meiotic P26: Biological Overview | References
Gene name - meiotic P26
Cytological map position 8C15-8C17
Function - regulation of translation
Symbol - mei-P26
FlyBase ID: FBgn0026206
Genetic map position - chrX:9057331-9083367
Classification - BBOX and NHL (NCL-1, HT2A and LIN-41) repeat
Cellular location - cytoplasmic
In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. This study shows that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism (Insco, 2012).
Adult stem cells act throughout life to replenish differentiated cells lost to turnover or injury. In many adult stem cell lineages, stem cell daughters destined for differentiation first undergo a limited number of mitotic divisions to amplify cell number prior to terminal differentiation. This transit amplifying (TA) division strategy may protect large long-lived animals from tumorigenesis by minimizing the number of stem cell divisions required for tissue homeostasis and preventing accumulation of oncogenic mutations in progenitor cells due to programmed differentiation. The mechanisms that limit the number of TA divisions and initiate terminal differentiation thus may provide tumor suppressor function, and defects may contribute to progression toward cancer in adult stem cell lineages (Insco, 2012).
This study investigated the mechanisms that force TA cells to stop proliferating and initiate terminal differentiation in the Drosophila male germline adult stem cell lineage. Drosophila male germline stem cells (GSCs) reside in a niche at the tip of the testis, attached to somatic hub cells and flanked by somatic cyst stem cells (CySCs). When a GSC divides, one daughter remains in the niche and self-renews, while the other is displaced away and initiates differentiation. The resulting gonialblast, which is enveloped by a pair of CySCs, proceeds through four synchronous TA divisions with incomplete cytokinesis, producing a clone of 16 interconnected germ cells. These 16 mitotic sisters normally stop proliferating, undergo premeiotic DNA synthesis in synchrony, and switch to the spermatocyte program of cell growth, meiosis, and terminal differentiation. Because TA sister cells are contained within a common somatic cell envelope, are joined by cytoplasmic bridges, and divide in synchrony, mutations that cause overproliferation of TA cells can be easily identified (Insco, 2012).
The bag of marbles (bam) gene is required cell autonomously for TA spermatogonia to stop proliferating and enter the spermatocyte differentiation program. Male germ cells mutant for bam undergo several extra rounds of mitotic TA division, fail to differentiate, and eventually die. The number of TA divisions appears to be set by the time required for Bam protein to accumulate to a critical threshold. Bam protein is normally first detected in 4-cell cysts, increases to a peak in 8-cell cysts, and is degraded in early 16-cell cysts immediately after premeiotic DNA replication. Lowering the bam dosage slowed Bam protein accumulation and delayed the transition to differentiation, whereas early accumulation of Bam protein caused a premature switch to differentiation (Insco, 2009; Insco, 2012).
Bam, a protein with no recognizable domains, acts with a partner, benign gonial cell neoplasm (Bgcn), discovered in a genetic screen for Drosophila tumor suppressors. bam and bgcn have similar mutant phenotypes, and Bam protein directly interacts with Bgcn in Drosophila ovaries or when coexpressed in cultured cells or yeast (Li, 2009; Shen, 2009). Bgcn is related to the DExH-box family of RNA-dependent helicases, indicating that Bgcn, and with it Bam, may regulate RNA (Insco, 2012).
Consistent with a role in translational repression, Bam protein binds the translation initiation factor eIF4A. Furthermore, expression of Bam and Bgcn in Drosophila cultured cells resulted in a 4-fold reduction in expression of a luciferase reporter coupled to the 3′ untranslated region (UTR) of e-cadherin messenger RNA (mRNA), and tethering Bam to the 3′ UTR induced translational repression of the attached reporter (Shen, 2009). In female germ cells, Bam and Bgcn allow the onset of differentiation through translational repression of nanos (nos) via the nos 3' UTR (Li, 2009). However, direct interaction of Bam or Bgcn protein with e-cadherin or nos mRNAs has not been demonstrated (Insco, 2012).
