26893479
Summary:
Cancer stem cells exert enormous influence on neoplastic behavior, in part by governing asymmetric cell division and the balance between self-renewal and multipotent differentiation. Mutation of a single gene in Drosophila, Brain Tumor (Brat), leads to disrupted asymmetric cell division resulting in dramatic neoplastic proliferation of neuroblasts and massive larval brain overgrowth. To uncover mechanisms relevant to deregulated cell division in human glioma stem cells, this study developed a novel adult Drosophila brain tumor model using brat-RNAi driven by the neuroblast specific promoter inscuteable. Suppressing Brat in this population leads to accumulation of actively proliferating neuroblasts and a lethal brain tumor phenotype. brat-RNAi causes upregulation of Notch signaling, a node critical for self-renewal, by increasing protein expression and enhancing nuclear transport of NICD. In human glioblastoma, it was demonstrated that the human ortholog of Drosophila Brat, TRIM3, similarly suppresses NOTCH1 signaling and markedly attenuates the stem cell component. TRIM3 was also found to suppress nuclear transport of active NOTCH1 (NICD) in glioblastoma, and these effects are mediated by direct binding of TRIM3 to the Importin complex. Together, these results support a novel role for Brat/TRIM3 in maintaining stem cell equilibrium and suppressing tumor growth by regulating NICD nuclear transport.

Luo, H., Li, X., Claycomb, J.M. and Lipshitz, H.D. (2016). The Smaug RNA-binding protein is essential for microRNA synthesis during the Drosophila maternal-to-zygotic transition. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27591754
Summary:
Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which maternal gene products are eliminated and the zygotic genome becomes transcriptionally active. During this process RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) target maternal mRNAs for degradation. In Drosophila, the Smaug (SMG), Brain tumor (BRAT) and Pumilio (PUM) RBPs bind to and direct the degradation of largely distinct subsets of maternal mRNAs. SMG has also been shown to be required for zygotic synthesis of mRNAs and several members of the miR-309 family of microRNAs (miRNAs) during the MZT. This study carried out global analysis of small RNAs both in wild type and in smg mutants. It was found that 85% all miRNA species encoded by the genome are present during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting RNAs (piRNAs) or small interfering RNAs (siRNAs), zygotic production of more than 70 species of miRNAs fails or is delayed in smg mutants. SMG is also required for the synthesis and stability of a key miRISC component, Argonaute 1 (AGO1), but plays no role in accumulation of the Argonaute-family proteins associated with piRNAs or siRNAs. In smg mutants, maternal mRNAs that are predicted targets of the SMG-dependent zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these miRNAs but not with SMG itself. The study hypothesizes that SMG controls the MZT, not only through direct targeting of a subset of maternal mRNAs for degradation but, indirectly, through production and function of miRNAs and miRISC, which act together with BRAT and/or PUM to control clearance of a distinct subset of maternal mRNAs.
BIOLOGICAL OVERVIEW

Brain tumor (Brat) is one of three NHL domain proteins found in Drosophila (Arama, 2000; Sonoda, 2001). The family name derives from three of the founding members: NCL-1, HT2A, and LIN-41 (Slack, 1998). All three factors have ties to RNA metabolism: the nucleoli in Caenorhabditis elegans ncl-1 mutants are enlarged (Frank, 1998); HT2A was identified by virtue of interaction with the RNA-binding protein HIV Tat (Fridell, 1995), and posttranscriptional regulation of lin-29 mRNA is abrogated in lin-41 mutants (Slack, 2000). Little is known of the biological roles of other family members, and no direct molecular mechanism has been described previously for any NHL domain protein (including Brat). The NHL domain of Brat mediates its recruitment to the 3' UTR of Hunchback (HB) mRNA. Recruitment occurs through protein-protein interactions with RNA-bound Pumilio and Nanos; formation of the resulting quaternary complex is essential for translational control of HB. These results suggest a general mechanism by which other NHL domain proteins may act to control posttranscriptional gene expression (Sonoda, 2001).

Maternally derived HB mRNA is uniformly distributed throughout the embryo; the mRNA is translationally repressed in the posterior, giving rise to an anterior-to-posterior gradient of Hb protein. Failure of this repression results in the abnormal accumulation of Hb in the posterior, which inhibits abdominal segmentation. Two conserved RNA-binding proteins, Pumilio (Pum) and Nanos (Nos), are specifically required to repress HB translation. Pum, which is distributed uniformly throughout the embryo, is the founding member of a large family of RNA-binding proteins. Pum binds to 32 nucleotide sites in the 3' UTR of HB (Nos Response Elements, NREs) to regulate HB translation. Nos, which initially is distributed as a gradient emanating from the posterior pole of the embryo, contains a conserved zinc finger that mediates nonspecific RNA binding. Nos is selectively recruited into a ternary complex on HB mRNA by NRE-bound Pum. The mechanism by which the resulting Nos/Pum/NRE complex regulates translation is not yet understood, although deadenylation is thought to play a role (Sonoda, 2001 and references therein).

To identify targets or cofactors of the Nos/Pum/NRE ternary complex, a yeast 'four-hybrid' experiment was performed; a Gal4 activation domain fusion library was screened for proteins that interact with the ternary complex. The bait contained the RNA-binding domain of Pum, full-length Nos, and NRE-bearing RNA. As anticipated, factors that interact with individual components in isolation were identified. However, one factor, which proved to be a fragment of Brat, interacts only with the ternary complex and not with either Nos alone, Pum alone, or a Pum/NRE binary complex. Deletion analysis revealed that recruitment of Brat is dependent on the conserved carboxy-terminal domain of Nos that mediates its interaction with Pum on HB mRNA (Sonoda, 1999), and not the amino-terminal domain of Nos that mediates interaction with Cup during early oogenesis (Verrotti, 2000). Mutational analysis further showed that a fragment of Brat consisting of little more than the NHL domain is recruited to the ternary complex. Protein-protein interaction experiments show that Nanos and Pumilio are required to recruit Brat to HB mRNA and genetic experiments show that Brat is required for repression of HB mRNA (Sonoda, 2001).

Analysis of mutant phenotypes has revealed that Nos and Pum are required for a variety of processes in addition to the development of abdominal segmentation. nos and pum are expressed in tissues other than the female germ line. More important, nos and pum mutants are subviable, revealing an (unknown) essential function for each factor in somatic cells. In the germ line, nos and pum mutants exhibit a number of defects including loss of germ-line stem cells in both sexes, failure of precursor cells to migrate into and populate the somatic gonad, and premature proliferation of precursor cells (pole cells) in the embryo. The premature proliferation appears to result from the inappropriate derepression of maternal Cyclin B (CycB) mRNA in the pole cells; in no other case is the molecular basis of Nos or Pum action currently understood (Sonoda, 2001 and references therein).

It was thus of interest to determine whether Nos and Pum also act in conjunction with Brat to regulate maternal Cyclin B mRNA. Using antibodies directed against different regions of the Brat protein, it has been found that Brat is distributed throughout the syncitial blastoderm stage embryo when HB mRNA is repressed, and is also present in the cytoplasm of the pole cells when maternal Cyclin B mRNA is regulated. However, Cyclin B mRNA is repressed normally in the pole cells of bratfs mutant embryos, but not in the pole cells of nos mutant embryos. Thus, Brat does not appear to play a role in repression of Cyclin B, although the possibility that the residual activity of Bratfs1 is sufficient to regulate Cyclin B but not hb cannot be ruled out (Sonoda, 2001).

The cis-acting signals that mediate Nos- and Pum-dependent regulation of Cyclin B have not yet been defined precisely. However, NRE-like sequences are present in the maternal isoform of the Cyclin B mRNA, which is regulated. If indeed Pum, Nos, and NRE-like sequences mediate its regulation, then why would repression of Cyclin B mRNA be Brat independent (Sonoda, 2001)?

To investigate this issue, an examination was made of the binding of Pum, Nos, and Brat to the Cyclin B NRE-like element in vitro. The RNA used in these experiments contains 136 nucleotides that include all of the NRE homologous elements as well as flanking sequences. Pum binds to this Cyclin B-derived RNA in gel mobility-shift experiments, but not to a derivative bearing mutations in the conserved NRE-like element, consistent with the idea that similar sequences in Cyclin B and HB are recognized. Bound Pum can recruit Nos into a ternary complex on Cyclin B RNA, much as it does on the HB NRE. However, the ternary complex assembled on Cyclin B RNA recruits Brat at least 10-fold less efficiently than the corresponding complex assembled on the hb NRE. This surprising observation may in part explain the Brat independence of Cyclin B regulation. Furthermore, it suggests that the RNA sequence specifies the geometry of the Pum/Nos complex, which in turn determines whether Brat is recruited or not (Sonoda, 2001).

Brat acts as a growth suppressor in the larval brain (Arama, 2000). Whether Brat acts by a similar molecular mechanism in the brain and in the early embryo (in regulating HB mRNA) is unclear; however, the observation that single amino acid substitutions in the NHL domain of the Bratfs mutant proteins disrupt both processes is consistent with such an idea. The role of Brat in the brain is not yet clear, since the phenotype has not been characterized in detail and regulatory targets have yet to be identified (Sonoda, 2001).

The role of Brat in the imaginal discs has been even less clear. Loss of brat function leads to no obvious defects in imaginal development (Arama, 2000), and rare escaper homozygous brat- flies appear morphologically normal. One role for Brat was revealed by experiments in which imaginal disc tissue was transplanted into the body cavity of adult hosts; brat- but not wild-type discs metastasize and kill the fly (Woodhouse, 1998). This observation suggests that Brat is expressed in the discs, which led to a consideration of the possibility that loss-of-function mutants exhibit no apparent phenotype due to the presence of a redundant activity (Sonoda, 2001).

To investigate this possibility, either Brat+ or Bratfs1 were misexpressed using an engrailed (en)-GAL4 driver line and UAS transgenes. Flies were examined for phenotypes resulting from the gain of Brat function. Endogenous Brat accumulates uniformly in the cytoplasm of cells in wing discs from third instar larvae. In either UASbrat+ or UASbratfs1 discs that also bear the enGAL4 driver, a modest excess of protein accumulates in the posterior compartment of the wing disc; analysis of Western blots suggests that the level of overproduction is less than 2-3 fold. At this stage of development, ectopic expression of either protein does not substantially alter the morphology of the discs (Sonoda, 2001).

However, misexpression of Brat+ causes an intriguing growth suppression phenotype that is evident in the wings of adults. Three observations stand out: (1) the en;brat+ wings are 24% smaller than control wings. They are also usually deformed, probably as a result of poor adhesion between the dorsal and ventral surfaces. (2) The reduction in wing size appears to be due to a reduction in the number of cells contributing to the wing rather than a reduction in the size of the cells. This conclusion is based on a measurement of the density of epidermal hairs, each secreted by a single cell. (3) The phenotype is nonautonomous, extending into the anterior compartment where the en promoter is not active. For example, the anterior-most sector of en;brat+ wings (bounded by the first and second longitudinal veins) is on average 22% smaller than the corresponding region of control wings (Sonoda, 2001).

Significantly, none of the phenotypes associated with misexpression of Brat+ is caused by misexpression of similar levels of Bratfs1. This supports the idea that Brat acts by a similar molecular mechanism to regulate abdominal segmentation in the embryo and growth of the wing imaginal disc (Sonoda, 2001).

A model is presented of how Nos, Pum, and Brat act to regulate gene expression. The model involves combinatorial interactions among cis-acting sequences in regulated mRNAs, proteins that recognize these sequences, and the NHL domain of Brat. Recruitment of Brat occurs through protein-protein interactions with RNA-bound Pum and Nos; formation of the resulting quaternary complex is essential for translational control of HB. Recruitment of Brat to the NRE jointly by Nos and Pum is essential for regulation of HB mRNA. Three lines of evidence show that the NHL domain plays a key role in this process: (1) the NHL domain is sufficient to mediate interaction with the Nos/Pum/NRE complex, thereby targeting Brat to HB mRNA; (2) single amino acid substitutions within the NHL domain attenuate interaction with the ternary complex and regulation of hb in vivo; (3) maternal expression of the wild-type NHL domain alone is sufficient to restore HB regulation in bratfs mutant embryos. This result suggests that the NHL domain contains intrinsic translation regulatory activity. However, activity of the isolated NHL domain is (necessarily) assayed in the presence of Bratfs mutant protein, and thus, the possibility that the amino-terminal BCC domain participates somehow in HB mRNA regulation cannot be ruled out (Sonoda, 2001).

Brat appears to play no role in regulating Cyclin B mRNA in the pole cells, although Nos and Pum are required for this process. This observation is perhaps not surprising, since translation of Cyclin B mRNA in the posterior region of the syncitial cleavage stage embryo appears to be uninhibited, even in the presence of Nos, Pum, and Brat. Only in the pole cells, which extrude from the posterior extreme of the embryo, is Cyclin B mRNA repressed. Perhaps the specialized pole plasm incorporated into these cells contains a Cyclin B-specific corepressor that acts in conjunction with Nos and Pum. Alternatively, the Nos/Pum complex on Cyclin B mRNA may be sufficient to regulate translation without a cofactor in the pole cells (Sonoda, 2001).

In either case, experiments with Cyclin B reveal an unanticipated complexity: the Nos/Pum complexes assembled on Cyclin B and HB mRNAs apparently have different conformations, as revealed by their ability to interact with Brat. Perhaps the RNA sequence acts as a scaffold, bringing Nos and Pum together on the RNA in different relative orientations in the two cases. Alternatively, the RNA might act as an allosteric effector, altering the conformation of Pum to allow interaction with different cofactors (Sonoda, 2001).

Brat acts as a growth suppressor in the larval brain and, upon modest overexpression, in the wing imaginal disc. Current evidence suggests that, in these tissues, Brat likely acts with cofactors other than Pum or Nos, although the supporting evidence is relatively weak. The brains of mutant larvae bearing the strongest extant alleles of pum do not exhibit a tumorous brat phenotype, consistent with the idea that some other factor acts in conjunction with Brat in this tissue. Attempts were made to test the role of Pum in mediating the en;brat+ phenotype, but flies of the appropriate genotype could not be recovered (presumably due to the subviability of both pum- and en;brat+ flies). The role of Nos in mediating Brat action is less easily assessed, since larvae bearing lethal nos alleles die before the third instar when both the brat- and en;brat+ phenotypes are evident. Weaker alleles, such as nosRC, have substantial residual activity (Sonoda, 2001).

Perhaps the most striking aspect of the en;brat+ phenotype is that ectopic Brat suppresses growth nonautonomously. This is in contrast to the action of other growth regulators that have been the focus of recent research in flies. Regulation of cell size and cell number in the imaginal discs is complex. One class of regulators is the extracellular signals of the EGF, TGF-ß, and Wg pathways that coordinately control pattern and growth. Another class consists of signals mediated by molecules such as Ras, Myc, TOR, and members of the insulin receptor pathway that primarily control cell size or number, but not pattern. For many of these 'pure' growth regulators, ectopic expression enhances (or suppresses) growth of the imaginal discs to an extent similar to that observed for Brat. However, in none of these cases is the effect on growth transmitted to surrounding cells, as is true for Brat. Thus, Brat appears to regulate either novel pathways or novel combinations of pathways that generate extracellular signals (Sonoda, 2001).

All three Drosophila NHL proteins regulate some aspect of growth, suggesting this may be a common role for NHL proteins in general. Mutations in mei-P26/CG12218 and dappled/CG1624 reveal that the proteins encoded by these loci suppress growth of melanotic and ovarian tumors, respectively (Rodriguez, 1996; Page, 2000). Mei-P26 is also required for a normal frequency and distribution of genetic exchange during meiosis. Given the structural similarity among the three fly proteins, it was assumed that Dappled and Mei-P26, like Brat, act by regulating translation or some other aspect of mRNA metabolism. Little else is currently known of the cell biological processes controlled by Brat, Dappled, or Mei-P26 (Sonoda, 2001).

Do other NHL proteins act in a manner similar to Brat? Relatively little is known about the molecular mechanism by which these factors act in vivo, and thus it is not clear whether they regulate translation or some other aspect of posttranscriptional gene expression. Nevertheless, an argument for analogous function can be made for three of the family members, based on current knowledge; (1) the HT2A human protein interacts with the site-specific RNA-binding Tat protein (Fridell, 1995), much as Brat interacts with Nos and Pum; (2) C. elegans NCL-1 appears to regulate growth, although the mutant worms have larger cells rather than more cells (Frank, 1998). (3) The most striking analogy with Brat function comes from C. elegans LIN-41, which acts in the penultimate step of the heterochronic pathway (Reinhart, 2000; Slack, 2000). Like Brat, LIN-41 is a posttranscriptional regulator. And like Brat, which acts in concert with Nos and Pum, LIN-41 appears to play a role in the switch from sperm to oocyte production in hermaphrodites that is governed by homologs of Nos and Pum. Thus, it seems likely that LIN-41 and Brat act by a similar mechanism, interacting with RNA-bound factors to repress translation (Sonoda, 2001 and references therein).

Brain tumor specifies intermediate progenitor cell identity by attenuating beta-catenin/Armadillo activity

During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. This study reports that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification (Komori, 2013).

Asymmetric stem cell division provides an efficient mechanism to simultaneously self-renew a stem cell and to generate a progenitor cell that produces differentiated progeny. Because self-renewal proteins segregate into both daughter progeny of the dividing parental stem cell through the inheritance of its cytoplasmic content, rapidly downregulating the activity of these proteins is essential for the specification of progenitor cell identity. Brat plays a central role in specifying INP identity in the Ase- immature INP by antagonizing the function of the self-renewal transcription factor Klu (Xiao, 2012). These previous findings have been extended to show that Brat specifies INP identity in the Ase- immature INP through two separable, but convergent, mechanisms. A novel Brat-dependent mode of Wnt pathway regulation was identified that prevents Ase- immature INPs from reverting into supernumerary neuroblasts. Brat specifies INP identity by attenuating the transcriptional activity of Arm through its N-terminal B-boxes. This negative regulation of Arm is achieved through the activity of Apc2 and the destruction complex. Because increased arm function alone is insufficient to induce supernumerary neuroblasts, the ability of Wnt signaling to promote neuroblast identity is dependent on other signaling mechanisms that act downstream of Brat. Indeed, Arm function is essential for Klu to induce supernumerary neuroblasts. These two Brat-regulated mechanisms function to safeguard against the accidental reversion of an uncommitted progenitor cell into a supernumerary stem cell and to ensure that an uncommitted progenitor cell can only adopt progenitor cell identity (Komori, 2013).

Physical interaction with the cargo-binding domain of Mira is essential for the unequal segregation of Brat into the immature INP following the asymmetric division of neuroblasts. Previous studies concluded that the NHL domain of Brat directly interacts with the cargo-binding domain of Mira, but the roles of the B-boxes and the coiled-coil domain in the asymmetric segregation of Brat were unknown due to a lack of specific mutant alleles. By combining a yeast two-hybrid interaction assay and in vivo functional validation, it is concluded that both the coiled-coil domain and the NHL domain are indeed required for the asymmetric segregation of Brat into the Ase- immature INP following the asymmetric division of neuroblasts. It is speculated that the coiled-coil domain and the NHL domain of Brat function cooperatively to provide a more stable binding platform for Mira to ensure efficient protein segregation (Komori, 2013).

The severity of the supernumerary neuroblast phenotype in various brat mutant allelic combinations correlates with the level of endogenous brat inherited by the Ase- immature INP. The brat DG19310 mutation carries a transposable P-element inserted in the 5′ regulatory region of the brat gene. The brat11 mutation, however, results in a premature stop codon at amino acid 779, leading to a truncated form of the protein that lacks most of the NHL domain and is predicted to be unable to interact with Mira. The brat DG19310 or brat DG19310/11allelic combination most likely reduces Brat expression without affecting its binding to Mira. Thus, the minimal threshold of Brat necessary for the proper specification of INP identity in Ase- immature INPs is met most of the time, leading to a mild supernumerary neuroblast phenotype in brat DG19310 or brat DG19310/11 brains. By contrast, the brat11 homozygous or brat11/Df mutant allelic combination impairs the binding of Brat to Mira, rendering the Mira-based asymmetric protein-sorting mechanism unable to segregate Brat into the Ase- immature INP. As such, the threshold of Brat necessary for proper specification of INP identity in Ase- immature INPs is rarely met, leading to a severe supernumerary neuroblast phenotype in brat11 or brat11/Df brains. Overexpression of the bratΔC-coil or bratΔNHL transgene using the UAS/Gal4 system almost certainly results in an abnormally high level of the transgenic protein in the cytoplasm of neuroblasts. Thus, inheriting a portion of the neuroblast cytoplasm containing an overwhelming abundance of the mutant transgenic protein is likely to be sufficient to reach the threshold of Brat necessary for proper specification of INP identity in Ase- immature INPs. It is concluded that the mechanism that causes Brat to asymmetrically segregate into the Ase- immature INP is functionally separable from the mechanism that specifies INP identity (Komori, 2013).

Could the asymmetric protein segregation mechanism promote the specification of INP identity by depleting Brat from the neuroblast? Type II neuroblasts overexpressing brat, bratΔC-coil or brat<ΔNHL maintained their identity and generated similar numbers of progeny as wild-type control neuroblast. Thus, it is unlikely that Brat-dependent specification of INP identity occurs through asymmetric depletion of Brat from the neuroblast. Whether Brat acts redundantly with other asymmetrically segregating determinants to specify INP identity in Ase- immature INPs was also tested. Numb also exclusively segregates into the immature INP during asymmetric divisions of type II neuroblasts. However, asymmetric segregation of Numb is not dependent on Brat, and Numb-dependent specification of INP identity also occurs independently of Brat. Thus, it is unlikely that Brat acts redundantly with other asymmetric segregating determinants to specify INP identity in Ase- immature INPs (Komori, 2013).