This study identified the microRNA (miRNA) regulator and TRIM-NHL (tripartite motif and Ncl-1, HT2a, and Lin-41 domain) family member Mei-P26 (Neumüller, 2008; Page, 2000) both as a regulator of Bam protein accumulation and, subsequently, as a direct target of translational repression by Bam and Bgcn in male germ cells. Mei-P26 function facilitates both the switch from mitosis to meiosis and spermatocyte differentiation. In mei-P26 mutant males, Bam protein failed to accumulate to its normal peak levels. The overproliferation of TA cells in mei-P26 mutant testes was suppressed by expression of additional Bam, suggesting that the continued TA cell proliferation in mei-P26 mutant males is due to the failure of Bam protein to reach the threshold required for the switch to the spermatocyte state. In turn, Bam specifically binds the mei-P26 3' UTR, and Bam and Bgcn function are required for translational repression of mei-P26 via its 3' UTR in vivo. Mutating two potential let-7 target sites within the mei-P26 3' UTR derepressed reporter expression in vivo and disrupted Bam binding in vitro. These data suggest that a stepwise progression in regulatory states from [Mei-P26 on/Bam off] to [Mei-P26 on/Bam on] to [Bam on/Mei-P26 off], choreographed by translational regulation, accompanies the switch from TA cell proliferation to terminal differentiation in the Drosophila male GSC lineage (Insco, 2012).
It is proposed that Mei-P26 and Bam act in a regulatory cascade based on translational control to affect the switch from TA cell proliferation to spermatocyte differentiation in the Drosophila male GSC lineage. First, wild-type function of Mei-P26 in TA cells facilitates accumulation of Bam protein. Consistent with this model, a mei-P26 hypomorphic allele enhanced the overproliferation of germ cell cysts in a bam/+ heterozygote (Page, 2000). Furthermore, the finding that adding one extra copy of bam is sufficient to rescue the early germ cell overproliferation phenotype of mei-P26 mutant males indicates that allowing normal accumulation of Bam is the major role of Mei-P26 in regulating proliferation of early male germ cells. Second, as Bam protein levels rise, Bam and Bgcn repress translation of mei-P26 via its 3' UTR in late TA cell cysts. As a result, GSCs, gonialblasts, and two-cell cysts begin with Mei-P26 expressed and Bam off and transition to 4-cell and early 8-cell cysts wherein both Mei-P26 and Bam protein are expressed. In late 8-cell and early 16-cell cysts, Bam protein levels are high, causing Mei-P26 protein to drop to very low levels. Finally, in early spermatocytes, Mei-P26 levels rise again after Bam protein disappears to facilitate normal differentiation of spermatocytes and spermatids (Insco, 2012).
Recent data suggest that Mei-P26 and related TRIM family proteins may function in the miRNA pathway. Mei-P26, two of its Drosophila homologs, and several mouse homologs have been shown to interact structurally with RISC effector proteins such as Ago-1 (Neumüller, 2008; Rybak, 2009; Schwamborn, 2009). Mei-P26 protein localized to cytoplasmic puncta in early male and female germ cells, similar to the punctate distribution of mouse TRIM71. Many of the Mei-P26 puncta colocalized with the RISC component GW182 (Li, 2012), which accumulates in processing bodies that consist of enzymes involved in mRNA translational repression and degradation. The action of Mei-P26 in early male germ cells may facilitate accumulation of Bam protein by repressing an intermediate negative regulator of Bam. For example, Mei-P26 may function in TA cells to facilitate the accumulation of Bam protein through decreasing the function of the RNA-binding protein HOW. Previous studies suggest that HOW represses Bam expression in early male germ cells. In wild-type testes, HOW protein was expressed in early cells, including GSCs, gonialblasts, and two-cell cysts. However, in mei-P26 mutant testes, HOW protein perdured throughout the overproliferating cysts. Alternatively, given that Mei-P26 also contains a RING domain, it could facilitate degradation of an intermediate that normally degrades Bam. Bam has a C-terminal PEST sequence, a motif that targets proteins for ubiquitination and turnover by the proteasome, and expression of Bam lacking the PEST sequence resulted in early accumulation of high levels of Bam protein and a premature switch to the spermatocyte state (Insco, 2009) (Insco, 2012).
As Bam protein peaks in late TA cells, it acts with its binding partner Bgcn to repress translation of mei-P26 mRNA via sequences in the mei-P26 3' UTR. Bam protein specifically binds the mei-P26 3' UTR, suggesting that Bam and Bgcn act directly as translational repressors. Translational regulation via 3' UTR sequences frequently blocks formation of the translation initiation complex by inhibiting interactions between the cap binding protein eIF4E and the 5′ cap or the rest of the eIF4F complex. Bam protein physically interacts with eIF4A independent of RNA (Shen, 2009), and eIF4A/+ partially suppressed the phenotype of bam mutants in both the male and female germline systems (Insco, 2009; Shen, 2009), raising the possibility that Bam, recruited to a target 3' UTR as part of a translational repressor complex, may block translation initiation by antagonizing eIF4A (Shen, 2009). Mutating two potential let-7 binding sites within the mei-P26 3' UTR led to derepression of the in vivo reporter and disrupted binding of Bam to the mei-P26 3' UTR. These data raise the possibility that let-7 may work with Bam and Bgcn to translationally repress Mei-P26 in TA cells. In addition, introducing the let-7-CGK1 loss-of-function allele into a bamΔ86/+ mutant background enhanced the bam heterozygous mutant phenotype, suggesting that let-7 and Bam may share additional targets within the testes (Insco, 2012).
Mei-P26 appears to play two distinct roles in the female germline as well: an early function in GSC maintenance and a later function required for cystocytes to switch to nurse cell and oocyte differentiation (Li, 2012; Neumüller, 2008). However, there are also important differences between the male and female germline. Although Mei-P26 protein levels decreased when Bam was expressed in female germ cells, low levels of Mei-P26 were still detected. Bam and Bgcn may inhibit mei-P26 translation in female germ cells, although probably not to the same degree as in males. Notably, the mei-P26 3' UTR cloned from testes lacked the Vasa binding sites shown to be important for Mei-P26 expression in female germ cells (Liu, 2009). In addition, Bam is active at an earlier stage in the ovary, wherein the function of Bam is necessary for female GSCs to initiate the TA divisions rather than exit the TA divisions, as in males. Finally, Bam and Bgcn may have different mRNA targets in the female germline. In the female, Bam action directly or indirectly represses translation of the translational repressor Nanos (Li, 2009), allowing the expression of proteins that initiate germ cell differentiation from the stem cell state. However, Nanos does not appear to play the same role in male as in female GSCs. Thus, the core machinery of Bam, Bgcn, and Mei-P26 probably acts through similar molecular mechanisms in female and male germ cell differentiation, but at a different point in the differentiation pathway, with different regulators and, most likely, on different targets (Insco, 2012).
Strikingly, as is shown in this study for the Drosophila male germline, the switch from mitosis to meiosis is also controlled by a regulatory network based on translational control in the C. elegans germline (Kimble, 2007). BLAST and ClustalW alignments revealed that Bgcn, a core component of the switch mechanism in the Drosophila germline, is a homolog of C. elegans proteins Mog1, Mog4, and Mog5, which are required for stopping mitosis and repressing target-mRNA translation via the 3' UTR (Insco, 2012).
The requirement for TRIM-NHL proteins to facilitate the switch from proliferation to differentiation may be a widely conserved feature in many adult stem cell lineages. In Drosophila, loss of the Mei-P26 homolog dappled causes large melanotic tumors, suggesting the continued proliferation of blood cells. Likewise, loss of the Drosophila Mei-P26 homolog brat in TA cells in certain neural lineages leads to brain tumors that are highly proliferative, invasive, transplantable, and lethal to the animal. In mammals, the mouse Mei-P26 homolog TRIM32 is necessary and sufficient for differentiation in neural lineages, and progenitor cells lacking TRIM32 retain proliferative status (Schwamborn, 2009). Thus, elucidating the mechanisms by which Mei-P26 homologs and their interacting structural and regulatory partners control the switch from proliferation to differentiation in adult stem cell lineages may uncover a new class of tumor suppressors that act at the level of the developmental program rather than cell-cycle progression (Insco, 2012).
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 (see Xia, 2010). 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 (Hammell, 2009; Schwamborn, 2009; 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 (Kedde, 2010). 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 (Kedde, 2010). 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 (Neumuller, 2008). 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).