A surprising finding revealed by the current study is that the B-boxes are uniquely required for the specification of INP identity. This raises a series of interesting questions. What are the roles of B-boxes in the function of Brat in embryonic neuroblasts? Embryos lacking both maternal and zygotic function of brat often lack RP2 neurons but never possess supernumerary neuroblasts. Since brat mutant alleles that specifically affect the function of B-boxes are unavailable, the roles of B-boxes in the function of Brat during the asymmetric division of embryonic neuroblasts remain unknown. Brat regulates embryonic pattern formation by repressing mRNA translation through the ternary complex that also contains Nanos and Pumilio. However, it is unlikely that Brat specifies INP identity through the Nanos-Pumilio-Brat translational repression complex for the following reasons. First, the NHL domain of Brat is required for binding to Pumilio and Nanos and for the assembly of the translational repressor complex. However, the NHL domain is dispensable for Brat-dependent specification of INP identity. Second, Nanos expression is undetectable in larval brains, and pumilio mutant larval brains do not possess supernumerary type II neuroblasts. Together, these results are consistent with the conclusion that Brat specifies INP identity via a novel Arm-mediated mechanism (Komori, 2013).

The amino acid sequence of the B-boxes is highly conserved among all TRIM family proteins, including Brat, and is predicted to adopt a 'RING-like' fold tertiary structure. The RING-like fold might facilitate protein-protein interactions. This is a particularly intriguing hypothesis in light of the fact that Apc2 and Brat both localize to the basal cortex in type II neuroblasts, and overexpression of brat, but not bratδB-boxes, can restore Apc2 protein localization in neuroblasts. However, epitope-tagged Brat and endogenous could not be coprecipitated Apc2 from the brain lysate extracted from brat null mutant larvae overexpressing a Myc-tagged Brat transgenic protein. Thus, Brat might maintain Apc2 protein localization indirectly through other factors. Future biochemical analyses of the Brat protein and identification of the proteins that directly interact with the B-boxes will provide insight into how Brat controls Apc2 localization (Komori, 2013).

The destruction complex targets β-catenin/Arm for degradation during canonical Wnt signaling, so reduced function of the destruction complex will lead to an increase in β-catenin/Arm, which forms a complex with Tcf/LEF family transcription factors to activate Wnt target gene expression. This study has concluded that the Brat-Apc2 mechanism specifies INP identity by preventing aberrant activation of Wnt target gene expression in Ase- immature INPs. The role of the Wnt ligand was tested in the Brat-dependent specification of INP identity by removing the function of the Wnt ligand using a temperature-sensitive mutant allele or by overexpressing a dominant-negative form of Frizzled (FzDN or GPIdFz2) in brat DG19310/11 mutant brains. Interestingly, neither of these manipulations modified the supernumerary neuroblast phenotype in the sensitized brat genetic background (data not shown). These results suggest that the Wnt ligand and its receptor Fz are irrelevant in the Brat-dependent specification of INP identity and that the Brat- Apc2 mechanism prevents Wnt target gene expression in Ase- immature INPs by negatively regulating the activity of Arm. However, these data do not exclude the possibility that a novel activating mechanism of Wnt signaling might be present in type II neuroblasts in Drosophila larval brains (Komori, 2013).

Attempts were made to directly demonstrate that loss of brat function indeed leads to derepression of Wnt target gene expression in supernumerary neuroblasts.The expression was examined of two distinct Wnt reporter transgenes, WRE-lacZ and Notum-lacZ in brat mutant brains. However, it was not possible to detect the expression of these transgenes in supernumerary neuroblasts in brat null mutant brains. Because genetic manipulations altering the activity of Arm efficiently modify the supernumerary neuroblast phenotype in brat mutant brains, these two transgenes are unlikely to have the necessary regulatory elements to reflect Wnt target gene activity in this tissue. Thus, it is proposed that the Brat-Apc2 mechanism specifies INP identity by antagonizing the transcriptional activity of Arm in Ase- immature INPs via a receptor-independent mechanism (Komori, 2013).

Wnt signaling regulation plays key roles in both stem cell renewal and the differentiation of progenitor cell types (Merrill, 2012; Habib et al., 2013). In the mammalian intestinal epithelium, for example, loss of Apc and activation of Wnt signaling results in the maintenance of stem cell properties in the progenitor cells, a failure to differentiate, and the production of intestinal polyps that progress to malignant tumors. In the intestine, the inappropriate activation of Wnt signaling is sufficient to elicit stem cell properties. In the progenitor cells of larval type II neuroblasts, the activation of Wnt signaling alone, through either the expression of stabilized Arm or the loss of Apc2, does not drive stem cell renewal in otherwise wild-type immature INPs. In this system, Brat is the key regulator attenuating self-renewal through two independent, but convergent, mechanisms in its regulation of both Klu and Wnt signaling. Although Arm activity is required for Klu-dependent self-renewal in immature INPs, its inability to promote self-renewal alone suggests that Wnt signaling is likely to be playing a permissive role rather than an instructive role in eliciting the neuroblast identity. It is proposed that Brat downregulates the function of Klu through both Arm-dependent and -independent mechanisms. Previous studies have demonstrated that TRIM32 and TRIM3, vertebrate orthologs of Brat, are essential regulators of neural stem cells during brain development and brain tumor formation (Boulay, 2009; Schwamborn, 2009). It will be interesting to test whether TRIM32 and TRIM3 regulate neural stem cells via a β-catenin-dependent mechanism (Komori, 2013).


REGULATION

Transcriptional Regulation

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis: Transcriptional regulation of brat

The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription. This study identifies the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. Genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. Evidence is provided that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. An initial characterization is presented of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes. This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development (Beckstead, 2005).

To identify genes that alter their expression in synchrony with the late third instar and prepupal pulses of 20E, RNA was isolated from w1118 animals staged at -18, -4, 0, 2, 4, 6, 8, 10, and 12 hours relative to pupariation, labeled, and hybridized to Affymetrix Drosophila Genome Arrays. The sensitivity and accuracy of the array data were determined by comparing the expression patterns of known 20E-regulated genes with previously published developmental Northern blot data. A subset of this analysis reveals that the temporal expression pattern of key regulatory genes - EcR, usp, E74A, DHR3, FTZ-F1, and DHR39 - are faithfully reproduced in the temporal arrays, as well as the 20E-regulated switch from Sgs glue genes to L71 late genes in the larval salivary glands, and the expression of representative IMP and Edg genes in the imaginal discs and epidermis. This comparison demonstrates that the microarrays accurately reflect the temporal patterns of 20E-regulated gene expression at the onset of metamorphosis and have sufficient sensitivity to detect rare transcripts such as EcR and E74A (Beckstead, 2005).

Roles for brat during metamorphosis were examined because, unlike the other six 20E primary-response genes described above, a brat mutant allele is available (bratk06028) that allows an assessment of its functions during later stages of development. The bratk06028 P-element maps to the fourth exon of the brat gene. Precise excisions of this transposon result in viable, fertile animals, demonstrating that the transposon is responsible for the mutant phenotype. Lethal phase analysis of bratk06028 mutants revealed that 61% of the animals survive to pupariation, with the majority of these animals pupariating 1 to 2 days later than their heterozygous siblings. Of those mutants that pupariated, 11% died as prepupae, 8% died as early pupae, 46% died as pharate adults, and the remainder died within a week of adult eclosion. Phenotypic characterization of bratk06028 mutant prepupae and pupae revealed defects in several ecdysone regulated developmental processes, including defects in anterior spiracle eversion (29%), malformed pupal cases (15%), and incomplete leg and wing elongation (12%). Northern blot hybridization of RNA isolated from staged bratk06028 mutant third instar larvae or prepupae revealed a disruption in the 20E-regulated transcriptional hierarchy. In wild type animals, brat mRNA is induced in late third instar larvae and 10 hour prepupae, similar to the temporal profile determined by microarray analysis, with reduced levels of brat mRNA in bratk06028 mutants, consistent with it being a hypomorphic allele. βFTZ-F1 is unaffected by the brat mutation in mid-prepupae, while E74 mRNA is reduced at 10 hours after pupariation. BR-C, E93, EcR, DHR3, and L71-1 are expressed at higher levels in late third instar larvae and early prepupae, with significant upregulation of BR-C. In addition, the smallest BR-C mRNA, encoding the Z1 isoform, is under-expressed in brat mutant prepupae. It is unlikely that brat exerts direct effects on transcription since it encodes a translational regulator. Nonetheless, these effects on 20E-regulated gene expression are consistent with the late lethality of bratk06028 mutants. In particular, the rbp function provided by the BR-C Z1 isoform is critical for developmental responses to 20E, and overexpression of BR-C isoforms can lead to lethality during metamorphosis. Thus, not only are the brat mutant phenotypes consistent with it playing an essential role during metamorphosis, but it may exert this function through the regulation of key 20E-inducible genes. Efforts are currently underway to address the roles of the remaining six new 20E primary-response regulatory genes in transducing the hormonal signal at the onset of metamorphosis (Beckstead, 2005).

Neural-specific elongation of 3' UTRs during Drosophila development

The 3' termini of eukaryotic mRNAs influence transcript stability, translation efficiency, and subcellular localization. This study reports that a subset of developmental regulatory genes, enriched in critical RNA-processing factors, exhibits synchronous lengthening of their 3' UTRs during embryogenesis. The resulting UTRs are up to 20-fold longer than those found on typical Drosophila mRNAs. The large mRNAs emerge shortly after the onset of zygotic transcription, with several of these genes acquiring additional, phased UTR extensions later in embryogenesis. These extended 3' UTR sequences are selectively expressed in neural tissues and contain putative recognition motifs for the translational repressor, Pumilio, which also exhibits the 3' lengthening phenomenon documented in this study. These findings suggest a previously unknown mode of posttranscriptional regulation that may contribute to the complexity of neurogenesis or neural function (Hilgers, 2011).

This study identified ~30 genes that exhibit developmental regulation of their 3' UTRs. As a class, the expressed transcripts contain some of the longest 3' UTRs in the Drosophila genome and are comparable to the largest 3' UTRs known in mammals. All of the genes undergo this posttranscriptional transition shortly after the onset of zygotic transcription, with the first detection of the long isoforms at 2-4 h AF. Perhaps the loss or gain of specialized RNA-processing factors during the MZT leads to the extension of the 3' UTRs. Alternatively, depletion of one or more components of the general mRNA poly(A) machinery at the MZT or in neural tissues could lead to weakened poly(A) and mRNA cleavage efficiency, therefore promoting the synthesis of longer transcripts. Such a mechanism, diminished levels of the essential poly(A) factor Cstf-64, promotes the formation of longer isoforms of IgM in B lymphocytes (Hilgers, 2011).

Previous studies suggest that Drosophila 3' UTRs are longest during early development. The genes identified in this study do not conform to this general trend but are consistent with recent whole-genome studies in vertebrates that suggest a statistical enrichment for longer 3' UTRs at later stages in development. In mammals, the expression of long 3' UTR isoforms has been correlated with the loss of cell proliferation and the onset of differentiation. The genes described in this study do not fit this model and may instead be responding to a specific developmental cue during neurogenesis. The key correlation for the large 3' extensions identified in this study is neural expression, irrespective of the state of proliferation. However, the occurrence of 3' elongation events at additional genes in other tissues cannot be excluded because the datasets used for this analysis made use of whole-embryo RNA samples at various developmental stages (Hilgers, 2011).

A significant fraction of the genes with extended 3' UTRs encode proteins implicated in RNA binding or processing, including ago1, adar, pumilio, brat, mei-P26, shep, imp, fne, and elav. Some of these genes, like ago1, are broadly expressed in a variety of tissues. Nonetheless, the extended isoforms of ago1 mRNAs are specifically enriched in neural tissues, a known hotbed of posttranscriptional regulation, including regulation by miRNAs and differential splicing. For example, Dscam is thought to produce tens of thousands of spliced isoforms in the Drosophila CNS. Furthermore, in Drosophila, directed transport of mRNAs, like bicoid, requires functional elements within the 3' UTR. Whether RNA binding factors such as Pum participate in a network of cross-regulation by repression, activation, or transport awaits further study (Hilgers, 2011).

It is currently unclear whether the long forms of mRNAs as seen in mammalian cells produce less protein than the short forms in Drosophila. However, enrichment of Pum recognition motifs in the extended 3' UTRs of elav, brat, and pumilio suggests regulation by repression because Pum and Brat are known to form localized translation repression complexes essential for anterior-posterior body patterning in early embryogenesis. Such regulation may have particular relevance in the Drosophila nervous system because Pum is required for dendrite morphogenesis. It is proposed that neural-specific isoforms of the genes identified in this study comprise elements of an interactive RNA-processing network that mediates some of the distinctive posttranscriptional processes seen in the nervous system (Hilgers, 2011).

Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling

In the Drosophila ovary, bone morphogenetic protein (BMP) ligands maintain germline stem cells (GSCs) in an undifferentiated state. The activation of the BMP pathway within GSCs results in the transcriptional repression of the differentiation factor bag of marbles (bam). The Nanos-Pumilio translational repressor complex and the miRNA pathway also help to promote GSC self-renewal. How the activities of different transcriptional and translational regulators are coordinated to keep the GSC in an undifferentiated state remains uncertain. Data presented in this study show that Mei-P26 cell-autonomously regulates GSC maintenance in addition to its previously described role of promoting germline cyst development. Within undifferentiated germ cells, Mei-P26 associates with miRNA pathway components and represses the translation of a shared target mRNA, suggesting that Mei-P26 can enhance miRNA-mediated silencing in specific contexts. In addition, disruption of mei-P26 compromises BMP signaling, resulting in the inappropriate expression of bam in germ cells immediately adjacent to the cap cell niche. Loss of mei-P26 results in premature translation of the BMP antagonist Brat in germline stem cells. These data suggest that Mei-P26 has distinct functions in the ovary and participates in regulating the fates of both GSCs and their differentiating daughters (Li, 2012).

Evidence is provided that Mei-P26 promotes GSC self-renewal in addition to its previously described role in negatively regulating the miRNA pathway during germline cyst development. Disruption of mei-P26 results in a bam-dependent GSC loss phenotype and further characterization reveals that Mei-P26 fosters BMP signal transduction within GSCs by repressing Brat protein expression. In addition, Mei-P26 also appears to participate in the miRNA-mediated silencing of orb mRNA in GSCs. These results indicate that Mei-P26 carries out multiple functions within the Drosophila ovary and might be at the center of a molecular hierarchy that controls the fates of GSCs and their differentiating daughters (Li, 2012).

Three observations suggest that mei-P26 functions within GSCs. First, the average number of GSCs per terminal filament decreases from an average of two to well below one in mei-P26 mutant ovaries. Second, mei-P26 mutant germline clones are rapidly lost from the GSC niche. Third, syncytial cysts and Bam-expressing cells are often observed immediately adjacent to the cap cells in mei-P26 mutant ovaries (Li, 2012).

Research over the last ten years has shown that BMP ligands emanating from cap cells at the anterior of the germarium initiate a signal transduction cascade in GSCs that results in the transcriptional repression of bam. Stem cell daughters one cell diameter away from the cap cell niche express bam, suggesting that a steep gradient of Dpp availability or responsiveness exists between GSCs and cystoblasts. Recent work has shed light on how various mechanisms antagonize BMP signaling in cystoblasts. For example, the ubiquitin ligase Smurf (Lack -- FlyBase) promotes germline differentiation and partners with the serine/threonine kinase Fused to reduce levels of the Dpp receptor Tkv in cystoblasts. The TRIM-NHL domain protein Brat also functions in cystoblasts, serving to translationally repress Mad expression. Notably, inappropriate expression of Brat within GSCs results in a stem cell loss phenotype. Brat itself is translationally repressed in GSCs by the Pumilio-Nanos complex. Mutant phenotypes and co-IP experiments presented in this study support a model in which Mei-P26 partners with Nanos to repress Brat expression in GSCs. This negative regulation of Brat expression protects the BMP signal transduction pathway in GSCs from inappropriate deactivation (Li, 2012).

Mei-P26 appears to enhance miRNA-dependent translational silencing within GSCs based on several lines of experimental evidence. First, co-IP experiments using ovarian extracts from c587-gal4>UAS-dpp and bam mutants suggest that Mei-P26 physically associates with Ago1 and GW182 in undifferentiated germ cells. Second, disruption of mei-P26 results in a GSC loss phenotype, similar to the effects of disrupting components of the miRNA pathway tested to date. Third, Mei-P26 and Ago1 can physically associate with the same target mRNA. Finally, disruption of either Ago1 or mei-P26 results in increased expression of this target in GSCs. The evidence that Mei-P26 promotes miRNA action in certain contexts is consistent with the established activities of its close homologs NHL-2 and TRIM32 (Li, 2012 and references therein).

It is proposed that Mei-P26 regulates GSC self-renewal and early germ cell differentiation through distinct mechanisms. In GSCs, Mei-P26 promotes self-renewal by repressing the expression of Brat and potentially other negative regulators of BMP signal transduction. Within stem cells, Mei-P26 also functions together with miRISC to attenuate the translation of specific mRNAs. miRISC does not appear to target brat mRNA based on clonal data. However, the possiblity cannot be ruled out that the enhancement of miRNA-mediated silencing of some mRNAs by Mei-P26 contributes to stem cell self-renewal. Interestingly, recent findings suggest that Pumilio can function together with the miRNA pathway in certain contexts In BJ primary fibroblasts, Pumilio 1, miR-221 and miR-222 regulate the expression of p27 in a 3' UTR-dependent manner. In response to growth factors, Pumilio 1 becomes phosphorylated, which in turn increases its RNA binding activity. Pumilio 1 binding to p27 mRNA results in a conformational change in the 3' UTR that allows miR-221 and miR-222 to bind more efficiently, resulting in greater repression of p27. Perhaps, together, Drosophila Pumilio, Nanos, Ago1 and Mei-P26 also silence specific messages in specific contexts. Identifying more direct in vivo targets for these proteins within GSCs will be crucial for testing this idea (Li, 2012).

In cystoblasts, Mei-P26 promotes germline cyst development by antagonizing the miRNA pathway. This study shows that Mei-P26 can also promote miRNA translational repression in another cell, the GSC. Evidence is provided that Mei-P26 physically associates with miRISC and co-regulates translation of at least one mRNA, orb, through specific elements within its 3′UTR. In cystoblasts and early developing cysts, the induction of Bam expression might cause Mei-P26 to switch from an miRISC-associated silencer to an miRNA antagonist. How Bam activates this switch is currently under investigation. The finding that Mei-P26 functions in both GSCs and differentiating cysts hints at a mechanism whereby different translational repression programs coordinate changes in cell fate (Li, 2012).

Further work will be needed to determine the specific biochemical function of Mei-P26 when it associates with either the Nanos complex or miRISC. Like other TRIM-NHL domain proteins, Mei-P26 contains a RING domain that may have E3 ubiquitin ligase activity. Based on results presented in this study, it is proposed that Mei-P26 and perhaps other TRIM-NHL domain proteins act as effectors for multiple translational repressor complexes. In this model, Mei-P26 is targeted to specific mRNAs through sequence-directed RNA-binding proteins. Specific protein substrates of Mei-P26 in the germline have not yet been determined but identifying these targets will provide key insights into how Mei-P26 and other related TRIM-NHL domain proteins regulate translational repression. Furthermore, the Mei-P26 complex is likely to target additional mRNAs for silencing in both GSCs and developing cysts. Identifying more of these mRNAs will further elucidate the complex translational regulatory hierarchies that control the balance between stem cell self-renewal and differentiation (Li, 2012).

The Smaug RNA-binding protein is essential for microRNA synthesis during the Drosophila maternal-to-zygotic transition

Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which maternal gene products are eliminated and the zygotic genome becomes transcriptionally active. During this process RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) target maternal mRNAs for degradation. In Drosophila, the Smaug (SMG), Brain tumor (BRAT) and Pumilio (PUM) RBPs bind to and direct the degradation of largely distinct subsets of maternal mRNAs. SMG has also been shown to be required for zygotic synthesis of mRNAs and several members of the miR-309 family of microRNAs (miRNAs) during the MZT. This study carried out global analysis of small RNAs both in wild type and in smg mutants. It was found that 85% all miRNA species encoded by the genome are present during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting RNAs (piRNAs) or small interfering RNAs (siRNAs), zygotic production of more than 70 species of miRNAs fails or is delayed in smg mutants. SMG is also required for the synthesis and stability of a key miRISC component, Argonaute 1 (AGO1), but plays no role in accumulation of the Argonaute-family proteins associated with piRNAs or siRNAs. In smg mutants, maternal mRNAs that are predicted targets of the SMG-dependent zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these miRNAs but not with SMG itself. The study hypothesizes that SMG controls the MZT, not only through direct targeting of a subset of maternal mRNAs for degradation but, indirectly, through production and function of miRNAs and miRISC, which act together with BRAT and/or PUM to control clearance of a distinct subset of maternal mRNAs (Luo, 2016).

To identify small RNA species expressed during the Drosophila MZT and to assess the role of SMG in their regulation 18 small-RNA libraries were produced and sequenced: nine libraries from eggs or embryos produced by wild-type females and nine from smg-mutant females. The 18 libraries comprised three biological replicates each from the two genotypes and three time-points: (1) 0-to-2 hour old unfertilized eggs, in which zygotic transcription does not occur and thus only maternally encoded products are present; (2) 0-to-2 hour old embryos, the stage prior to large-scale zygotic genome activation; and (3) 2-to-4 hour old embryos, the stage after to large-scale zygotic genome activation. After pre-alignment processing, a total of ~144 million high quality small-RNA reads was obtained and 110 million of these perfectly matched the annotated Drosophila genome (Luo, 2016).

Loss of SMG had no significant effect on piRNAs and siRNAs, or on the Argonaute proteins associated with those small RNAs: Piwi, Aubergine (AUB), AGO3, and AGO2, respectively. In contrast, loss of SMG resulted in a dramatic, global reduction in miRNA populations during the MZT as well as reduced levels of AGO1, the miRISC-associated Argonaute protein in Drosophila (Luo, 2016).

A pre-miRNA can generate three types of mature miRNA: (1) a canonical miRNA, which has a perfect match to the annotated mature miRNA; (2) a non-canonical miRNA, which shows a perfect match to the annotated mature miRNA but with additional nucleotides at the 5'- or 3'- end that match the adjacent primary miRNA sequence, and (3) a miRNA with non-templated terminal nucleotide additions (an NTA-miRNA), which has nucleotides at its 3'-end that do not match the primary miRNA sequence (Luo, 2016).

In these libraries a total of 364 distinct miRNA species were identified that mapped to miRBase, comprising 85% (364/426) of all annotated mature miRNA species in Drosophila. Thus, the vast majority of all miRNA species encoded by the Drosophila genome are expressed during the MZT. Overall, in wild type, an average of 75% of all identified miRNAs fell into the canonical category. The remaining miRNAs were either non-canonical (10%) or NTA-miRNAs (15%) (Luo, 2016).

To validate these sequencing results, those mature miRNA species identified in the data that perfectly matched the Drosophila genome sequence (i.e., canonical and non-canonical) were compared with a previously published miRNA dataset from 0 to 6 hour old embryos. To avoid differences caused by miRBase version, data sets from previous study were remapped to miRBase Version 19 and f99% of their published miRNA species were found to be on the miRNA list (176/178 mature miRNA species comprising 161 canonical miRNA s and 94 non-canonical miRNA s) . There were an additional 181 mature miRNA species in the library that had not been identified as expressed in early embryos in the earlier study (Luo, 2016).

As a second validation, the list of maternally expressed miRNA species (those present in the 0-to-2 hour wild-type unfertilized egg samples) were compared with the most recently published list of maternal miRNAs, which had been defined in the same manner. 99% of the 86 published maternal miRNA species were on this study's maternal miRNA list (85/86). An additional 144 maternal miRNA species in the library were identified that had not been observed in the previous study. Identification of a large number of additional miRNA species in unfertilized eggs and early embryos can be attributed to the depth of coverage of the current study. The current dataset, therefore, provides the most complete portrait to date of the miRNAs present during the Drosophila MZT (Luo, 2016).

Next, global changes in miRNA species during the MZT were analyzed in wild-type embryos. A dramatic increase was observed in the proportion of miRNAs relative to other small RNAs that was due to an increase in absolute miRNA amount rather than a decrease in the amount of other types of small RNAs. In wild-type 0-to-2 hour unfertilized eggs, the proportion of the small RNA libraries comprised of canonical and non-canonical miRNAs was 12.8%. These represent maternally loaded miRNAs since unfertilized eggs do not undergo zygotic genome activation. The proportion of small RNAs represented by miRNAs increased dramatically during the MZT, reaching 50.7% in 2-to-4 hour embryos. The other abundant classes of small RNAs underwent either no change or relatively minor changes over the same time course. It is concluded that there is a large amount of zygotic miRNA synthesis during the MZT in wild-type embryos (Luo, 2016).

For more detailed analysis of the canonical, non-canonical and NTA isoforms focus was placed on 154 miRNA species that possessed an average of > 10 reads per million (RPM) for all three isoform types in one or more of the six sample sets. A focus was placed on changes in wild type. Among all miRNAs, in wild type the proportion of canonical isoforms increased over the time-course from 69% to 83%, the proportion of non-canonical miRNAs remained constant (from 9% to 10%) , and the proportion of the NTA-miRNAs decreased (from 22% to 7%). These results derive from the fact that, during the MZT, the vast majority of newly synthesized miRNAs were canonical, undergoing a more than seven-fold increase from 103,105 to 744,043 RPM; that non-canonical miRNAs underwent a comparable, nearly seven-fold, increase from 13,902 to 92,199; whereas NTA-miRNAs underwent a less than two-fold increase, from 32,840 to 63,847, thus decreasing in relative proportion (Luo, 2016).

Whereas the proportion of the small-RNA population that was comprised of miRNAs increased fourfold over the wild-type time-course, concomitant with increases in overall miRNA abundance, there was no such increase in the smg mutant embryos: 21.9% of the small RNAs were miRNAs in 0-to-2 hour unfertilized smg mutant eggs (mean RPM = 203,415) and 20.5% (mean RPM = 196,110) were miRNAs in 2-to-4 hour smg mutant embryos (Luo, 2016).

This difference between wild type and smg mutants could have resulted from the absence of a small number of extremely highly expressed miRNA species in the mutant. Alternatively, it may have been a consequence of a widespread reduction in the levels of all or most zygotically synthesized miRNAs in smg mutants. To assess the cause of this difference, canonical miRNA reads were graphed in scatter plots. These showed that a large number of miRNA species had significantly reduced expression levels in 0-to-2 and in 2-to-4 hour smg-mutant embryos relative to wild type. Most of the down-regulated miRNA species exhibited a more than four-fold reduction in abundance relative to wild type. Furthermore, this reduction occurred for miRNA species expressed over a wide range of abundances in wild type (Luo, 2016).

Box plots were then used to analyze the canonical, non-canonical and NTA isoforms of the 154 miRNA species identified in the previous section. These showed that, in wild type, the median abundance of canonical, non-canonical and 3' NTA miRNAs increased significantly in 0-to-2 and in 2-to-4 hour embryos relative to 0-to-2 hour unfertilized eggs. In contrast, there was no significant increase in the median abundance of any of the three isoforms of miRNAs in the smg-mutant embryos. Also for all three isoform types, when each time point was compared between wild type and smg mutant, there was no difference between wild type and mutant in 0-to-2 hour unfertilized eggs but there was a highly significant difference between the two genotypes at both of the embryo time-points. Whereas the abundance of miRNAs differed between wild-type and mutant embryos, there was no difference in length or first-nucleotide distribution of canonical miRNAs, nor in the non-templated terminal nucleotides added to NTA-miRNAs (Luo, 2016).

As described above, during the wild-type MZT canonical miRNAs comprised the major isoform that was present (69% to 83% of miRNAs). It was next asked whether miRNA species could be categorized into different classes based on their expression profiles during the wild-type MZT. 131 canonical miRNA species that had > 10 mean RPM in at least one of the six datasets were analyzed. Hierarchical clustering of their log 2 RPM values identified five distinct categories of canonical miRNA species during the MZT. The effects of smg mutations on each of these classes were analyzed (Luo, 2016).

The data are consistent with a model in which SMG degrades its direct targets without the assistance of miRNAs whereas a large fraction of the indirectly affected maternal mRNAs in smg mutants fails to be degraded by virtue of being targets of zygotically produced miRNA species that are either absent or present at significantly reduced levels in smg mutants. Thus, SMG is required both for early, maternally encoded decay and for late, zygotically encoded decay. In the former case SMG is a key specificity component that directly binds to maternal mRNAs; in the latter case SMG is required for the production of the miRNAs (and AGO1 protein) that are responsible for the clearance of an additional subset of maternal mRNAs (Luo, 2016).

In Drosophila, the stability of miRNAs is enhanced by AGO1 and vice versa. Since miRNA levels are dramatically reduced in smg mutants, Ago1 mRNA and AGO1 protein levels were assessed during the MZT both in wild type and in smg mutants. In wild type, AGO1 levels were low in unfertilized eggs and 0-to-2 hour embryos but then increased substantially in 2-to-4 hour embryos. These western blot data are consistent with an earlier, proteomic, study that reported a more than three-fold increase in AGO1 in embryos between 0-to-1.5 hours and 3-to-4.5 hours. In contrast to AGO1 protein, it was found using RT-qPCR that Ago1 mRNA levels remained constant during the MZT. Taken together with a previous report that Ago1 mRNA is maternally loaded, the increase in AGO1 protein levels in the embryo is, therefore, most likely to derive from translation of maternal Ago1 mRNA rather than from newly transcribed Ago1 mRNA (Luo, 2016).

Next, AGO1, AGO2, AGO3, AUB and Piwi protein levels were analyzed in eggs and embryos from mothers carrying either of two smg mutant alleles: smg1 and smg47. The smg mutations had no effect on the expression profiles of AGO2, AGO3, AUB or Piwi. In contrast, in smg-mutant embryos, the amount of AGO1 protein at both 0-to-2 and 2-to-4 hours was reduced relative to wild type and this defect was rescued in embryos that expressed full-length, wild-type SMG from a transgene driven by endogenous smg regulatory sequences. The reduction of AGO1 protein levels in smg mutants was not a secondary consequence of reduced Ago1 mRNA levels since Ago1 mRNA levels in both the smg-mutant and the rescued-smg-mutant embryos were very similar to wild type (Luo, 2016).

A plausible explanation for the decrease in AGO1 levels in smg mutants is the reduced levels of miRNAs, which would then result in less incorporation of newly synthesized AGO1 into functional miRISC and consequent failure to stabilize the AGO1 protein. To assess this possibility, a time-course in wild-type unfertilized eggs was analyzed in which zygotic genome activation and, therefore, zygotic miRNA synthesis, does not occur. It was found that AGO 1 levels were reduced in 2-to-4 hour wild-type unfertilized eggs compared with wild-type embryos of the same age. This result is consistent with a requirement for zygotic miRNAs in the stabilization of AGO1 protein (Luo, 2016).

Next, wild-type unfertilized egg and smg-mutant unfertilized egg time-courses were compared, and AGO1 levels were found to be further reduced in the smg mutant relative to wild type. This suggests that SMG protein has an additional function in the increase in AGO1 protein levels that is independent of SMG's role in zygotic miRNA production (since these are produced in neither wild-type nor smg-mutant unfertilized eggs) (Luo, 2016).

To assess whether this additional function derives from SMG's role as a post-transcriptional regulator of mRNA, smg1 mutants were rescued either with a wild-type SMG transgene driven by the Gal4:UAS system (SMGWT) or a GAL4:UAS-driven transgene encoding a version of SMG with a single amino-acid change that abrogates RNA-binding (SMGRBD) and, therefore, is unable to carry out post-transcriptional regulation of maternal mRNAs. It was found that, whereas AGO1 was detectable in both unfertilized eggs and embryos from SMGWT-rescued mothers, AGO1 was undetectable in unfertilized eggs from SMGRBD-rescued mothers and was barely detectable in embryos from these mothers. Thus, SMG's RNA-binding ability is essential for its non-miRNA-mediated role in regulation of AGO1 levels during the MZT (Luo, 2016).

Since the abundance of SMGWT and SMGRBD proteins is very similar, the preceding result excludes the possibility that it is physical interaction between SMG and AGO1 that stabilizes the AGO1 protein. It was previously shown that the Ago1 mRNA is not bound by SMG. Thus, SMG must regulate one or more other mRNAs whose protein products, in turn, affect the synthesis and/or stability of AGO1 protein. It is known that turnover of AGO1 protein requires Ubiquitin-activating enzyme 1 (UBA1) and is carried out by the proteasome . It was previously shown that the Uba1 mRNA is degraded during the MZT in a SMG-dependent manner and that both the stability and translation of mRNAs encoding 19S proteasome regulatory subunits are up-regulated in smg-mutant embryos. It is speculated that increases in UBA1 and proteasome subunit levels in smg mutants contribute to a higher rate of AGO1 turnover and, thus, lower AGO1 abundance than in wild type (Luo, 2016).

AGO1 physically associates with BRAT. It is not known whether AGO1 interacts with PUM but it has been reported that, in mammals and C. elegans , Argonaute-family proteins interact with PUM/PUF-family proteins. Recent studies identified direct target mRNAs of the BRAT and PUM RBPs in early Drosophila embryos and showed through analysis of brat mutants that, during the MZT, BRAT directs late (i.e., after zygotic genome activation) decay of a subset of maternal mRNAs. These data permitted asking whether the maternal mRNAs that are predicted to be indirectly regulated by SMG via its role in miRISC production might be co-regulated by BRAT and/or PUM (Luo, 2016).

A highly significant overlap was found between the predicted miRNA-dependent indirect targets of SMG and both BRAT-and PUM-bound mRNAs in early embryos. This suggests that BRAT and PUM might function together with miRISC during the MZT to direct decay of maternal mRNAs (Luo, 2016).

Given that BRAT and PUM bind to largely non-overlapping sets of mRNAs during the MZT, there are three types of hypothetical BRAT-PUM-miRISC-containing complexes: one with both BRAT and PUM, one with BRAT only, one with PUM only. To assess this possibility for a specific set of zygotically produced miRNAs, the lists of mRNAs stabilized in 2-to-3 hour old embryos from miR-309 deletion mutants were compared to the lists of BRAT and PUM direct-target mRNAs. There was no significant overlap of PUM-bound mRNAs with those up-regulated in miR-309 mutants. However, there was a highly significant overlap of mRNAs up-regulated in miR-309-mutant embryos with BRAT-bound mRNAs. These results lead to the hypothesis that BRAT (but not PUM) co-regulates clearance of miR-309-family miRNA target maternal mRNAs during the MZT (Luo, 2016).

Protein Interactions

Nanos and Pumilio are required to recruit Brat to Hunchback mRNA. To test the biological significance of the interaction between Brat and the Nos/Pum/NRE ternary complex, it was asked whether substitutions in Pum and Nos that interfere with Brat recruitment also abrogate HB mRNA regulation in vivo. The impetus for these experiments derives from two properties of the Pum680 mutant, which bears the G1330D substitution in the seventh repeat of its RNA-binding domain. (1) PumG1330D binds RNA normally and recruits Nos into a ternary complex, but is defective in regulating HB in embryos; (2) when tested in a yeast four-hybrid experiment, PumG1330D does not recruit Brat. Seven additional Pum mutations were engineered by site-directed mutagenesis into both yeast and Drosophila expression vectors. Residues adjacent to 1330 or at analogous positions in other repeats within the RNA-binding domain were chosen for mutagenesis. The capacity of each Pum mutant to recruit Nos to the NRE or to recruit Brat to the Pum/Nos/NRE complex was assayed in transformed yeast. And the capacity of each Pum mutant to regulate HB mRNA translation in embryos (and thereby direct the development of abdominal segmentation) was assayed in transgenic flies. Brat recruitment in yeast correlates with HB mRNA regulation in embryos, suggesting that contacts between Pum and Brat are essential in vivo (Sonoda, 2001).

To test the role of Nos in Brat recruitment, mutations in the Nos carboxy-terminal domain were screened that were identified originally in a genetic screen for defective nos alleles. Most of the Nos mutants are not recruited by Pum into a ternary complex with the NRE. However, one mutant, NosM379K, is incorporated into a ternary complex normally, but this complex does not interact with Brat. When expressed in appropriately engineered transgenic embryos, the M379K derivative is stable but inactive, consistent with the idea that contacts between Nos and Brat are also essential for HB mRNA regulation in vivo (Sonoda, 2001).

All of the protein-protein interaction experiments described above involve indirect assays performed in yeast. To determine whether the interaction between Brat and the ternary complex is direct and independent of yeast factors, binding experiments in vitro were performed using purified components. Ternary complexes containing the Pum RNA-binding domain, the carboxy-terminal domain of Nos and the wild-type NRE can be captured on glutathione agarose beads (which bind to the GST moeity attached to Pum). Under the same reaction conditions, Brat is recruited into a quaternary complex that, by three criteria, has the same properties as the complex detected in yeast experiments. (1) Retention of either Nos or Brat is substantially reduced (~10-fold and 6-fold, respectively) if the NRE bears a mutation that abrogates Nos binding; (2) Brat is not detectably retained by a binary Pum/NRE complex in the absence of Nos; (3) the PumG1330D mutant captures Nos but not Brat. Taken together, these results demonstrate that Nos and Pum act jointly and directly to recruit Brat to the NRE (Sonoda, 2001).

All of the recessive brat alleles are associated to a greater or lesser extent with a variety of phenotypes, including a dramatic (~10-fold) overgrowth of the larval brain (Arama, 2000); early oogenesis defects; metastasis of transplanted brain and imaginal tissue, and a maternal effect on embryonic viability. This last class of 'female sterile' (fs) alleles appears to interfere preferentially with function in the female germ line, although class members also exhibit the other brat phenotypes. Unlike lethal alleles that encode truncated proteins, bratfs1 and bratfs3 encode proteins with single amino acid substitutions at conserved residues within the NHL domain (Sonoda, 2001 and Arama, 2000).

It was of interest to determine whether the substitutions in the Bratfs1 and Bratfs3 mutant proteins interfere with recruitment to the Nos/Pum/NRE ternary complex. To this end, ternary complexes were assembled in vitro with purified GST-Pum, Nos, and NRE-bearing RNA; these were subsequently incubated with embryonic extracts prepared from embryos derived from either wild-type or bratfs mutant females (henceforth referred to as bratfs mutant embryos). Complexes were captured on glutathione-agarose beads, and bound proteins displayed on a Western blot probed with Brat-specific antibodies (Sonoda, 2001).

About 5% of full-length Brat+ is retained under the conditions of the experiment, but neither Bratfs1 nor Bratfs3 binds appreciably to the ternary complex. The mutant proteins accumulate to normal levels and are stable in vivo. Thus, the single amino acid substitutions in the Bratfs mutant proteins prevent efficient recruitment to the Nos/Pum/NRE complex (Sonoda, 2001).

The consequences of altered Brat function on embryonic development were examined. bratfs mutant embryos have defects in abdominal segmentation that are essentially indistinguishable from those in embryos with reduced nos or pum function. These defects arise as a result of incomplete translational repression of HB mRNA in the posterior of the preblastoderm embryo, as is the case for nos or pum mutants. The level and distribution of Nos, Pum, and HB mRNA appear to be normal in bratfs mutant embryos, suggesting that Brat does not act indirectly to regulate abdominal segmentation. Taken with the interaction data, it is concluded that recruitment of Brat jointly by Nos and Pum to the NRE is required for the normal regulation of HB mRNA in the early embryo (Sonoda, 2001).

The Brain Tumor (Brat) protein is recruited to the 3' untranslated region (UTR) of hunchback mRNA to regulate its translation. Recruitment is mediated by interactions between the Pumilio RNA-binding Puf repeats and the NHL domain of Brat, a conserved structural motif present in a large family of growth regulators. The crystal structure of the Brat NHL domain is described and a model is presented of the Pumilio-Brat complex derived from in silico docking experiments and supported by mutational analysis of the protein-protein interface. A key feature of the model is recognition of the outer, convex surface of the Pumilio Puf domain by the top, electropositive face of the six-bladed Brat ß-propeller. In particular, an extended loop in Puf repeat 8 fits in the entrance to the central channel of the Brat ß-propeller. Together, these interactions are likely to be prototypic of the recruitment strategies of other NHL-containing proteins in development (Edwards, 2003).

One feature of the Brat-Pum model is common to protein complexes formed by other ß-propellers: interaction along the top surface, particularly around the central channel. The WD40 domain of the transcriptional corepressor Tup1 interacts with the DNA-binding factor Matalpha2 to regulate mating-type genes in budding yeast. Although the structure of this complex is unknown, all the mutations in Tup1 that interfere with Tup1-Matalpha2 interaction are located on the top surface of its seven-bladed ß-propeller around the central channel, analogous to the mutations described for Brat. The ubiquitin-conjugating enzyme Cdc4 similarly uses the top surface of its eight-bladed ß-propeller to bind a peptide ligand derived from the Cdk inhibitor, Sic1. In the case of Cdc4, the peptide-binding site is relatively small (buried surface area of ~750 Å2) when compared to the large interface in the docked Brat-Pum complex (2,900 Å2). However, in each case, a flexible peptide (or a loop in the case of Pumilio) docks in and around the central pore, suggesting an emerging recognition theme for ß-propeller molecules (Edwards, 2003).

Brat is normally recruited not to Pum alone, but to a ternary complex of Pum and Nos bound to the NRE. The model of the Pum-Brat subassembly suggests that the 'edge' of the Brat ß-propeller is available to interact with Nos, much as Gß and the scaffolding protein clathrin use the sides of their seven-bladed ß-propellers to bind cofactors. Although its location in the repressor complex is not yet well defined, Nos is probably recruited to the Pum-RNA complex via contacts made by the C terminus of the Pum RNA-binding domain. The proximity of the NHL domain to the presumptive Nos-binding site on Pum extends the likelihood of cooperative Brat-Nos interactions, and may explain why Brat is only recruited subsequent to Pum and Nos binding to the hb 3' UTR (Edwards, 2003).

The Drosophila proteome contains two additional NHL domain proteins [MeiP-26 and Dappled (Dpld)] that, based on genetic evidence, appear to be tumor suppressors and growth regulators like Brat. It is tempting to speculate they interact with cofactors or regulatory targets much as Brat interacts with Pum. However, neither seems likely to use Pum as a cofactor. The NHL domains of Brat and MeiP-26 are very similar: There are no major insertions or deletions in the DA loops of MeiP-26, and the top surface of its ß-propeller, like that of Brat, is electropositive. Thus, based on structural considerations, MeiP-26 might interact with Pum; however, genetic experiments suggest it does not do so in vivo. In contrast, structural considerations suggest the NHL domain of Dpld, which governs the growth of larval organs, is unlikely to bind Pum due to substantial differences in its predicted surface charge distribution and the presence of large insertions in the DA loops on the top surface. Therefore, although Brat, MeiP-26, and Dpld may use their NHL domains in a similar manner, each probably binds to distinct partners (Edwards, 2003).

Based on the analysis of loss- and gain-of-function experiments, Brat appears to regulate abdominal segmentation (via hb translation), brain size, cell size in the imaginal discs, and the accumulation of rRNA. Strikingly, substitutions that abrogate many of these processes map to the 'same' top surface of the NHL domain, near the central channel. This suggests that Brat may recognize protruding, flexible loops in a number of protein cofactors or regulatory targets, much as it recognizes the loop in Puf repeat 8 that constitutes the core of the Brat-Pum interaction surface (Edwards, 2003).

Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal

An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. The Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. It is concluded that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation (Lee, 2006).

The translational repressor Brat directly interacts with the Miranda central domain and is a Miranda cargo protein specifically partitioned into the GMC daughter cell during neuroblast asymmetric cell division. Brat is the first Miranda cargo protein identified since the original finding that Prospero and Staufen were shown to be Miranda cargo proteins over 8 years ago. Prospero is a homeodomain transcriptional repressor, and Staufen is an RNA binding protein that interacts with prospero mRNA. It is unknown whether Miranda has other cargo proteins in addition to Brat, Prospero, and Staufen, and it is unclear whether all three known cargo proteins can associate with a single Miranda protein (Lee, 2006).

It is unknown how Brat promotes Prospero basal localization. A model is favored in which Brat protein stabilizes Prospero/Miranda interactions, so that Prospero protein is cytoplasmic in the absence of Brat. An obviously elevated level of cytoplasmic Prospero is not seen in brat mutant neuroblasts, but delocalization of Prospero protein from the basal crescent might not be visible over background. Alternatively, brat mutant neuroblasts may fail to transcribe or translate prospero in neuroblasts. This would most likely be an indirect effect, since Brat has been shown to only have translational repressor function. It has not been possible to detect prospero mRNA in wild-type larval neuroblasts, despite robust levels in GMCs, so this possibility has not been tested (Lee, 2006).

Some brat mutant neuroblasts show expanded aPKC cortical crescents, in some cases reaching the basal cortex. This phenotype appears specific for aPKC, because other apical cortical proteins (e.g., Baz, Pins) are unaffected. Brat might repress aPKC translation, leading to increased aPKC protein levels in brat mutants. Alternatively, the absence of Prospero or other basal cortical proteins may indirectly affect aPKC localization (Lee, 2006).

brat mutant brains show a dramatic increase in the number of large, proliferating Dpn+ neuroblasts between 48 and 96 hr ALH. Where do these hundreds of extra neuroblasts come from? They are unlikely to come from outside the brain, or from dedifferentiation of neurons or glia, although these models can't formally be ruled out. They are likely to derive from the pool of Dpn+ neuroblasts in the brain, because these are the primary pool of proliferating cells in the larval central brain, and thus the best candidates to generate the thousands of extra cells found in the hypertrophied brat mutant brains (Lee, 2006).

A model is proposed in which a subset of brat mutant “GMCs” enlarge into proliferative neuroblasts. This model is supported by several lines of evidence. (1) brat mutant GMCs maintain neuroblast-specific gene expression (Dpn, Miranda, Worniu); (2) brat mutants show an inverse relationship between increasing neuroblast number and decreasing neuronal number over time, consistent with GMCs forming neuroblasts instead of neurons; (3) brat mutant GMCs can be labeled by a BrdU pulse at their birth, yet most lose BrdU incorporation during the chase interval, showing that they either reenter the cell cycle or undergo cell death, and that cell death is not consistent with the brain overgrowth phenotype; (4) brat mutant telophase profiles show that all GMCs are born as small Miranda+ cells, ruling out physically or molecularly symmetric neuroblast divisions as a mechanism for increasing the neuroblast population; and (5) brat mutants show cell enlargement in other tissues, and a similar cell growth phenotype has been observed in mutants in the C. elegans brat ortholog (Lee, 2006).

What is the cellular origin of the brat mutant phenotype? brat mutant GMCs are compromised in three ways: they lack Brat translational repression activity, lack Prospero, and some may have ectopic aPKC. Loss of Brat translational repression activity could well play a role in the ectopic neuroblast self-renewal phenotype, because all brat mutants disrupting the NHL translational repression domain exhibit a brain tumor phenotype, and Brat has been previously shown to negatively regulate cell growth. Loss of Prospero also plays a role in the brat phenotype: prospero mutant GMCs have a failure to downregulate neuroblast gene expression and a failure in neuronal differentiation, similar to brat mutants. prospero null mutant embryos also show a slight delay in neuronal differentiation, although they appear to undergo normal neuroblast self-renewal. Finally, ectopic aPKC can also mimic aspects of the brat phenotype, including formation of supernumerary large Dpn+ neuroblasts. Interestingly, the mammalian paralogs of Drosophila aPKC (aPKCλ/ζ) are expressed in neural progenitors of the ventricular zone, and the mammalian Prospero ortholog Prox1 is expressed in differentiating neurons of the subventricular zone. Thus, identifying Prospero transcriptional targets and aPKC phosphorylation targets may provide further insight into the molecular mechanism of neural stem cell self-renewal in both Drosophila and mammals (Lee, 2006).

Cap-dependent translational inhibition establishes two opposing morphogen gradients in Drosophila embryos

In the early Drosophila embryo, asymmetric distribution of transcription factors, established as a consequence of translational control of their maternally-derived mRNAs, initiates pattern formation. For instance, translation of the uniformly distributed maternal hunchback (hb) mRNA is inhibited at the posterior to form an anterior-to-posterior protein concentration gradient along the longitudinal axis. Inhibition of hb mRNA translation requires an mRNP complex (the NRE-complex) that consists of Nanos (Nos), Pumilio (Pum) and Brain tumor (Brat) proteins, and the Nos responsive element (NRE) present in the 3' UTR of hb mRNA. The identity of the mRNA 5' effector protein that is responsible for this translational inhibition remained elusive. This study shows that d4EHP, a cap-binding protein which represses caudal (cad) mRNA translation (Cho, 2005), also inhibits hb mRNA translation by interacting simultaneously with the mRNA 5' cap structure (m7GpppN, where N is any nucleotide) and Brat. Thus, by regulating Cad and Hb expression, d4EHP plays a key role in establishing anterior-posterior axis polarity in the Drosophila embryo (Cho, 2006).

Transcription is globally repressed in the rapidly-dividing nuclei of early Drosophila embryos, and therefore gene expression is largely regulated by translational control of maternally-provided mRNAs. Translation is often regulated at initiation, which occurs in multiple steps starting with the recruitment of the 40S ribosomal subunit to the 5' end of an mRNA and resulting in the correct positioning of the 80S ribosome at the initiation codon. Recognition of the cap structure by eIF4F (composed of three subunits: eIF4E, eIF4A and eIF4G) is an integral part of this process. Moreover, eIF4G interacts both with eIF4E and the poly(A)-binding protein (PABP), thus circularizing the mRNA, which in turn is believed to promote re-initiation. Consistent with their importance, eIF4E and PABP have emerged as major targets of translational regulatory mechanisms mediated by such modulator proteins as 4E-BPs and Paip2 (Cho, 2006).

Embryonic development in many metazoans requires the activity of various maternal determinants called morphogens, whose spatial and temporal expression is tightly regulated. In Drosophila, local morphogen concentrations are important for the establishment of polarity and subsequent organization of both the antero-posterior and dorso-ventral axes of the embryo. A key morphogen for antero-posterior patterning is the transcription factor Hunchback (Hb); when maternal Hb is allowed to accumulate inappropriately, posterior segmentation is blocked. Two modes of translational control have been proposed for the establishment of the maternal Hb gradient: translational silencing via deadenylation and inhibition at the initiation step in a cap-dependent manner (Cho, 2006 and references therein).

d4EHP, an eIF4E-like cap-binding protein that does not interact with deIF4G and d4E-BP, inhibits the translation of cad mRNA by interacting simultaneously with the cap and Bicoid (Bcd) (Cho, 2005). While many embryos (~41%) produced by females homozygous for the d4EHPCP53 mutation showed anterior patterning defects consistent with mislocalized Cad, some (~7%) also exhibited patterning defects such as missing abdominal segments that cannot be readily explained by ectopic Cad expression. Since inhibition of hb mRNA translation has been linked in one study to the cap structure (Chagnovich, 2001) and since these additional phenotypes could be consistent with inappropriate regulation of Hb, this study investigated the role of d4EHP in Hb expression. Embryos (0-2h) from females homozygous for the d4EHPCP53 mutation (Cho, 2005) were collected and immunostained using anti-Hb antibody. DNA was stained with DAPI to highlight the nuclei). For simplicity, embryos will subsequently be referred to by their maternal genotype. To evaluate the extent of the Hb gradient its signal intensity was measured at 38-50 locations along the anterior-posterior axes of 6-16 embryos of each genotype. The values were corrected for overall signal intensity and then normalized the data for embryo length (EL, anterior pole = 0%, posterior pole = 100%). The normalized values were plotted and average intensity values were calculated to obtain an average trend. It was observed that in OreR embryos, Hb signal intensity drops steeply in the middle of the embryo and reaches 50% maximum intensity at 48% EL. In d4EHPCP53 embryos the Hb expression domain extended substantially further toward the posterior and signal intensity remained at approximately 50% of the maximum throughout the region between 50-75% EL. Normal Hb distribution was restored to d4EHPCP53 mutant embryos by transgene-derived expression of wild-type d4EHP (d4EHPwt, but not by expression of a mutant form of d4EHP (d4EHPW114A), which is unable to bind the cap structure. Expression of another form of d4EHP (d4EHPW85F) which cannot bind Bcd, fully rescued the defective Hb gradient. The expression levels of the wild-type and mutant d4EHP transgenes are essentially equal. Distributions of Nos, Pum, and Brat were unaffected in d4EHPCP53 mutant embryos. Taken together, these data demonstrate that d4EHP plays a key role in establishing the posterior boundary of Hb expression in a manner that requires its cap-binding activity but not an association with Bcd (Cho, 2006).

It was reasoned that Brat might be a candidate partner protein for d4EHP since both are relevant for hb regulation. Thus whether d4EHP and Brat physically interact was investigated in vivo. Extracts prepared from 0-2h Oregon-R (OreR) embryos were treated with RNase and used to examine the interaction between Brat and d4EHP. Western blotting analysis using antibodies against d4EHP and Brat demonstrates that, while anti-d4EHP co-immunoprecipitated endogenous Brat, pre-immune serum did not. To further demonstrate the specificity of this interaction, HA-tagged deIF4EI and the RNA-binding protein La (negative controls) were transfected in HEK293 cells along with FLAG-tagged full-length Brat. While anti-FLAG antibody immunoprecipitated wild-type HA-d4EHP together with FLAG-Brat, deIF4EI and La failed to co-immunoprecipitate. Similarly, other RNA-binding proteins such as hnRNP U and HuR, and a d4EHP mutant (W173A), in which a tryptophan residue that is part of the hydrophobic core and thus affects protein folding is replaced, also failed to interact with Brat, demonstrating that Brat interacts specifically with d4EHP. Since a cell transfection system was used to assay for the d4EHP:Brat interaction, it is possible that other bridging proteins are required for the d4EHP-Brat association (Cho, 2006).

To identify the Brat-interacting domain of d4EHP, a number of individual residues located on its convex dorsal surface were mutated, and co-immunoprecipitation was tested with Brat. From this work no point mutant of d4EHP was identified that abrogated the interaction. As an alternative approach, chimeric proteins were created in which different domains of d4EHP were replaced with their counterparts from deIF4EI, taking advantage of the knowledge that, unlike d4EHP, deIF4EI does not interact with Brat. Mhree mutant forms of d4EHP were produced, with each one of its three dorsal α-helices replaced with that of deIF4EI. It was found that, while helix 1 and 2 mutants failed to disrupt binding to Brat, replacement of d4EHP helix 3 (residues 179 to 194) significantly reduced the interaction with Brat. Consistent with these observations, α-helix 3 is the most divergent between d4EHP and deIF4EI. The overall structure of d4EHP is not affected by the replacement of helix 3 with its deIF4EI counterpart, since the chimeric protein still binds to the cap. Thus, these data demonstrate that Brat interacts with d4EHP on its convex dorsal surface and that this interaction is mediated by the third α-helix of d4EHP (Cho, 2006).

A C-terminal domain of Brat termed the NHL domain is both necessary and sufficient to inhibit hb mRNA translation. The NHL domain contains two large surfaces (defined as top and bottom), that can support protein-protein interactions. While the top surface of the NHL domain binds to Pum and Nos, the bottom surface does not interact with any known protein. Although the Brat NHL domain contains an amino acid sequence that conforms to the YxxxxxxLΦ d4EHP-binding motif (Cho, 2005), the d4EHP:Brat interaction does not require this motif, since a Brat deletion mutant that lacks it can still interact with both d4EHP and the d4EHP W85F mutant. This sequence is most probably masked from interaction with d4EHP because it is located in the hydrophobic core of the NHL domain. To determine whether the d4EHP:Brat interaction requires the NHL domain, a Brat mutant that lacks the domain (Brat ΔNHL) was engineered and used in a co-immunoprecipitation experiment. While wild-type Brat was readily co-immunoprecipitated with d4EHP, the Brat ΔNHL mutant was not. Thus, it is concluded that the NHL domain is the site of d4EHP interaction. To further characterize this interaction, point mutations were designed to replace residues on the two surfaces of the NHL domain, and the mutant proteins were tested for their ability to interact with d4EHP. Mutation of a top surface residue that affects Brat interaction with Pum (G774A) did not affect the d4EHP:Brat interaction. However, when residues on the bottom surface were mutated, the d4EHP:Brat interaction was either significantly reduced (G860D and KE809/810AA), or abrogated (R837D and K882E). Importantly, the Brat NHL R837D mutant can assemble into an NRE-complex, demonstrating that this mutation specifically affects the d4EHP interaction and not the interactions with Pum and Nos (Cho, 2006).

Brat inhibits hb mRNA translation by interacting with the NRE-complex (Sonoda, 2001). Since d4EHP interacts physically with Brat, it was asked whether d4EHP can be co-purified with the NRE complex in vitro. Incubation of recombinant components of the NRE-complex (Brat, Pum, Nos and NRE) together with HA-tagged d4EHP resulted in the retention of d4EHP on glutathione-Sepharose beads through the GST-Pum RNAB fusion protein. The association of Brat with d4EHP was dependent on the ability of d4EHP to bind to Brat, since addition of Pum/Nos/NRE alone or in combination with the Brat R837D mutant failed to capture it. Thus, by interacting with Brat, d4EHP can associate with the NRE complex (Cho, 2006).

To investigate the biological significance of the d4EHP:Brat interaction, the effects of Brat mutants, which are defective for d4EHP binding, were examined in Drosophila embryos. bratfs1 mutant embryos exhibit a significant expansion of the Hb expression domain towards the posterior and display severe abdominal segmentation defects. When a bratWT transgene is expressed in the bratfs1 mutant background, normal Hb distribution and a wild-type segmentation pattern is restored). To investigate whether interaction with d4EHP is essential for the function of Brat in embryonic patterning, transgenes were introduced encoding mutant forms of Brat that affect the d4EHP:Brat interaction (bratR837D and bratK882E) into the bratfs1 mutant background. Despite being expressed at levels similar to the bratWT transgene, these mutant forms fail to fully rescue the normal Hb gradient and, importantly, do not fully rescue the bratfs1 mutant phenotype. Taken together, these data strongly argue that the d4EHP:Brat interaction contributes significantly to hb regulation (Cho, 2006).

Through its interaction with Brat, d4EHP defines and sharpens the posterior boundary of Hb expression. Based on the hypomorphic d4EHPCP53 phenotype, its activity appears most relevant to hb regulation in the region of the embryo from 50-75% EL, although it is possible that a null d4EHP allele would have more drastic effects. The d4EHP:Brat interaction is mediated via residues on the bottom surface of the Brat NHL domain. Thus, as in the established for cad (Cho, 2006), a simultaneous interaction of d4EHP with the cap and Brat results in mRNA circularization and renders hb translationally inactive. Since the interaction between Brat and d4EHP does not involve the previously described 4EHP-binding motif (YxxxxxxLΦ), it is possible that d4EHP interacts with Brat through a bridging protein (Cho, 2006).

The data support a model for the requirement for the 5' cap structure in regulation of endogenous hb mRNA. This is consistent with an earlier study that assessed translation of NRE-containing mRNAs after injection into Drosophila embryos and concluded that the cap structure is functionally significant (Chagnovich, 2001). In contrast, another study reported that Nos and Pum repressed the expression of an engineered transgene containing an internal ribosome entry site (IRES) and a hairpin loop designed to block cap-dependent translation. These results were used to conclude that hb translational repression is cap-independent. However, the phenotypic assay used in that study was indirect and the observed results could also be caused by RNA destabilization. Furthermore, Nos-dependent deadenylation was also shown to be important in establishing the Hb gradient. It is difficult to reconcile all these data without concluding that multiple distinct post-transcriptional mechanisms regulate Hb expression, including two that require Nos. The novel d4EHP-dependent mechanism defined in this study appears important for repressing hb in more central regions of the embryo, while cap-independent regulation involving deadenylation of hb mRNA may predominate in more posterior regions of the embryo. It is noted that mutant forms of Brat that are abrogated for d4EHP interaction retain substantial (but not complete) activity in repressing hb, suggesting some redundancy between these two mechanisms. Analogous overlapping translational control mechanisms have recently been reported for Bruno, which represses Oskar (Osk) expression both through cap-dependent translational regulation and through packaging osk mRNA into translationally silent RNP complexes (Cho, 2006).

Identification of a common inhibitory mechanism which regulates cad and hb mRNA translation simplifies the understanding of how the anterior-posterior axis is organized during early Drosophila embryogenesis. By regulating two classical maternal morphogenetic gradients, d4EHP plays a critical role in early Drosophila embryonic development. It is noteworthy that d4EHP is recruited to these mRNAs through different RNA binding proteins that presumably recognize different sequence elements. In the case of cad, d4EHP becomes associated by binding directly to Bcd, which in turn recognizes a defined 3’UTR element, the BBR. In the case of hb, Bcd binding is not involved in d4EHP recruitment and no element similar to the BBR is present. It remains uncertain whether the interaction between d4EHP and Brat is direct or indirect; since d4EHP and Brat are both uniformly distributed in early embryos, a non-uniformly distributed bridging protein mediating this interaction may be the basis of the spatially-restricted requirement for d4EHP in hb repression. Since d4EHP and some of its interacting partners are evolutionarily conserved in higher eukaryotes and because cap-dependent translation regulation plays such an important role in eukaryotic gene expression, it is predicted that 4EHP-dependent translational inhibitory mechanisms are widespread throughout the animal kingdom (Cho, 2006).

The crystal structure of the NHL domain in complex with RNA reveals the molecular basis of Drosophila brain-tumor-mediated gene regulation

TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, this study identified Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. Brat-mediated repression and direct RNA-binding were shown to depend on the identified motif; binding of the localization factor Miranda to the Brat-NHL domain was shown to inhibit Brat activity. Finally, to unravel the sequence specificity of the NHL domain, the Brat-NHL domain was crystalized in complex with RNA and a high-resolution protein-RNA structure of this fold is presented (Loedige, 2015).

The identification of binding sites of RBPs and the elucidation of how binding specificity and selectivity are brought about are key questions to understand post-transcriptional gene regulatory networks. This study reports the identification of the target RNAs and the binding motif of the TRIM-NHL protein Brat. By solving the crystal structure of the Brat-NHL domain in complex with its consensus sequence, molecular insights are provided into how sequence specificity and selectivity in RNA binding by Brat-NHL is accomplished. Only a few RBDs, including RNA recognition motifs (RRMs), K homology (KH) domains, cold shock domains (CSDs), and DEAD box RNA helicases, are well characterized, and their RNA specificity has been studied. The six-bladed β propeller of the NHL domain provides a compact platform (~47-Å diameter), and the RNA runs across the entire positively charged top surface. Sequence specificity is provided by surface complementary (three preformed binding clefts accommodate the six bases of the consensus motif) and base-specific hydrogen bonds to the protein main and side chain. Interestingly, all pockets are formed by two neighboring blades. It is therefore tempting to speculate that β propeller structures might be ideal platforms for generating sequence-specific RNA contacts, depending on the loops and side chains protruding into the inter-blade space. WD40 domains, which are highly abundant in eukaryotic proteomes, fold into β propeller structures as well. Although their role in protein-protein interactions is well established, it is conceivable that WD40 domains are widespread sequence-specific RBDs. Supporting this idea, the WD40 domain of Gemin5 has been recognized as RBD interacting with small nuclear RNAs (snRNAs), and recent large-scale screens have identified several WD40 domain containing proteins as direct mRNA binders (Loedige, 2015).

Using RNAcompete, this study identified RNA-binding motifs for several members of the TRIM-NHL protein family, corroborating the hypothesis that TRIM-NHL proteins constitute a conserved family of RBPs. The observation that different TRIM-NHL proteins bind distinct sequence motifs suggests their association with different (m)RNA targets and their engagement in different biological processes. Evolutionary related NHL domains, however, use similar RNA-binding sites, indicating that the processes these TRIM-NHL proteins regulate might be conserved (Loedige, 2015).

Brat-NHL binds single-stranded RNA, and its binding affinity seems to be impaired when nucleotides of the motif are engaged in RNA-RNA base-pairing, illustrating the importance of the structural context and target site accessibility. Even though the Pum HD is a single-stranded RBD as well, high-affinity binding is still observed when nucleotides of its motif base pair. Within the context of the NRE, where Brat and Pum sites are in close proximity, Pum binding changes the RNA structure, resulting in a more accessible Brat-binding site. It should be noted that the observed binding mechanisms are in an in vitro setting. The secondary structure of the full-length 3' UTR in vivo might be different and involve different regions as well as additional proteins being bound to the RNA. Nevertheless, for longer RNAs, the amount of secondary structure is probably larger and more dynamic (Loedige, 2015).

Although this study identified several examples of close proximity of Brat and Pum binding sites, no evidence was found for a general correlation of the two RBPs by computational analysis, indicating that, for the majority of targets, Brat acts independently of Pum. Nevertheless it seems conceivable that, in addition to Pum, other RBPs might affect Brat RNA binding activity as well. Close proximity of binding sites for two distinct RBPs can increase affinity and specificity as the cognate RNA sequence is elongated, as has been shown recently for the cooperative RNA recognition by Sxl and UNR binding to msl-2 mRNA (Loedige, 2015).

Brat's most prominent function is its role in repressing tumor formation in the larval brain. Brat-mutant brain tumors originate from immature progenitor cells of type II NBs that fail to downregulate self-renewal factors and revert into over-proliferating NBs, potentially involved in tumor formation, or identified in a genome-wide RNAi screen for factors controlling NB self-renewal and differentiation are on the list of Brat targets. These include, for example, the self-renewal transcription factors dpn, klu, and kni but also many genes that are still uncharacterized. In cases where functional data are available, knockdown of most of the putative Brat targets leads to NB loss or decreased proliferation, phenotypes that would be expected for physiologically relevant Brat targets. This study provides a molecular and structural understanding of how Brat mediates the downregulation of these NB-specific factors (Loedige, 2015).

Asymmetric segregation of Brat into progenitor cells is accomplished by the adaptor protein Mira , and this study show sthat Mira not only ensures correct segregation of Brat but also inhibits Brat function by preventing Brat RNA binding. This provides an elegant mechanism to have sufficient Brat available after cytokinesis when Mira is degraded but to prevent pre-mature downregulation of NB-specific factors in the NB (Loedige, 2015).

In addition to NB-specific factors, the list of Brat-associated mRNAs contains numerous genes characteristic for postmitotic neurons, including the known Brat target para, which encodes a voltage-gated Na+ channel. Brat is expressed in mature neurons and has been reported to regulate membrane excitability, synaptic size (Shi, 2013), and axon maintenance (Marchett, 2014). Strikingly, gene ontology analysis of the Brat targets reveals a strong enrichment of categories associated with synaptic transmission, neurotransmitter regulation, secretion, and transport, and many Brat targets encode for proteins that localize to the plasma membrane and/or synaptic vesicle, including many ion channels and membrane-bound transporters. This strongly suggests that Brat regulates membrane-associated processes and might be involved in mRNA sorting and/or localization, a function that has not been described previously. In agreement with a function for brain-specific TRIM-NHL proteins in mRNA localization is the identification of the mammalian Brat orthologs TRIM2 and TRIM3 as components of mRNA transport granules, their association with kinesin and myosin motor proteins, and their enrichment in synaptic fractions, although their association with RNA still needs to be established. TRIM-NHL proteins have been implicated in the control of cell fate decisions in various tissues and across species. Therefore, conferring robustness and directionality to cell fate decisions through mRNA regulation might be a common mechanism of TRIM-NHL protein action (Loedige, 2015).



DEVELOPMENTAL BIOLOGY

The brat gene is expressed in the embryonic central and peripheral nervous systems including the embryonic brain. In third instar larva brat expression is detected in the larval central nervous system including the brain and the ventral ganglion, in two glands - the ring gland and the salivary gland, and in parts of the foregut - the gastric caecae and the proventriculus. Accumulated data suggests that Brat may regulate proliferation and differentiation by secretion/transport-mediated processes (Arama, 2000). Brat accumulates uniformly in the cytoplasm of cells in wing discs from third instar larvae (Sonoda, 2001).

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).

The expression of the brain tumor (brat) decreased 5.5 fold from 24 to 40 hrs. This gene has been studied primarily due to the neural tumor phenotype seen in loss of function mutants. The wings of bratts/Df brat flies raised at semi-permissive conditions were examined. No hair phenotype was seen but the occasional loss of sensory bristle shaft cells (principally distally along the anterior margin) was seen and occasional duplicated bristle cells (principally in the costa). These phenotypes are suggestive of a role for brat in specifying cell fate or in Notch mediated lateral inhibition (Ren, 2005).

klumpfuss distinguishes stem cells from progenitor cells during asymmetric neuroblast division

Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. This study used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. The transcription factor Klumpfuss (Klu) was identified as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led to a proposal that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division (Xiao, 2012).

Asymmetric stem cell division provides an efficient mechanism to preserve a steady stem cell pool while generating differentiated progeny within the tissue where the stem cells reside. Precise spatial control of the stem cell determinants inherited by both sibling cells in every asymmetric cell division ensures that a daughter cell maintains the stem cell characteristics while the sibling progeny acquires the progenitor cell identity. In mitotic type II neuroblasts, the basal proteins Brat and Numb segregate into immature INPs and are required for the formation of INPs. This study significantly extends the findings from previous studies and showed that Brat and Numb function in immature INPs to prevent them from acquiring a neuroblast fate while promoting the INP identity. Identification and characterization of the klu gene led to a proposal that Brat and Numb converge to exert precise control of Klu to distinguish an immature INP from its sibling type II neuroblast. Numb also prevents a GMC from reverting to a type I neuroblast by inhibiting Notch signaling in the type I neuroblast lineage. Interestingly, although overexpression of klu was insufficient to induce supernumerary type I neuroblasts, increased function of klu can drastically enhance the reversion of GMCs to type I neuroblasts in the presence of activated Notch signaling. Thus, it is proposed that aberrant activation of Notch signaling induces reversion of GMCs by activating multiple downstream genes including klu. Together, these data lead to the conclusion that precise regulation of klu by multiple signaling mechanisms distinguishes a progenitor cell from its sibling stem cell during asymmetric stem cell division (Xiao, 2012).

The essential role of Brat and Numb in regulating the formation of INPs is well established, but lack of insight into maturation has hindered investigation into the mechanisms by which these two proteins distinguish an immature INP from its sibling type II neuroblast. A previous study defined immature INPs by the following criteria: (1) being immediately adjacent to the parental type II neuroblast, (2) lacking Dpn expression and (3) displaying a very low level of CycE expression. Based on these criteria, analyses of the spatial expression pattern of various cell fate markers in the type II neuroblast lineage clones in wild-type brains revealed that onset of Ase expression correlates with an intermediate stage of maturation. In 16-hour clones, one type II neuroblast (Dpn+ Ase- CycE+), two to three Ase- immature INPs (Dpn- Ase- CycE-), two to three Ase+ immature INPs (Dpn- Ase+ CycE-) and INPs (Dpn+ Ase+ CycE+) were reproducibly observed. Furthermore, it was shown that Ase- immature INPs maintain expression of the type II neuroblast-specific marker PntP1, whereas Ase+ immature INPs showed virtually undetectable PntP1 expression. Thus, onset of Ase expression should serve as a useful marker for an intermediate stage during maturation (Xiao, 2012).

The data lead to a proposal that Brat distinguishes an immature INP from its sibling type II neuroblast by indirectly antagonizing the function of Klu based on the following evidence. First, Klu was undetectable in Ase immature INPs in the brat single-mutant or brat and numb double-mutant type II neuroblast clones. Thus, a Brat-independent mechanism must exist to downregulate Klu in immature INPs. Second, overexpression of a truncated Brat transgenic protein lacking the NHL domain, which is required for repression of mRNA translation, completely suppresses the formation of supernumerary neuroblasts. Thus, it is unlikely that downregulation of Klu in immature INPs occurs via a Brat-dependent translational repression of klu mRNA. It is proposed that Brat might suppress the expression of a co-factor necessary for the function of Klu, just as WT1 requires co-factors in order to regulate the expression of its target genes in vertebrates (Roberts, 2005). Further investigation will be necessary to discern how Brat establishes restricted developmental potential in immature INPs by antagonizing the function of Klu (Xiao, 2012).

WT1 requires its zinc-finger motifs to regulate transcription of its target genes and can function as an activator or a repressor of transcription in a context-dependent manner (Roberts, 2005). A previous study showed that overexpression of Klu can partially suppress the expression of a lacZ reporter transgene containing the cis-regulatory elements from the even-skipped gene, a putative direct target of Klu, in the fly embryonic central nervous system. Since Klu and WT1 display extensive homology in zinc-fingers 2-4, Klu is likely to recognize a similar DNA binding sequence as WT1. The even-skipped cis-regulatory element contains three putative WT1 binding sites, but nucleotide substitutions in these sites that were predicted to abolish Klu binding failed to render the lacZ reporter transgene unresponsive to overexpression of klu. These data led to a speculation that Klu might recognize a distinct consensus DNA binding sequence to WT1. To test this hypothesis, two UAS-WT1 transgenes were generated that encode the two most prevalent isoforms of the WT1 protein, WT1 −KTS and WT1 +KTS. Interestingly, neither WT1 transgene, when overexpressed by wor-GAL4, triggered the formation of supernumerary type II neuroblasts in larval brain. This is consistent with Klu recognizing a distinct consensus DNA binding sequence to WT1. However, it cannot be ruled out that the inability of the WT1 transgenic protein to induce supernumerary type II neuroblasts is simply due to the absence of necessary co-factors in the fly, as repression of target gene transcription by WT1 requires additional co-factors in vertebrates. More studies will be necessary to elucidate the molecular function of Klu in promoting type II neuroblast identity (Xiao, 2012).

Restricted developmental potential functionally defines progenitor cells and allows them to generate differentiated progeny through limited rounds of cell division without impinging on the homeostatic state of the stem cell pool. Despite their importance, the molecular mechanisms by which progenitor cells acquire restricted developmental potential remain experimentally inaccessible in most stem cell lineages. However, studies from various groups have paved the way for using fly larval brain neuroblast lineages as an in vivo model system for investigating how progenitor cells acquire restricted developmental potential (Xiao, 2012).

This study describes the expression pattern of additional molecular markers that allow unambiguous identification of two distinct populations of immature INPs. Furthermore, experimental evidence is provided strongly suggesting that these two groups of immature INPs possess distinct functional properties. More specifically, Ase- immature INPs readily revert to type II neuroblasts in response to misexpression of Klu, whereas Ase+ immature INPs appear much less responsive to Klu. These data lead to a proposal that the genome in immature INPs becomes reprogrammed during maturation such that these cells become progressively less responsive to neuroblast fate determinants such as Klu. As a consequence, an INP becomes completely unresponsive to Klu following maturation. Further experiments will be required to validate this model in the future (Xiao, 2012).


EFFECTS OF MUTATION

Inactivation of both alleles of the brat gene results in the production of a tumor-like neoplasm in the larval brain, and lethality in the larval third instar and pupal stages. Sequence analysis of four brat alleles reveal that all of them are mutated at the beta-propeller domain. The clustering of mutations in this domain strongly suggests that it has a crucial role in the normal function of Brat, and defines a novel protein motif involved in tumor suppression activity (Arama, 2000).

Many NHL domain proteins also share three other motifs: a Ring-finger, one or two B-box motifs, and a coiled-coil (RBCC) (Slack, 1998). All of the evidence of work with Brat points to the central role of the NHL domain in mediating Brat activity. Analysis of lin-41 alleles also suggests that the NHL domain plays an important role in Lin-41 function (Slack, 2000). However, another report shows that expression of the RBCC domain of human BERP (brain expressed Ring-finger protein) in PC12 cells blocks a response to nerve growth factor (El-Husseini, 1999), suggesting an essential role for this region of BERP and, by extension, other family members (Sonoda, 2001).

To test the role of these other motifs in HB mRNA regulation, transgenic flies that express wild-type Brat, the amino-terminal BCC domain (Brat lacking a Ring-finger), and the carboxy-terminal NHL domain were prepared. As controls, similar transgenic flies that express full-length Bratfs1 (NHLfs1 proteins have a single amino acid substitution at a conserved residue within the NHL domain) and NHLfs1 derivatives were also prepared. Expression of each transgene is controlled by the Gal4-binding site's upstream activating sequence (UAS); using appropriate genetic crosses, each Brat derivative was expressed during oogenesis under the control of a nos;GAL4-VP16 transgene in a bratfs1 mutant background (Sonoda, 2001).

Expression of full-length Brat+ but not Bratfs1 rescues the abdominal defects of bratfs1 embryos. For reasons that are not understood, overexpression of either protein severely disrupts oogenesis and females produce very few eggs. Although the basis of this phenotype is not understood, the segmentation pattern was analyzed among larvae derived from rare fertilized eggs. Expression of the amino-terminal BCC domain has no effect in wild-type females and does not rescue the defects of bratfs1 embryos, although the protein is stable in vivo and accumulates to a higher level than the endogenous mutant Brat protein. Somewhat surprisingly, expression of the wild-type NHL domain almost completely rescues the bratfs1 embryonic phenotype. In contrast, expression of the NHLfs1 mutant domain to essentially the same level, does not. Thus, these experiments suggest the Brat NHL domain is necessary and sufficient to regulate HB mRNA translation in the early embryo (Sonoda, 2001).

The regulation of ribosome synthesis is likely to play an important role in the regulation of cell growth. The ncl-1 gene in C. elegans functions as an inhibitor of cell growth and ribosome synthesis. The Drosophila tumor suppressor brain tumor (brat) is an inhibitor of cell growth and is a functional homolog of the C. elegans gene ncl-1. The brat gene is able to rescue the large nucleolus phenotype of ncl-1 mutants. brat mutant cells are larger, have larger nucleoli, and have more ribosomal RNA than wild-type cells. Furthermore, brat overexpressing cells contain less ribosomal RNA than control cells. These results suggest that the tumorous phenotype of brat mutants may be due to excess cell growth and ribosome synthesis (Frank, 2002).

Because NCL-1 protein is most highly expressed in cells with low rates of rRNA and protein synthesis, such as cells of the early embryo and neurons in the adult, it was predicted that brat expression would be highest in cells with low levels of biosynthetic activity. The brat expression pattern was examined using RNA in situ hybridization. Using a BRAT RNA probe, high level brat expression was observed in brains from wild-type third instar larvae. This expression is quite uniform throughout the entirety of the brain hemispheres, including the optic lobe. Weaker but fairly uniform expression is also seen in virtually all cells of the imaginal discs. In the eye disc, higher brat expression levels were observed in small clusters of cells along the morphogenetic furrow. These are most likely the neuronal preclusters, the first cells in the eye disc to exit the cell cycle and differentiate into relatively metabolically inactive cells. This expression pattern is consistent with a hypothesis that brat, like ncl-1, functions as an inhibitor of cell growth (Frank, 2002).

Given that brat mutant cells are larger than wild-type cells, it was hypothesized that brat functions to inhibit cell growth, such that overexpression of brat would be expected to lead to a decrease in cell and organ size. Because ubiquitous overexpression of brat results in lethality, the Gal4-UAS system was used to overexpress a wild-type brat cDNA specifically in the developing eye using the eyeless-Gal4 line (ey-Gal4). The eyeless enhancer directs expression in actively proliferating cells of the eye disc. Expression of brat in the developing eye, using two different UAS-brat lines, results in a dramatic decrease in organ size (Frank, 2002).

The Gal4-UAS system to overexpress brat in the developing wing using the decapentaplegic-Gal4 line (dpp-Gal4). In this line, Gal4 is expressed between wing veins LIII and LIV. Overexpression of brat leads to an obvious decrease in the size of this intervein region using two different UAS-brat lines. To quantitate this growth inhibition, the wing blade area bounded by veins LIII and LIV was measured and compared with the area bound by veins LII and LIII, which serves as an internal control since it is affected only slightly. brat overexpression results in a 36% decrease in wing area relative to the control. The decrease in eye and wing size caused by brat overexpression is probably due to a combination of cell growth inhibition and cell death. To determine if there was an effect on cell size in the wing, the number of bristles was counted in a defined area. Since each cell in the wing is associated with a single bristle, the inverse of the number of bristles in a region of a defined area gives a relative estimate of cell size. Surprisingly, the UAS-brat line appears to have increased cell sizes in the wing (Frank, 2002).

Because overexpression of brat in the wing appears to cause an increase in cell size while inhibiting organ growth, the effect of brat overexpression in clones of cells was examined, thus allowing a comparison of overexpressing and control cells directly in the same tissue. The flip-out technique was used to overexpress brat and GFP in clones of cells. Wing discs were dissociated from staged larvae in which overexpression was induced and analyzed by flow cytometry. Overexpression of brat results in a slight increase in cell size with no effect on cell cycle phasing. Microscopic examination of clones reveals that overexpression of brat leads to cell death as evidenced by pycnotic nuclei visualized by DAPI staining of clones. To overcome this effect, the cell death inhibitor P35 was co-expressed with brat in the clones; an even larger increase in cell size was observed. P35 expression appears to be somewhat deleterious to cells, as on its own it causes a small but reproducible decrease in cell size. The increased cell size in the presence of P35 is probably due to the fact that P35 expression inhibits the cell death caused by brat overexpression, thus allowing a greater proportion of the brat overexpressing cells to be analyzed (Frank, 2002).

Although brat overexpression results in enlarged cells, analysis of clone areas shows that brat overexpression actually inhibits total clone growth. The areas of wing imaginal disc clones expressing brat, GFP and P35 was compared with control clones expressing only GFP and P35; brat overexpression leads to a significant decrease in clone area (Frank, 2002).

Because brat overexpression inhibits clone growth yet results in enlarged cells, it was hypothesized that brat might be causing a slowing of cell division. To address this possibility, clones were induced to express brat, P35 and GFP at 72 hours AED, and the number of cells per clone was counted 43 hours later. Clones expressing brat have significantly fewer cells than control clones expressing only P35 and GFP. These cells have 50% longer doubling times than control cells. Thus, overexpression of brat results in a slowing of cell division. Since cell size is controlled by the rates of both cell growth and cell division, the fact that brat overexpressing cells are larger than control cells is interpreted to mean that the inhibition of cell division rate is more severe than the inhibition of cell growth (Frank, 2002).

How might brat and ncl-1 affect ribosome synthesis? It has been proposed that in E. coli and in Drosophila rRNA synthesis is regulated by the polysome to free ribosomal subunit ratio. When this ratio is high, rRNA synthesis is upregulated. Conversely, when translation, and therefore this ratio, are low, rRNA synthesis is inhibited. Since NCL-1 and BRAT are both cytoplasmic proteins, one possibility is that brat and ncl-1 serve as sensors of this ratio. Alternatively, they could directly affect this ratio by serving as translational repressors. Future work should provide insight into the specific mechanism of brat and ncl-1 action (Frank, 2002).

Asymmetric segregation of the tumor suppressor Brat regulates self-renewal in Drosophila neural stem cells

How stem cells generate both differentiating and self-renewing daughter cells is unclear. This study shows that Drosophila larval neuroblasts - stem cell-like precursors of the adult brain - regulate proliferation by segregating the growth inhibitor Brat and the transcription factor Prospero into only one daughter cell. Like Prospero, Brat binds and cosegregates with the adaptor protein Miranda. In larval neuroblasts, both Brat and Prospero are required to inhibit self-renewal in one of the two daughter cells. While Prospero regulates cell-cycle gene transcription, Brat acts as a posttranscriptional inhibitor of dMyc. In brat or prospero mutants, both daughter cells grow and behave like neuroblasts leading to the formation of larval brain tumors. Similar defects are seen in lethal giant larvae (lgl) mutants where Brat and Prospero are not asymmetric. This study has identified a molecular mechanism that may control self-renewal and prevent tumor formation in other stem cells as well (Betschinger, 2006).

These data reveal a molecular mechanism that controls self-renewal in Drosophila larval neuroblasts. The growth regulator Brat segregates asymmetrically during neuroblast division and inhibits self-renewal in one of the two daughter cells. Together with the asymmetrically segregating transcription factor Prospero, Brat ensures that this daughter cell will stop growing, exit the cell cycle, and differentiate into neurons. In brat or prospero mutants, or in lgl mutants, where Brat and Prospero are not asymmetrically segregated, both daughter cells proliferate leading to the formation of a brain tumor and death of the animal. These tumors are neoplastic and can be transplanted into the abdomen of wild-type flies where they overgrow, invade other tissues, and eventually kill the host (Betschinger, 2006).

Asymmetric cell division has been studied in the Drosophila central and peripheral nervous systems. In the peripheral nervous system, the determinants Numb and Neuralized segregate into one of the two daughter cells, and in their absence, this cell is transformed into its sister cell. In the embryonic central nervous system, Prospero acts as a segregating determinant, but in prospero mutants, many GMCs are still correctly specified. The data suggest that this is because Prospero acts partially redundant with Brat. In embryos double mutant for prospero and brat, most GMCs expressing the marker Eve are missing and neuronal differentiation in the embryonic CNS is greatly impaired. These observations suggest that Brat and Prospero act together to specify GMC fate in Drosophila embryos (Betschinger, 2006).

Although cell-cycle markers are expressed longer and stronger in prospero and brat, prospero mutant embryos, uncontrolled overproliferation has not been described in Drosophila embryos so far. In larvae, however, both brat and prospero mutant neuroblasts can initiate tumor formation. It is proposed that this difference is due to distinct mechanisms of cell growth during the two stages. During embryogenesis, cell number increases dramatically but the total volume of the embryo remains constant. Embryonic neuroblasts therefore shrink with each division and they might exit the cell cycle simply because they become too small. Support for this model comes from mutations affecting cell size asymmetry during neuroblast divisions, like Gβ13F (Fuse, 2003) or Ric-8 (Hampoelz, 2005): in these mutants, GMCs are larger, neuroblasts shrink faster and, as a consequence, divide less often. In larval neuroblasts, the situation is quite different. Several results indicate that larval neuroblasts grow significantly while cell growth is inhibited in GMCs. First, the total volume of clones generated from individual neuroblasts is several times more than the initial volume of the neuroblast. Second, the size of 'old' and 'young' (earlier and more recently generated) GMCs is approximately the same, indicating that GMCs do not grow significantly during clone formation. Third, larval neuroblasts do not become progressively smaller during development indicating that the loss of cytoplasm from each division must be compensated for by growth. Taken together, these results suggest that larval neuroblasts might be more appropriate as a model for the control of self-renewal in stem cells (Betschinger, 2006).

These experiments show that the restriction of cell growth in the GMC requires the genes lgl, brat, and prospero. While lgl seems to be required indirectly due to its role in asymmetric protein segregation, Prospero and Brat act in the GMC to regulate several important events: They repress neuroblast fate, inhibit cell-cycle progression, and prevent cell growth. Prospero is a homeodomain transcription factor, and the cell-cycle genes Cyclin A, Cyclin E, and Dacapo (the fly homolog of the CDK inhibitor p21) were shown to be among its transcriptional targets. Similar to Drosophila Prospero, its vertebrate homolog Prox-1 has been shown to regulate cell-cycle genes, and loss of prox-1 leads to increased proliferation of retinal progenitor cells (Betschinger, 2006).

For Brat, two different functions have been described: First, it acts as a translational regulator of the gap-gene hunchback. Hunchback is expressed in the embryonic nervous system but is not present in wild-type or brat mutant larval neuroblasts and is unlikely to be relevant for the growth regulatory activity of Brat. More likely, Brat acts through its well-described inhibitory activity on ribosomal RNA synthesis. Cells mutant for brat or its C. elegans homolog ncl-1 have larger nucleoli, more ribosomal RNA, and higher rates of protein synthesis, and these activities have been made responsible for the cell size increase that is observed in C. elegans and Drosophila brat mutant cells. These data suggest that this second function of Brat is also linked to posttranscriptional gene regulation. It is proposed that Brat downregulates dMyc in one of the two daughter cells and thereby inhibits protein synthesis and cell growth. Whether Brat controls dMyc translation, protein stability, or RNA stability is unclear. Interestingly, the C. elegans Brat homolog ncl-1 has been identified as one of the genes required for RNAi (Kim, 2005). Since the microRNA pathway was shown to be involved in regulation of Drosophila stem cell proliferation (Hatfield, 2005), differential regulation of this pathway in neuroblasts and GMCs by Brat could provide another explanation for its mutant phenotype (Betschinger, 2006).

Brat is part of a protein family that is characterized by a C-terminal NHL domain, several zinc-finger like B boxes, and a coiled-coil region. While the vertebrate members of this family (TRIM-2, TRIM-3, and TRIM-32) are not well characterized, the mutant phenotype of the two other Drosophila members (Dappled and Mei-P26) suggests a common function as tumor suppressors. Mutations in dappled cause melanomic tumors of the fat body, and mei-P26 mutations lead to ovarian tumors. While dappled tumors have not been well characterized, the mei-P26 phenotype has been attributed to overproliferation of undifferentiated germ cells. It is similar to-and genetically interacts with-bag of marbles, a well-characterized repressor of proliferation in the daughter cells of germline stem cells. Thus, it is conceivable that proliferation control in stem cells is a common activity of NHL domain proteins (Betschinger, 2006).

Recent evidence suggests that some human brain tumors contain stem cell-like neural progenitors that are responsible for tumor formation. Together with the identification of stem cell-like subpopulations in leukaemia, multiple myeloma, and breast cancer, this has led to the so-called cancer stem cell hypothesis which proposes that only a small population of cells in a tumor have the ability to proliferate and self-renew. This discovery suggests mechanisms for tumorigenesis other than the simple loss of proliferation control, in particular dedifferentiation of cells into additional stem cells and symmetric division of stem cells. Animal models for tumor stem cells are essential for developing new therapeutic approaches that target these mechanisms. Although Drosophila can only mimic some aspects of tumorigenesis, it might contribute to the identification of the molecular pathways operating in tumor stem cells. Human Lgl has already been implicated in tumor progression, and the characterization of Brat homologs will verify the relevance of Drosophila as a tumor stem cell model (Betschinger, 2006).

The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila

Brain development in Drosophila is characterized by two neurogenic periods, one during embryogenesis and a second during larval life. Although much is known about embryonic neurogenesis, little is known about the genetic control of postembryonic brain development. This study used mosaic analysis with a repressible cell marker (MARCM) to study the role of the brain tumor (brat) gene in neural proliferation control and tumour suppression in postembryonic brain development of Drosophila. The findings indicate that overproliferation in brat mutants is due to loss of proliferation control in the larval central brain and not in the optic lobe. Clonal analysis indicates that the brat mutation affects cell proliferation in a cell-autonomous manner and cell cycle marker expression shows that cells of brat mutant clones show uncontrolled proliferation, which persists into adulthood. Analysis of the expression of molecular markers, which characterize cell types in wild-type neural lineages, indicates that brat mutant clones comprise an excessive number of cells, which have molecular features of undifferentiated progenitor cells that lack nuclear Prospero (Pros). pros mutant clones phenocopy brat mutant clones in the larval central brain, and targeted expression of wild-type pros in brat mutant clones promotes cell cycle exit and differentiation of brat mutant cells, thereby abrogating brain tumour formation. Taken together, these results provide evidence that the tumour suppressor brat negatively regulates cell proliferation during larval central brain development of Drosophila, and suggest that Prospero acts as a key downstream effector of brat in cell fate specification and proliferation control (Bello, 2006).

Previous studies suggested that brat loss-of-function mutants lead to massive cellular overgrowth and tumour formation in larval optic lobes of Drosophila. These studies also indicated an embryonic requirement for brat to suppress tumour formation. By contrast, the current analysis showed that the brat overproliferation phenotype is due to loss of proliferation control in the larval central brain; the optic lobes initially appear wild-type-like but subsequently are overgrown by neoplastic central brain brat mutant tissue. This conclusion is further supported by MARCM clonal analysis which demonstrated that loss of brat function causes overproliferation in the larval central brain only (Bello, 2006).

In vivo mosaic analysis reveals a cell-autonomous, larval requirement for brat to limit cell proliferation in the brain. Although brat is expressed in all parts of the nervous system both in the embryo, induction of brat mutant clones in the first larval instar is sufficient to cause massive overproliferation in the central brain but not the ventral ganglia. This may suggest that either unknown compensatory mechanisms actively suppress a brat mutant phenotype in the larval ventral ganglia, or that this reflects region-specific differences in cell cycle control. Indeed, transcriptional activity of the mitotic regulator string/Cdc25 is regulated by a plethora of cis-acting elements, most of which are devoted to differential control of cell proliferation during embryonic and larval neurogenesis (Bello, 2006).

During postembryonic neurogenesis, intense proliferation takes place in the brain. This analysis shows that central brain brat mutant clones display sustained cell cycle marker expression, indicating that mutant cells are unable to withdraw from the cell cycle. This is further supported by the presence of enormous brat mutant clones with pronounced proliferative activity even in 3-week-old adult brains, an observation that contrasts with the postmitotic adult wild-type brain. Previous studies have shown that cessation of proliferation in the developing Drosophila brain occurs during metamorphosis, although the underlying genetic mechanisms are currently unknown. The elevated and aberrant cell cycle activity of central brain brat mutant cells suggests that these cells are either able to escape or that they lack cell cycle termination signals (Bello, 2006).

Mosaic analysis demonstrates that enlarged brat mutant clones comprise cells that display sustained expression of neural progenitor cell markers, and simultaneously lack marker gene expression specific for differentiating ganglion cells. Indeed, lack of axonal processes suggests that brat mutant clones comprise an excessive number of mutant cells that are unable to exit the cell cycle and hence do not differentiate into ganglion cells but rather continue to proliferate. These data indicate that brat mutation impairs proliferation control of neural progenitor cells, namely either neuroblasts and GMCs or only one of these progenitors, since in the wild-type central brain only these two cell types are actively engaged in the cell cycle. Based on this analysis it is not possible to distinguish unambiguously between the two possibilities and the underlying mechanisms. The possibility that differentiating ganglion cells de-differentiate due to brat mutation was excluded, because lack of differentiation was consistently observed right after clone induction and also at any later stages of mutant clone development. This was especially exemplified by the lack of nuclear Pros expression, which in the wild type is unambiguously detectable in differentiating progeny of larval neuroblast lineages, namely GMCs as well as ganglion cells (Bello, 2006).

Moreover, loss-of-function analysis indicates that brat mutant MARCM clones lack Pros and also phenocopy pros mutant clones. Thus, enlarged pros mutant clones consist of cells that are devoid of Elav expression, that lack axonal processes but display sustained expression of Grh and Mira as well as cell cycle markers such as CycE, CycB and PH3. These data suggest that mutant clones are essentially devoid of terminally differentiating postmitotic ganglion cells, indicating that Pros functions like Brat in terminating neural progenitor cell proliferation and inducing ganglion cell differentiation. In the embryonic CNS, Pros functions to terminate cell proliferation by repression of cell-cycle activators and simultaneously to induce a differentiation program, effectively coupling the two events. This Pros function appears to be warranted by its localization in the basal cortex of asymmetrically dividing neuroblasts and hence its distribution to only one daughter cell, the GMC. Upon completion of mitosis, Pros translocates from cytoplasm into the nucleus where it executes its transcriptional program ensuring both terminal division of the GMC and cell differentiation of its progeny. In the larval CNS nuclear localisation of Pros is observed in GMCs and ganglion cells but not in the neuroblast, suggesting that Pros has comparable functional features in larval central brain neurogenesis (Bello, 2006).

In addition, the results provide evidence that Pros acts downstream of Brat in neural proliferation control. The following points support this notion: (1) brat mutant clones lack nuclear Pros; (2) brat and pros mutant clones are indistinguishable both at the morphological and at the molecular level; (3) Brat expression is unaltered in pros mutant clones, which together with point no. 1 strongly suggests that Brat is epistatic over Pros; and (4) trans-activation of wild-type pros in brat mutant clones is sufficient to promote both cell cycle exit and differentiation. The experiments, however, do not provide any evidence about the direct or indirect nature of their interaction. Since overexpressed Pros is detected specifically in brat mutant clones in a wild-type-like pattern, the possibility that brat acts as a translational repressor of Pros, comparable to its role in hunchback repression during embryonic abdominal segmentation, is excluded. In addition, brat mutation apparently does not affect pros transcription, since pros RNA in situ hybridization in zygotic brat mutants produced a pattern indistinguishable from wild-type controls. Thus, Brat and Pros may act indirectly in the same pathway, regulating progenitor cell proliferation control in the brain. Alternatively, Brat may act in a process required to cargo Pros, comparable to the function of its mammalian homolog BERP (Bello, 2006).

In vivo mosaic analysis demonstrates that a single mutation in either brat or pros is sufficient to cause brain tumour formation in a cell-autonomous manner, suggesting that indefinite proliferation of brat and pros mutant cells is a cell intrinsic property. GFP-labelled MARCM cells each derive from a common precursor cell, implying that brat and pros mutant cells all descend from individual tumour cells of origin and hence lead to brain tumour formation in a clonally related manner. Moreover, the data indicate that pros and brat mutant clones in the larval central brain are composed of an excessive number of mutant progenitor cells that are unable to differentiate into ganglion cells but rather continue to proliferate. In this sense the results provide in vivo support for the notion that the initiating event in the formation of a malignant tumour is an error in the process of normal differentiation (Bello, 2006).

In addition, the unlimited capacity to generate undifferentiated, proliferating progeny suggests that cells mutant for brat or pros retain self-renewing capacities. In human, brain cancers are thought to arise either from normal stem cells or from progenitor cells in which self-renewal pathways have become activated, however the underlying mechanisms are elusive. The results in Drosophila may therefore provide a rationale and genetic model for the origin of brain cancer stem cells. Although parallels to human tumour formation are speculative, it is noteworthy that TRIM3, a human homolog of brat is located on chromosome 11p15, a region frequently deleted in brain tumours. Moreover, functional studies have shown that the pros homologue Prox1 regulates proliferation and differentiation of neural progenitor cells in the mammalian retina. These data may indicate that brat and pros function in cell differentiation and tumour suppression in an evolutionarily conserved manner (Bello, 2006).

Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons

Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. The translational repressor Pumilio (Pum) is a regulator of sodium current [I(Na)] and excitability in Drosophila motoneurons. This study shows that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel Paralytic (Para). A putative binding site for Pum was identified in the 3' end of the para open reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I(Na) in motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. Pum is able to reduce the level of nanos mRNA and as such a potential negative-feedback mechanism has been identified that protects neurons from overactivity of Pum. Finally, coupling was shown between I(Na) (para) and I(K) (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3'-untranslated region of target transcripts (Muraro, 2008).

Identification of the molecular components that underlie homeostasis of membrane excitability in neurons remains a key challenge. This study shows that the translational repressor Pum binds para mRNA, which encodes the Drosophila voltage-gated Na+ channel. This observation provides a mechanistic understanding for the previously documented ability of Pum to regulate INa and membrane excitability in Drosophila motoneurons (Mee, 2004). Thus, alteration in activity of Pum, in response to changing exposure to synaptic excitation, enables neurons to continually reset membrane excitability through the translational control of a voltage-gated Na+ channel (Muraro, 2008).

Previous studies report several mRNAs subject to direct Pum regulation including hb, bicoid (bcd), CycB, eIF4E, and possibly the transcript destabilization factor smaug (smg). The majority of these identified transcripts concentrate the roles of Pum to the establishment of the embryonic anterior-posterior axis (hb and bcd) and germ-line function/oogenesis (CycB). However, in the last few years, new findings have expanded the role of Pum to encompass predicted roles in memory formation, neuron dendrite morphology, and glutamate receptor expression in muscle. Indeed, the role of Pum is likely to be very much more widespread given that Pum pull-down assays followed by microarray analysis of bound mRNAs have now identified a plethora of possible additional targets of translational regulation (Gerber, 2006). The ~1000 or so genes identified are implicated to be involved in various cellular functions, suggesting that Pum-dependent translational repression might be a mechanism used in different stages of development and in diverse tissue function. To date, para is the first confirmed Pum target encoding a voltage-gated ion channel (Muraro, 2008).

Pum-binding motifs have been identified in the 3'-UTRs of many mRNAs known to bind to this protein. Analysis of 113 such genes expressed in adult Drosophila ovaries has identified a consensus 8 nt binding motif [UGUAHAUA]. This sequence contains the UGUA tetranucleotide that is a defining characteristic of the NRE-like motif described in the 3'-UTR of hb mRNA. Such an 8 nt motif has been identified within the ORF of para at the 3' end of the transcript. The biochemical binding data support the notion that this motif is indeed sufficient to bind Pum and as such represents the first such site to be localized to an ORF of any transcript. However, to translationally repress para mRNA, the data also show a requirement for regions of Pum in addition to the RBD. Interestingly, this kind of requirement has also been shown for another Pum target, eIF4E. The translational silencing of mRNAs is a complex mechanism on which only little information is available. It could involve deadenylation and degradation of the mRNA and/or the circularization of the mRNA and the recruitment of factors that would preclude translation. The fact that different Pum targets may require only the RBD (hb) or the full-length protein (eIF4E and para) suggests that Pum-mediated translational repression may follow complex target mRNA-specific mechanisms, most probably involving the interaction of other domains of Pum with additional, so far unknown, factors. In this regard, it is interesting to note that the N terminus of Pum has regions of low complexity including prion-like domains rich in Q/R. These domains may provide a platform for other proteins that influence the fate of Pum targets (Muraro, 2008).

The putative Pum binding motif lies within an exon that is common to all para splice variants identified (at least in the embryo) but is possibly subject to editing by adenosine deamination. Thus, in an analysis of splicing of para, a number of individual cDNA clones were sequenced and one splice variant was recovered that shows A-to-I editing in this motif. Together with a differential requirement for specific cofactors, editing of this motif might serve to influence how para is affected by Pum and, as such, further increase diversity in level of expression of INa in differing neurons or disease states (Muraro, 2008).

The known mechanism of action of Pum-dependent translational repression is absolutely dependent on additional cofactors. The most studied example, that of hb mRNA during early embryogenesis, requires the presence of both Nanos and Brat. However, the requirement for these two cofactors is seemingly transcript dependent. Thus, Pum-mediated repression of CycB mRNA requires Nanos but not Brat. However, Pum-dependent repression of bcd is apparently Nanos independent, because levels of Nanos in the anterior of the early embryo are undetectable. Although it was clearly shown that Pum-dependent repression of para mRNA in the Drosophila CNS requires Nanos, the requirement for Brat is less clear and seems to be neuronal cell type specific. A requirement for a different combination of cofactors for Pum-dependent translational regulation of a single gene transcript has not been reported previously, but clearly might represent an additional level of regulation. Such differential regulation might be required to spatially restrict the effect of Pum to certain cell types within the CNS. Voltage-gated Na+ currents are responsible for the initiation and propagation of the action potential and determine, together with other voltage-gated ion conductances, the membrane excitability of a neuron. Despite para being the sole voltage-gated sodium channel gene in Drosophila [compared with at least nine different genes in mammals, neuronal subpopulations nevertheless exhibit distinctive INa characteristics. To achieve this, para is known to undergo extensive alternative splicing and, additionally, RNA editing. It is highly likely that both alternative splicing and RNA editing generate mRNAs that encode channels with differing electrophysiological properties. It is also conceivable that these mechanisms might yield para transcripts that contain differing arrangements of Pum/Nanos binding sites, which may, or may not, recruit Brat. Indeed, it has been proposed that variations of the NRE consensus sequence may result in Pum-NRE-Nanos complexes with different topographies, resulting in altered recruitment abilities for additional cofactors such as Brat. Additional work is necessary to clarify where, in para mRNA, the binding sites for the Pum/Nanos complex are localized and how the recruitment of Brat is facilitated in only some neurons. In the hb repression complex, Brat has been shown to interact with the cap-binding protein d4EHP. Therefore, additional cofactors might be necessary for Pum-dependent para repression in the Brat-independent neuronal cell subtypes (Muraro, 2008).

In contrast to translational repression of hb, the data show that Nanos is unlikely to be a limiting factor of Pum-dependent repression of para translation. Consistent with this finding is the observation that overexpression of pum is sufficient to downregulate (and probably translationally repress) nanos mRNA. However, the opposite is not true; overexpression of nanos does not affect levels of pum mRNA. These data suggest that Pum is at least a principal orchestrating factor (if not the prime factor) in regulation of para translation. Moreover, the demonstration that overexpression of pum is sufficient to greatly downregulate nanos mRNA (relative to para mRNA), together with a requirement of Nanos for Pum-dependent para mRNA repression, implicates the existence of a protective negative-feedback mechanism that prevents overrepression of para mRNA. In the absence of such feedback, it is conceivable that excessive overrepression of para mRNA might lead to neurons falling silent as their membrane excitability drops below a critical threshold. Were this to happen, then signaling in the affected neuronal circuit would be severely compromised (Muraro, 2008).

Overexpression of full-length Pum in aCC/RP2 motoneurons not only causes a decrease in INa but also a significant decrease in IKfast. Additionally, pan-neuronal overexpression of Pum causes a significant decrease in Shal mRNA, a gene encoding a potassium channel known to contribute to IKfast. This result was surprising given that Shal was not identified as a Pum target from microarray analysis. That this mechanism might, therefore, be indirect is corroborated by the finding that IKfast and Shal mRNA remain at wild-type levels when Pum is overexpressed in a para-null background. It is, perhaps, counterintuitive that a reduction in INa, to achieve a reduction in membrane excitability, should be accompanied by a similar decrease in outward IKfast. However, changes in ionic conductances should not be considered in isolation and such a relationship might serve to maintain action potential kinetics within physiological constraints. Covariation of INa and IK as a mechanism for changing neuronal excitability has been described in these motoneurons previously. Moreover, there is precedent for coupling between transcripts: injection of Shal mRNA into lobster PD (pyloric dilator) neurons results in an expected increase in IA but also an unexpected linearly correlated increase in Ih, an effect that acts to preserve membrane excitability. Injection of a mutated, nonfunctional, Shal mRNA is also sufficient to increase Ih indicative that this coregulation is activity independent (MacLean, 2003). It remains to be shown whether genetic manipulation of para mRNA levels in Drosophila motoneurons will similarly evoke compensatory changes in Shal expression (Muraro, 2008).

In a previous study, it was shown that blockade of synaptic release, through pan-neuronal expression of tetanus toxin light chain, is sufficient to evoke a compensatory increase in membrane excitability in aCC/RP2 that was accompanied by increases in INa, IKfast, and also IKslow (Baines, 2001). In contrast, the current study showed that overexpression of pum is sufficient to decrease INa and IKfast but does not significantly affect IKslow (although there is a small nonsignificant reduction in this current). Clearly, the complete absence of synaptic input is a more severe change that likely elicits a greater compensatory change in these neurons than when Pum is overexpressed. However, whether removal of synaptic excitation also invokes additional compensatory mechanisms that act preferentially on IKslow remains to be determined. What is consistent, however, is that change in synaptic excitation of these motoneurons is countered by Pum-dependent regulation of both para mRNA translation and magnitude of INa (Muraro, 2008).

A key question remains as to what the mechanism is that transduces changes in synaptic excitation to altered Pum activity. Perhaps the most parsimonious mechanism will be one linked to influx of extracellular Ca2+. Indeed, experimental evidence supports a role for Ca2+, because blocking its entry can preclude changes in neuronal excitability observed as a result of activity manipulation. In addition, changes of gene expression resulting from activity-mediated Ca2+ entry have been described both in vitro and in vivo after plasticity changes such as long-term potentiation. Whether Ca2+ influx influences translation and/or transcription of Pum remains to be shown. Stimulation of mammalian neurons in culture with glutamate, after a preconditioning period of forced quiescence, results in an increase of Pum2 protein levels after just 10 min. The rapidity of this response suggests that it is mediated by a posttranscriptional mechanism. This study examined the role of Pum on Ca2+ channel activity. Neither IBa(Ca) nor levels of the voltage-gated calcium channel coded by Dmca1A (cacophony, Calcium channel α1 subunit, type A) are affected in aCC/RP2 motoneurons in which pum [full length (FL)] is overexpressed. The fact that Pum does not affect Ca2+channel activity directly could reinforce the idea of its serving as a primary sensor of activity changes (Muraro, 2008).

In summary, this study has shown that Pum is able to bind to para mRNA, an effect that is sufficient to regulate both INa and membrane excitability in Drosophila motoneurons. This mechanism requires the cofactor Nanos but does not obligatorily require Brat. Given that mammals express two Pum genes, Pum1 and Pum2, it will be of importance to determine whether this protein is also able to regulate sodium channel translation in the mammalian CNS (Muraro, 2008).

Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila

Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).

The differential cell growth rates observed between ectopic NBs and normal or primary NBs and the correlation between cell growth defects and NB fate loss prompted a test of whether slowing down cell growth might selectively affect the formation of ectopic NBs. Attenuation of TOR signaling, a primary mechanism of cell growth regulation, through NB-specific overexpression of TSC1/2, a strong allele of eIF4E antagonist 4EBP [4EBP(LL)s], or a dominant-negative form of TOR (TOR.TED) all partially suppressed ectopic NB formation in α-adaptin (ada) mutants without affecting normal or primary NBs. Interestingly, RNAi-mediated knockdown of eIF4E, a stimulator of oncogenic transformation and a downstream effector of TOR signaling, showed a better suppression than manipulating other TOR pathway components, suggesting that eIF4E might play a more important role in ectopic NB formation. Strikingly, the brain tumor phenotypes caused by overactivation of N signaling - as in lethal giant larvae (lgl) mutant, aPKCCAAX overexpression, or N overexpression conditions - were also fully suppressed by eIF4E knockdown. Furthermore, the brain tumor phenotypes of brat mutants were also completely rescued by eIF4E RNAi (Song, 2011).

In contrast, normal NB formation or maintenance was not affected by eIF4E knockdown. NBs with eIF4E knockdown remained highly proliferative, as evidenced by the mitotic figures, and displayed relatively normal apical basal cell polarity. There are several other eIF4E-like genes in the fly genome (Hernandez, 2005), which may play partially redundant roles in normal NB maintenance. eIF4E knockdown appeared to specifically block ectopic NB formation caused by the dedifferentiation of IPs in type II NB lineages, since it did not affect ectopic type I NB formation in cnn or polo mutants that are presumably caused by symmetric divisions of type I NBs. In addition, cell fate transformation induced by N overactivation in the SOP lineage was not affected by eIF4E RNAi, supporting the idea that eIF4E is particularly required for type II NB homeostasis. Supporting the specificity of the observed eIF4E RNAi effect, another eIF4E RNAi transgene (eIF4E-RNAi-s) also prevented ectopic NB formation. Moreover, a strong loss-of-function mutation of eIF4E also selectively eliminated ectopic NBs induced by N overactivation without affecting normal NBs, reinforcing the hypothesis that ectopic NBs exhibit higher dependence on eIF4E (Song, 2011).

To further support the notion that the ectopic NBs are particularly vulnerable to eIF4E depletion, a conditional expression experiment was carried out in which eIF4E-RNAi-s was turned on in brat mutants using the 1407ts system, after ectopic NBs had been generated. Whereas the brain tumor phenotype exacerbated over time in the brat mutants, 1407-GAL4-driven eIF4E-RNAi-s expression in brat mutants effectively eliminated ectopic NBs, leaving normal NBs largely unaffected (Song, 2011).

In normal type II NB lineage, eIF4E protein was enriched in the NBs. Ectopic NBs induced by N overactivation in ada mutants also expressed eIF4E at high levels, whereas spdo mutant NBs exhibited reduced eIF4E expression. Thus, eIF4E up-regulation correlates with N-induced ectopic NB formation in a dedifferentiation process that likely involves elevated cell growth (Song, 2011).

Given the coincidence of nucleolar size change with ectopic NB formation, the involvement of the growth regulator dMyc was tested. dMyc protein levels were up-regulated in normal or N overactivation-induced ectopic NBs, but were down-regulated in spdo mutant NBs. Furthermore, dMyc transcription, as detected with a dMyc-lacZ transcriptional fusion reporter, was also up-regulated in both normal and ectopic NBs in ada mutants. A previous study in Drosophila S2 cells identified dMyc as a putative N target. In vivo chromatin immunoprecipitation (ChIP) experiments were carried out to assess whether dmyc transcription is directly regulated by N signaling in NBs. Using chromatin isolated from wild-type larval brains and a ChIP-quality antibody against the N coactivator Suppressor of Hairless [Su(H)], specific binding was demonstrated of Su(H) to its putative binding sites within the second intron of dmyc (dmyc-A). No binding to an internal negative control region proximal to the first exon of dmyc (dmyc-B) or to the promoter region of the rp49 gene was detected. N signaling thus directly activates dMyc transcription in the NBs. Similar to eIF4E RNAi, knockdown of dMyc strongly suppressed ectopic NB formation induced by Brat or Ada inactivation or N overactivation. Intriguingly, the strong tumor suppression effect of eIF4E knockdown was partially abolished by dMyc overexpression. Furthermore, dMyc function, as reflected by its promotion of nucleolar growth in IPs, was attenuated by eIF4E RNAi, although eIF4E RNAi alone had no obvious effect. Different from the reported eIF4E regulation of Myc expression in mammalian cells (Lin, 2008), dMyc promoter activity or protein levels remained unaltered under eIF4E RNAi conditions, suggesting that eIF4E may modulate dMyc activity without altering its expression. One possibility is that eIF4E may enter the nucleus to interact with Myc and promote its transcriptional activity. To test this hypothesis, HEK293T cells were transfected with Flag-tagged human eIF4E alone or in combination with HA-tagged dMyc. Indeed, both Drosophila dMyc and endogenous human c-Myc specifically coimmunoprecipitated with human eIF4E from nuclear extracts, indicating a conserved interaction between eIF4E and Myc within the nuclei of proliferating cells. Consistent with these biochemical data, dMyc transcriptional activity within NBs, which could be monitored with an eIF4E-lacZ reporter, was drastically reduced upon eIF4E knockdown (Song, 2011).

In contrast, eIF4E transcription, as detected with an eIF4E-lacZ transcriptional fusion reporter, as well as eIF4E protein levels detected by immunostaining were up-regulated upon dMyc overexpression and down-regulated by dMyc RNAi. It is unlikely that the changes in eIF4E-lacZ activity were due to global increases or decreases in β-galactosidase (β-gal) translation caused by altered dMyc levels, since lacZ expression from a dMyc-lacZ reporter was unaffected under similar conditions. Furthermore, like dMyc protein, eIF4E-lacZ reporter expression was up-regulated in normal NBs or ectopic NBs in ada mutants, further supporting the notion that dMyc may up-regulate eIF4E transcription. Moreover, ChIP experiments using chromatins isolated from wild-type larval brains and a ChIP-quality antibody against dMyc demonstrated specific binding of dMyc to an eIF4E promoter region harboring a cluster of adjacent noncanonical E boxes, supporting a direct regulation of eIF4E transcription by dMyc. dMyc and eIF4E thus appeared to form a regulatory feedback loop that promoted NB growth and renewal. Consistent with this model, while knocking down either dMyc or eIF4E had no noticeable effect on type II NB maintenance and only a mild effect on NB nucleolar size in the case of dMyc RNAi, their simultaneous knockdown led to a significant reduction in nucleolar size, premature neuronal differentiation, and loss of NBs (Song, 2011).

If the dMyc-eIF4E axis of cell growth control is a crucial downstream effector of N signaling in regulating NB maintenance, its up-regulation might be able to rescue the type II NB depletion phenotype resulting from reduced N signaling. Indeed, the loss of NBs associated with reduced Notch signaling was preventable when cell growth was boosted by dMyc overexpression. Thus, while N-IR directed by 1407-GAL4 led to complete elimination of type II NBs, the coexpression of dMyc, but not CD8-GFP or Rheb, an upstream component of the TOR pathway, resulted in the preservation of approximately half of type II NBs with apparently normal cell sizes, cell fate marker expression, and lineage composition. A similar effect was observed when dMyc was coexpressed with N-IR using the conditional 1407ts system, with transgene expression induced at the larval stage. While both dMyc and Rheb promote cell growth, they do so through distinct mechanisms, with the former increasing nucleolar size and the latter expanding cytoplasmic volume. These results thus provide compelling evidence that control of cell growth, particularly nucleolar growth, is a critical component in the maintenance of NB identity by N signaling (Song, 2011).

The differential responses of normal and tumor-initiating stem cells to functional reduction of eIF4E prompted a test of whether chemicals that specifically inhibit eIF4E function might have therapeutic potential in preventing CSC-induced tumorigenesis. Indeed, the brain tumor phenotypes induced by N overactivation or ada loss of function were effectively suppressed by feeding animals with fly food containing Ribavirin, an eIF4E inhibitor that interferes with eIF4E binding to mRNA 5' caps and promotes the relocalization of eIF4E from the nucleus to the cytoplasm (Kentsis, 2004; Assouline, 2009) (Song, 2011).

The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has showen that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).

This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML) (Assouline, 2009), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. The drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by this biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis (Ruggero, 2004; Wendel, 2007), but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).

The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).

While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. This study usded detailed clonal analyses of NSCs over multiple time points to provide direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. These studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation (van Riggelen, 2010) as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).

The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The current results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc. Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).

Combinatorial use of translational co-factors for cell type-specific regulation during neuronal morphogenesis in Drosophila

The translational regulators Nanos (Nos) and Pumilio (Pum) work together to regulate the morphogenesis of dendritic arborization (da) neurons of the Drosophila larval peripheral nervous system. In contrast, Nos and Pum function in opposition to one another in the neuromuscular junction to regulate the morphogenesis and the electrophysiological properties of synaptic boutons. Neither the cellular functions of Nos and Pum nor their regulatory targets in neuronal morphogenesis are known. This study shows that Nos and Pum are required to maintain the dendritic complexity of da neurons during larval growth by promoting the outgrowth of new dendritic branches and the stabilization of existing dendritic branches, in part by regulating the expression of cut and head involution defective. Through an RNA interference screen a role was uncovered for the translational co-factor Brain Tumor (Brat) in dendrite morphogenesis of da neurons, and it was demonstrated that Nos, Pum, and Brat interact genetically to regulate dendrite morphogenesis. In the neuromuscular junction, Brat function is most likely specific for Pum in the presynaptic regulation of bouton morphogenesis. Thess results reveal how the combinatorial use of co-regulators like Nos, Pum and Brat can diversify their roles in post-transcriptional regulation of gene expression for neuronal morphogenesis (Olesnicky, 2012).

Post-transcriptional mechanisms of gene regulation such as translational control play a fundamental role in the development and function of the nervous system. Genetic studies have identified roles for the translational repressors Nos and Pum in sensory neuron and NMJ morphogenesis, NMJ function, and motor neuron excitability, and Pum has been implicated in long-term memory. Understanding the selectivity of these regulators for different mRNA targets is essential to identify the cellular processes they regulate for neuronal morphogenesis and neural function. This study shows that different combinations of Nos, Pum, and the co-factor Brat confer cell type-specific regulation during morphogenesis of Drosophila da sensory neurons and the NMJ (Olesnicky, 2012).

In Drosophila class IV da neurons, dendritic arbors grow rapidly during the first larval instar to establish nonredundant territories that cover the larval body wall. During the second and third larval instars, da neuron dendrites add and lengthen higher order branches to maintain body wall coverage as the larva undergoes dramatic growth. Results from live imaging analysis place the requirement for Nos and Pum during the third larval instar, indicating that Nos and Pum are not involved in the establishment of dendritic territories but rather in maintaining the density of terminal branches during late larval growth by promoting branch extension and preventing branch retraction. The possibility cannot be ruled that branch stabilization depends on Nos and Pum activity earlier during larval development. Evidence is provided that this maintenance function of Nos and Pum depends on their regulation of the proapoptotic protein Hid. Nos has previously been proposed to repress hid mRNA translation in developing germ cells to suppress apoptosis, although requirements for Pum and Brat were not tested. Together, these data showing that Hid is elevated in nos and pum mutant da neurons and that both the upregulation of Hid and the loss of terminal branches in nos mutants are suppressed by reduction of hid gene dosage suggest that repression of hid mRNA translation by Nos and Pum is also crucial for dendrite morphogenesis. Biochemical analysis will be required to test this model directly (Olesnicky, 2012).

In cultured Drosophila cells, Hid localizes to mitochondria and this localization is required for full caspase activation. By contrast, Hid protein is detected in the nucleus in nos and pum mutants. A similar nuclear accumulation has been proposed to sequester Hid in larval malphigian tubules and prevent apoptosis of this tissue during metamorphosis (Shukla, 2011). The nuclear accumulation of Hid may indeed explain why upregulation of Hid in nos and pum da mutants does not cause cell death. Nuclear Hid sequestration in nos and pum mutant neurons is also consistent with the apparent absence of activated caspase. How Hid causes dendrite loss in nos and pum mutant neurons remains to be determined but could involve activation of a pathway similar to injury induced dendrite degeneration, which resembles pruning but is caspase-independent (Olesnicky, 2012).

Nos and Pum were initially identified because of their role in translational repression of hb mRNA in the posterior region of the early embryo. There, the two proteins form an obligate repression complex, with Pum conferring the RNA-binding specificity and Nos, which is synthesized only at the posterior pole of the embryo, providing the spatial specificity. More recent studies have shown that Nos and Pum are not obligate partners, however. In the ovary, Pum functions together with Nos in germline stem cells to promote their self-renewal, while Pum acts independently of Nos in progeny cystoblasts to promote their differentiation (Harris, 2011). In the NMJ, Pum and Nos work in opposition to one another to regulate both morphological and electrophysiological characteristics of synaptic boutons. While Hid levels are similarly elevated in nos and pum mutant da neurons, the differential effects on cut expression observed in the two mutants suggest that in addition to working together, Nos and Pum participate in separate complexes that target different mRNAs even within the same cell type. Presumably, additional factors that associate selectively with Nos or Pum drive the formation of distinct complexes with different binding specificities. Pum represses eIF4E translation in the post-synaptic NMJ independently of Nos, suggesting that some of Pum's effects in da neurons could be through more global effects on translation (Olesnicky, 2012).

A third cofactor, Brat, is required for Nos/Pum-dependent repression of hb mRNA in the early embryo and paralytic mRNA in motorneurons. However, Brat is not required for Nos/Pum-mediated repression of cyclin B mRNA in primordial germ cells or for Nos/Pum function in germline stem-cell maintenance. Structural and molecular analyses have shown that Brat is recruited to the Nos/Pum/NRE ternary complexes through an interaction between its conserved NHL (NCL-1, HT2A, and LIN-41) domain and Pum. The Brat NHL domain also mediates interaction of Brat with the eIF4E-binding protein d4EHP and mutations in Brat that abrogate this interaction partially disrupt translational repression of hb, suggesting a mechanism by which the Pum/Nos/Brat/NRE complex could repress cap-dependent initiation. The results indicate that Brat also collaborates with Nos and Pum to regulate dendrite morphogenesis by a mechanism involving d4EHP interaction and that this requirement is cell type-specific. While genetic analysis suggests that Brat is required for Nos/Pum-mediated regulation of dendrite complexity and Hid expression in class IV da neurons, it is dispensible for Nos and Pum functions in class III da neurons. A similar cell type-specific requirement for Brat function in Nos/Pum-mediated repression within the CNS has been proposed based on the ability of brat mutants to counteract repression of paralytic mRNA due to Pum overexpression. Since Brat is expressed throughout the dorsal cluster of larval sensory neurons and CNS, it is unclear whether the recruitment of Brat to the complex occurs only in certain cell types or whether its function in the complex is target dependent. In contrast to nos and pum mutants, however, brat mutants have no effect on cut expression, suggesting that Brat's role in translational regulation is in fact limited to a subset of Nos/Pum-dependent processes (Olesnicky, 2012).

The findings that Brat functions presynaptically in bouton formation and that brat and pum mutant NMJs exhibit similar defects in bouton formation suggest that Brat is selectively recruited by Pum, but not by Nos, to regulate distinct target mRNAs in bouton development. Similarly, Brat functions selectively with Pum in ovarian cystoblasts to promote differentiation, suggesting that a Pum/Nos/NRE ternary complex is not essential for recruitment of Brat. Pum and many of its homologs in other organisms, members of the large Puf (Pum/FBF) protein family, typically recognize sequences that contain a core UGUA motif, although features beyond the core element also influence target mRNA recognition. Pum has been shown to also recognize a UGUG motif that is found in binding sites for the C. elegans Puf protein FBF (Menon, 2009). Thus, it is possible that the interaction of Pum with different binding sites dictates the assembly of the particular repression complex. Interactors like Brat might add an additional layer of regulation by altering the specificity or affinity of Pum for particular targets, thereby generating diverse cellular and morphological outputs within a particular cell type (Olesnicky, 2012).

Ecdysone and mediator change energy metabolism to terminate proliferation in Drosophila neural stem cells

Stem cells are highly abundant during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. This study shows that changes in energy metabolism induced by the steroid hormone ecdysone and the Mediator (see Med19) initiate an irreversible cascade of events leading to cell-cycle exit in Drosophila neural stem cells. The timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple the cell cycle from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Brain tumor mutant neuroblasts fail to undergo this shrinkage process and continue to proliferate until adulthood. These findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation in stem cells (Homem, 2014).

Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila

Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. The mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. This study reports the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. Seventeen putative modifiers involved in various function or process were isolated: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. The brat gene belongs to a group of suppressors, which is implicated in cell growth, proliferation or death. Other identified genes are involved in carbohydrate metabolism, such as Gpo-1. This result is in agreement with the evidence that Bcl-2 family proteins, in addition to their well characterized function in cell death, also play roles in metabolic processes in particular at the level of energetic metabolism. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation (Colin, 2015).

Major executioners of programmed cell death by apoptosis are relatively well conserved throughout evolution. However, the control of commitment to apoptosis exhibits some differences between organisms. During mammalian cells apoptosis, various key pro-apoptotic factors are released from the inter-membrane space of mitochondria. These factors include cytochrome c, Apoptosis Inducing Factor (AIF), Endonuclease G, Smac/DIABLO (Second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease Omi/HtrA2. Once released in the cytosol, cytochrome c binds to the WD40 domain of Apaf-1 and leads to the formation of a cytochrome c/Apaf-1/caspase-9 complex called 'apoptosome', in which caspase-9 (a cysteinyl aspartase) auto-activates to initiate a caspase activation cascade that will lead to cell death. Mitochondrial permeabilization is under the control of the Bcl-2 family of proteins. These proteins share one to four homology domains with Bcl-2 (named BH1-4) and exhibit very similar tertiary structures. However, while some of these proteins (such as Bcl-2) are anti-apoptotic, the others are pro-apoptotic and assigned to one of the following sub-classes: BH3-only proteins (such as Bid) and multi-domain proteins (such as Bax). During apoptosis, Bax translocates to the mitochondrial outer membrane, undergoes conformational changes, oligomerizes and finally allows the release of pro-apoptotic factors from the intermembrane space. Anti-apoptotic proteins of the Bcl-2 family oppose this Bax-mediated mitochondrial release of apoptogenic factors while BH3-only proteins can activate Bax or inhibit anti-apoptotic proteins of the family (Colin, 2015 and references therein).

In C. elegans, activation of the caspase CED-3 requires CED-4, the homologue of Apaf-1 but no cytochrome c. The Bcl-2 family protein CED-9 constitutively interacts with CED-4 and thereby prevents the activation CED-3. This repression of cell death is released upon binding of CED-9 to the BH3-only protein EGL-1, which induces a conformational change in CED-9 that results in the dissociation of the CED-4 dimer from CED-9. Released CED-4 dimers form tetramers, which facilitate auto-activation of CED-3. Although CED-9 appears bound to mitochondria, these organelles seem to play a minor role in apoptosis in C. elegans, contrarily to mammals (Colin, 2015 and references therein).

The role of mitochondria in Drosophila programmed cell death remains more elusive. Cytochrome c does not seem crucial in the apoptosome activation, which is mediated by the degradation of the caspase inhibitor DIAP1 by proteins of the Reaper/Hid/Grim (RHG) family. The apoptotic cascade appears somehow inverted between flies and worm/mammals. In these two last organisms, apoptosis regulators are relocated from mitochondria to the cytosol. Contrarily, Drosophila apoptosis regulators are concentrated at or around mitochondria during apoptosis. Indeed, targeting the RHG proteins Reaper (Rpr) and Grim to mitochondria seems to be required for their pro-apoptotic activity. Furthermore, Hid possesses a mitochondrial targeting sequence and is required for Rpr recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo (Colin, 2015).

The important role played in Drosophila by the mitochondria in apoptosis is also suggested by the mitochondrial subcellular localization of Buffy and Debcl, the only two members of the Bcl-2 family identified, so far, in this organism. Buffy was originally described as an anti-apoptotic Bcl-2 family member, but it can also promote cell death. Debcl (death executioner Bcl 2 homolog), is a multidomain death inducer that can be inhibited by direct physical interaction with Buffy. When overexpressed in mammalian cells, debcl induces both cytochrome c release from mitochondria and apoptosis. This protein interacts physically with anti-apoptotic members of the Bcl-2 family, such as Bcl-2 itself, in mammals. In Drosophila, Debcl is involved in the control of some developmental cell death processes as well as in irradiation-induced apoptosis (Colin, 2015).

Previous studies have shown in Drosophila that mammalian Bcl-2 inhibits developmental and irradiation-induced cell death as well as rpr- and bax-induced mitochondrial membrane potential collapse . Interestingly, bax-induced cell death has been shown to be mitigated by loss-of-function (LOF) mutations in genes encoding some components of the TOM complex which controls protein insertion in the outer mitochondrial membrane. These results suggest that Bax mitochondrial location remains important for its activity in Drosophila. Therefore, flies provide a good animal model system to study Bax-induced cell death in a simple genetic background and look for new regulators of Bcl-2 family members (Colin, 2015).

This study reports the results of the screening of P[UAS]-element insertion (UYi) library, performed in order to identify modifiers of bax-induced phenotypes in Drosophila. Among 1475 UYi lines screened, 17 putative modifiers were isolated, that include genes involved in various cellular functions. This paper presents a more detailed study of one of these modifiers, UY1039, and shows that glycerophosphate oxidase-1 (Gpo-1) [EC 1.1.5.3] participates in debcl-induced apoptosis by increasing reactive oxygen species (ROS) production (Colin, 2015).

This screen provided 17 suppressors of phenotypes induced by the expression of bax under control of the wing specific vg-GAL4 driver (lethality and wing notches). The possibility that these suppressors affect GAL4 synthesis or that the selected insertions titrate the GAL4 transcription factor is unlikely, since the number of suppressors is limited (1.6% of the collection). Moreover, UYi insertions were isolated that were not identified in other screens performed using the same collection and the UAS/Gal4 system. Finally, the specificity of one of the suppressors, UY3010, which corresponds to a gain-of-function of the Ubiquitin activating enzyme-encoding gene Uba1 has been reported. Indeed, Uba1 overexpression allows the degradation of Bax and Debcl, thanks to the activation of the ubiquitin/proteasome pathway. This study also showed that Debcl is targeted to the proteasome by the E3 ubiquitin ligase Slimb, the β-TrCP homologue (Colin, 2015).

Nine of the bax-modifiers also behaved as suppressors of debcl-induced wing phenotype while 4 showed no significant effect on this phenotype. Three hypotheses could explain this discrepancy. One possibility is that these bax modifiers are context artifacts and do not represent bona fide Bax interactors. The second possible explanation involves the difference in the driver used in each assay (vg-GAL versus ptc-GAL). Indeed, UY3010 did not significantly suppress debcl-induced apoptosis while another Uba1 overexpression mutant (Uba1EP2375) did. Third, although Bax and Debcl, share similarities in their mode of action and regulation, some signaling pathways could be specific of bax-induced apoptosis. Indeed, a LOF of brat mitigates neither debcl -- (this paper) nor hid -- or Sca3-induced cell death(Colin, 2015).

The brat gene belongs to a group of suppressors, which is implicated in cell growth, proliferation or death. Mutations in this type of genes could compensate cell loss due to ectopic apoptosis induction. Results observed for this group of modifiers can generally be easily interpreted with the literature data. UY1131 corresponds to an insertion in the brat (for brain tumor) gene that could allow the expression of a truncated form of the protein. To check whether this insertion leads to a LOF or a GOF of brat, the effect of the characterized LOF allele bratk0602 on bax-induced phenotypes was tested. This mutation strongly suppressed the wing phenotype showing that UY1131 is a LOF of brat. Brat belongs to the NHL family of proteins, represses translation of specific mRNAs and is a negative regulator of cell growth. The suppression of bax-induced phenotypes by a LOF of brat could suggest that this gene also regulates cell death, which seems unlikely according to its inability to suppress other cell death pathways. Alternatively brat could regulate somehow compensatory proliferation in this system (Colin, 2015).

Some candidate suppressors encode proteins involved in secretion or components of the extra-cellular matrix. The effect of these genes could rely on cell signaling. Change in levels of secreted proteins could modify cell-extracellular matrix interactions and thus affect viability via processes similar to anoikis (Colin, 2015).

Several suppressors are implicated in pathfinding (comm, comm3, hat, scratch and lola). Two hypotheses can be formulated. Either neurons are of particular importance in bax-induced phenotypes or a more general role of these proteins in signaling is responsible for these suppressions. If the neuronal death could explain the decreased survival of bax expressing flies, it could hardly explain the wing phenotypes. Therefore, these suppressor genes may have a more general role in signaling and in particular in cell death regulation. For example, UY2669 corresponds to a GOF mutant of scratch (scrt). This gene is a Drosophila homologue of C. elegans ces-1, which encodes a snail family zinc finger protein involved in controlling programmed death of specific neurons. Interestingly, a mammalian homologue of scratch, named Slug, is involved in a survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage. Slug also antagonizes p53-mediated apoptosis by repressing the bcl-2-family pro-apoptotic gene puma. More recently, a regulatory loop linking p53/Puma with Scratch has been described in the vertebrate nervous system, not only controlling cell death in response to damage but also during normal embryonic development (Colin, 2015).

Another possibility is that these modifiers could affect some extracellular survival and/or death factors. For example, sugarless, which was found twice in the screen, has been shown to interact with several survival pathways such as Wingless, EGF and FGF pathways that can play a role in defining shape and size of tissues and organs. This result can be paralleled with the suppressive effect of mutations in hephaestus and lola, both of which interact with the Notch/Delta signaling. Notably, lola, a gene encoding a Polycomb group epigenetic silencer, has been shown to be required for programmed cell death in the Drosophila ovary. Lola has also been identified for its role in normal phagocytosis of bacteria in Drosophila S2 cells and as a component of the Drosophila Imd pathway that is key to immunity. In contrast, Lola is required for axon growth and guidance in the Drosophila embryo. This indicates that lola could play a role in cell adhesion and motility. Accordingly, when coupled with overexpression of Delta, misregulation of pipsqueak and lola induces the formation of metastatic tumors associated with a downregulation of the Rbf (Retinoblastoma-family) gene (Colin, 2015).

Bcl-2 family proteins, in addition to their well characterized function in cell death, also play roles in metabolic processes in particular at the level of energetic metabolism. In particular, Bcl-2 regulates mitochondrial respiration and the level of different ROS through a control of cytochrome c oxidase activity. Study of heterologous bax expression in yeast has provided clues on Bax function in relation to ROS and yeast LOF mutants of genes involved in oxidative phosphorylation show increased sensitivity to Bax cytotoxicity. In agreement, Bcl-xL complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism. Furthermore, both the anti-apoptotic effect of LOF mutations in Gpo-1 and the GOF in transketolase genes can be related to a protective effect against oxidative stress. This result suggests that the cell death process induced by Bax involves, at least in part, the modulation of different ROS levels (Colin, 2015).

Indeed, this study reports that the suppressor effect of a null allele of Gpo-1 is associated with a decreased ability of Debcl to induce ROS production. This result is in agreement with the observation that 70% of the total cellular H2O2 production was estimated to stem from Gpo-1 in isolated Drosophila mitochondria. This enzyme has also been implicated in ROS production in mammalian brown adipose tissue mitochondria when glycerol-3-phosphate was used as the respiratory substrate and, more recently, in prostate cancer cells. In this latter case, ROS production seems to be beneficial to cancer cells, whereas this study show that it favors cell death in Drosophila wing disc cells. This apparent contradiction could be related to the abnormal ROS production occurring during the oncogenic transformation and the shift to a glycolytic metabolism (Colin, 2015).

In conclusion, this study shows that Gpo-1 contributes to debcl-induced apoptosis by increasing reactive oxygen species (ROS) production and provides a substantial resource that will aid efforts to understand the regulation of pro-apoptotic members of the Bcl-2 family proteins (Colin, 2015).


EVOLUTIONARY HOMOLOGS

Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-41 3'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. It is suggested that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29 (Slack, 2000).

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here it has been shown that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. It is proposed that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing (Reinhart, 2000).

Regulation of ribosome synthesis is an essential aspect of growth control. Thus far, little is known about the factors that control and coordinate these processes. The C. elegans gene ncl-1 encodes a zinc finger protein and may be a repressor of RNA polymerase I and III transcription and an inhibitor of cell growth. Loss of function mutations in ncl-1, previously shown to result in enlarged nucleoli, result in increased rates of rRNA and 5S RNA transcription and enlarged cells. Furthermore, ncl-1 adult worms are larger, have more protein, and have twice as much rRNA as wild-type worms. Localization studies show that the level of NCL-1 protein is independently regulated in different cells of the embryo. In wild-type embryos, cells with the largest nucleoli have the lowest level of NCL-1 protein. Based on these results it is proposed that ncl-1 is a repressor of ribosome synthesis and cell growth (Frank, 1998).

Transcriptional activation of HIV-1 gene expression by the viral Tat protein requires the interaction of a cellular cofactor with the Tat activation domain. This domain has been shown to consist of the cysteine-rich and core motifs of HIV-1 Tat and is functionally conserved in the distantly related Tat proteins of HIV-2 and EIAV. Using the yeast two-hybrid system, a novel human gene product, termed HT2A, has been identified that specifically and precisely binds to the activation domain of HIV-1 Tat and that can also interact with the HIV-2 and EIAV Tat proteins in vivo. The interaction between the activation domain of HIV-1 Tat and the HT2A protein can be readily detected in the mammalian cell nucleus. Sequence analysis demonstrates that HT2A is a novel member of the C3HC4 or ring finger family of zinc finger proteins that includes several known oncogenes and transcription factors. Overall, these data suggest that HT2A may play a significant role in mediating the biological activity of the HIV-1 Tat protein in vivo (Fridell, 1995).

A novel protein (BERP) has been identified that is a specific partner for the tail domain of myosin V. Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles. BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain. A yeast two-hybrid screening indicates that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb). This interaction has been confirmed by immunoprecipitation, which also demonstrates that BERP could associate with myosin Va, the product of the dilute gene. Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells. The RBCC domain of BERP is involved in protein dimerization. Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain results in defective PC12 cell spreading and prevents neurite outgrowth in response to nerve growth factor. These studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport (El-Husseini, 1999).

Mei-P26, a novel P-element-induced exchange-defective female meiotic mutant in Drosophila, has been cloned and characterized. Meiotic exchange in females homozygous for mei-P26 mutation is reduced in a polar fashion, such that distal chromosomal regions are the most severely affected. Additional alleles generated by duplication of the P element reveal that mei-P26 is also necessary for germline differentiation in both females and males. To further assess the role of mei-P26 in germline differentiation, double mutant combinations of mei-P26 and bag-of-marbles (bam), a gene necessary for the control of germline differentiation and proliferation in both sexes, were tested. A null mutation at the bam locus acts as a dominant enhancer of mei-P26 in both males and females. Interestingly, meiotic exchange in mei-P26;bam/+ females is also severely decreased in comparison to mei-P26 homozygotes, indicating that bam affects the meiotic phenotype as well. These data suggest that the pathways controlling germline differentiation and meiotic exchange are related and that factors involved in the mitotic divisions of the germline may regulate meiotic recombination (Page, 2000).

Altering the number of surface receptors can rapidly modulate cellular responses to extracellular signals. Some receptors, like the transferrin receptor (TfR), are constitutively internalized and recycled to the plasma membrane. Other receptors, like the epidermal growth factor receptor (EGFR), are internalized after ligand binding and then ultimately degraded in the lysosome. Routing internalized receptors to different destinations suggests that distinct molecular mechanisms may direct their movement. This study reports that the endosome-associated protein hrs is a subunit of a protein complex containing actinin-4, BERP, and myosin V that is necessary for efficient TfR recycling but not for EGFR degradation. The hrs/actinin-4/BERP/myosin V (CART [cytoskeleton-associated recycling or transport]) complex assembles in a linear manner and interrupting binding of any member to its neighbor produces an inhibition of transferrin recycling rate. Disrupting the CART complex results in shunting receptors to a slower recycling pathway that involves the recycling endosome. The novel CART complex may provide a molecular mechanism for the actin-dependence of rapid recycling of constitutively recycled plasma membrane receptors (Yan, 2005).

The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors

In the mouse neocortex, neural progenitor cells generate both differentiating neurons and daughter cells that maintain progenitor fate. This study shows that the TRIM-NHL protein TRIM32 regulates protein degradation and microRNA activity to control the balance between those two daughter cell types. In both horizontally and vertically dividing progenitors, TRIM32 becomes polarized in mitosis and is concentrated in one of the two daughter cells. TRIM32 overexpression induces neuronal differentiation while inhibition of TRIM32 causes both daughter cells to retain progenitor cell fate. TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific microRNAs. Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. TRIM32 is the mouse ortholog of Drosophila Brat and Mei-P26 and might be part of a protein family that regulates the balance between differentiation and proliferation in stem cell lineages (Schwamborn, 2009).

The data suggest that the increased levels of TRIM32 in one of the two daughter cells contribute to the decision of this cell to undergo neuronal differentiation. Like Brat, TRIM32 localizes asymmetrically in mitosis. Brat is localized by binding to Miranda, which, in turn, is recruited to the basal side by the protein Lgl and excluded from the apical side by aPKC (Knoblich, 2008). In fly neuroblasts, aPKC promotes self-renewal whereas Lgl inhibits proliferation. Although Miranda is not conserved, mouse Lgl and aPKC have similar effects on neural progenitor proliferation. In Lgl knockout mice, neural precursors overproliferate and eventually die by apoptosis. Removing one of the two aPKC mouse homologs does not affect the rate of neurogenesis, but depletion of its binding partner Par-3 results in premature cell-cycle exit of cortical progenitors. Despite these similarities, the precise mechanism by which TRIM32 localizes may be quite distinct. In Drosophila, the apical Par-3/6/aPKC complex directs the basal localization of Brat and Miranda but also orients the mitotic spindle along the apical-basal axis. In mice, however, the vast majority of progenitor divisions do not occur along the apical-basal axis. TRIM32 is asymmetric even in those planar divisions and provides a suitable explanation for how unequal fates can be generated independently of cleavage plane orientation. Therefore, the relevance of TRIM32 segregation is independent of the somewhat conflicting results that have been reported for the fraction of horizontal versus vertical divisions. Since TRIM32 asymmetry does not follow the polarity set up by Par-3/6/aPKC, however, it is likely that it is established by mechanisms distinct from Drosophila (Schwamborn, 2009).

What could those mechanisms be? TRIM32 often concentrates in the retracting basal fiber, a structure that is not present in Drosophila neuroblasts. TRIM32 might be present in the cytoplasm of the fiber and could be retained in the basal part of the cell during mitosis, when the fiber becomes extremely thin and its cytoplasm flows into the dividing progenitor. This would explain why TRIM32 is asymmetric even when the spindle is not oriented along the apical-basal axis. Since TRIM32-GFP expression prevents mitosis even at low levels, this observation cannot be verified by live imaging. The model would predict that the cell inheriting the basal fiber preferentially undergoes neuronal differentiation. This is in good agreement with some previous live-imaging studies, but other studies have actually proposed that the fiber is maintained in mitosis and serves as a guide for migration of the newly formed neuron. At the moment, it cannot be excluded that other mechanisms contribute to the asymmetric localization of TRIM32 (Schwamborn, 2009).

How does TRIM32 affect proliferation and differentiation? The data suggest that TRIM32 acts through two distinct pathways. Through its N-terminal RING finger, TRIM32 ubiquitinylates c-Myc and targets it for proteasome-mediated degradation. High levels of c-Myc are important for the ability of NSCs to self-renew and make NSCs relatively easy targets for reprogramming into ES cells. Furthermore, the bFGF–SHP2–ERK–c-Myc–Bmi-1 pathway is critical for the self-renewal capacity of neural progenitor cells, and Myc overexpression is known to promote neural progenitor proliferation in the mouse CNS. Therefore, a TRIM32-mediated reduction in the levels of c-Myc may well serve as a first step to induce neuronal differentiation. In agreement with this, overexpression of c-Myc in GFAP-positive astrocytes promotes formation of less differentiated Nestin-positive progenitor-like cells while a conditional ablation of the c-Myc ortholog N-Myc in mouse neuronal progenitor cells dramatically increases neuronal differentiation (Schwamborn, 2009).

Through its C-terminal NHL domain, TRIM32 acts as a potent activator of certain microRNAs. Although Drosophila Mei-P26 also binds Ago1, it inhibits rather than enhances microRNAs, and the mechanisms by which TRIM32 and its invertebrate homologs regulate microRNAs may actually be quite distinct. This is consistent with the observation that microRNAs support self-renewal in Drosophila stem cells while they potentiate differentiation in mammalian stem cells. In particular, Let-7a has an antiproliferative effect, and its expression reduces tumor growth and can prevent self-renewal in breast cancer cells. In NSCs, Let-7a is expressed and upregulated during differentiation. It is interesting to note that one of the targets for Let-7a is Myc. Protein degradation and concomitant translational inhibition through microRNAs might be the key strategy through which TRIM32 induces differentiation in NSCs (Schwamborn, 2009).

Although brat and mei-P26 mutant flies develop tumors, TRIM32 has not been described as a tumor suppressor. In fact, several reports have even suggested that TRIM32 might induce rather than prevent tumor formation. TRIM32 is mutated in patients carrying limb girdle muscular dystrophy type 2H. Since TRIM32 expression is upregulated during myogenic differentiation, the muscular dystrophy in these patients could be explained by a differentiation defect in the satellite cell lineage analogous to the one found in NSC lineages. TRIM32 has also been described as a gene potentially responsible for Bardet-Biedl syndrome and therefore has also been named BBS11. Distinct TRIM32 mutations are responsible for the two diseases, but none of them seems to cause cancer since an increase in tumor formation is not described for any of the two diseases. Since TRIM32 is a bifunctional molecule, mutating only the RING or the NHL domain might not be sufficient to prevent the antiproliferative function of TRIM32. In Drosophila, tumors only form in a small subset of brat mutant neuroblasts (Bowman, 2008). In other neuroblasts, redundancy with other tumor suppressors prevents overproliferation. Should a similar degree of redundancy exist in vertebrates, this might explain why TRIM32 is not a common target for oncogenic mutations. A similar lack of a human tumor phenotype has been shown for the Drosophila tumor suppressor Lgl. In Drosophila, lgl mutant neuroblasts overproliferate and form brain tumors. In mice, however, lgl mutant neural progenitors overproliferate initially but then die by apoptosis. A vertebrate-specific mechanism that prevents tumorigenesis in response to stem cell overproliferation could provide an alternative explanation for the lack of tumor formation when TRIM32 function is compromised. Although such a mechanism has been suggested previously the underlying mechanism remains unclear (Schwamborn, 2009).

These data establish TRIM-NHL proteins as a family of conserved stem cell regulators. The fact that Mei-P26 regulates stem cell proliferation in Drosophila ovaries (Neumuller, 2008) suggests that the function of this protein family might extend way beyond the brain. If this is the case, the presence of a catalytically active RING finger domain that could be inhibited by pharmaceutical compounds might make these proteins attractive targets for the manipulation of stem cell proliferation and the stimulation of regeneration in vivo (Schwamborn, 2009).


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Biological Overview

date revised: 2 December 2016

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