In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. This study presents data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam (Li, 2013).
This study presents data that Mei-P26 cooperates with Bam, Bgcn and Sxl to control the translation of nanos mRNA in the Drosophila female germline. Co-immunoprecipitation experiments indicate Mei-P26 physically associates with the differentiation factors Bam, Bgcn and Sxl and yeast 2-hybrid assays suggest the interaction between Mei-P26 and Bgcn may be direct. Disruption of mei-P26, or snf, which disrupts sxl expression in the germline, results in the upregulation of Nanos protein expression in early differentiating cysts. Both Mei-P26 and Sxl protein associate with nanos mRNA (Chau, 2012). In light of the recently published study that shows mutating Sxl binding sites within the 3′UTR of nanos mRNA leads to mis-regulation of the gene (Chau, 2012), these results suggest that Mei-P26 may be part of a Sxl, Bgcn and Bam complex that serves to promote cyst development by directly repressing the expression of Nanos. However, despite repeated attempts, direct interactions between Bam and Bgcn with nanos mRNA could not be detected. While various technical issues may prevent the detection of these specific interactions, the inability to observe direct association between Bam/Bgcn and nanos mRNA leaves open the possibility that interactions between the components of the Mei-P26, Sxl, Bam and Bgcn complex and its target mRNAs may be dynamic in nature. For instance, Bam and Bgcn may help to prepare Sxl and Mei-P26 for mRNA binding but do not themselves directly interact or only transiently interact with these targets. Further experiments will be needed to clarify the more specific molecular mechanisms that underlie Bam/Bgcn function with respect to the translational repression of nanos mRNA (Li, 2013).
Two other recent studies investigated the role of mei-P26 during germline development. Liu (2009) showed that the RNA helicase Vasa directly regulates the translation of mei-P26 mRNA through poly (U) elements within its 3' UTR. Mutations in each gene strongly enhance the phenotype of the other, resulting in the formation of cystic germline tumors. Neumuller (2008) focused on the function of Mei-P26, showing that it negatively regulates the activity of the miRNA pathway. It is now proposed that Mei-P26 functions in both GSCs and early differentiating germ cells. Within GSCs, Mei-P26 is in a complex with miRISC proteins and enhances miRNA-mediated silencing. In addition, Mei-P26 associates with Nanos protein and promotes BMP signaling within GSCs by repressing the expression of the negative regulator Brat. GSC daughters displaced away from the cap cell niche experience less BMP signaling, allowing for the expression of Bam (Li, 2013).
It is speculated that upon Bam expression, Mei-P26 switches its activity and/or its mRNA targets. This switch allows Mei-P26 to promote germline differentiation by both negatively regulating the miRNA pathway and cooperating with Bam, Bgcn and Sxl to repress the translation of specific mRNAs such as nanos. However the complex functional relationships between Mei-P26, Sxl, Bam and Bgcn remain incompletely understood. While evidence is provided that these factors can physically associate with each other under certain conditions, disruption of these genes results in two discrete phenotypes. mei-P26 and snf mutants exhibit a cystic tumorous phenotype marked by the accumulation of undifferentiated cysts that do not express A2BP1, a molecular marker present in 4-, 8- and 16-cell cysts in wild-type samples. In contrast, disruption of bam or bgcn results in the formation of single cell germ cell tumors. These phenotypic differences suggest that Bam and Bgcn carry out additional functions independent of Mei-P26 and Sxl. A more complete characterization of the regulatory networks that govern the very early steps of germline cyst differentiation will have to await a better biochemical characterization of Bam and Bgcn function (Li, 2013).
Together these data suggest that Mei-P26 has a variety of molecular functions inside and outside of the germline. It remains unclear whether Mei-P26 exhibits the same biochemical activity when complexed with different proteins or whether its function completely changes depending on context. Based on the presence of a RING domain, Mei-P26 may act as an ubiquitin ligase. However this specific enzymatic activity has not been demonstrated nor have any direct in vivo substrates been identified. In regards to the translational repression of specific mRNAs, a model is favored in which Mei-P26 exhibits the same molecular activity within GSCs and their early differentiating daughters. It is further speculated that association of Mei-P26 with different mRNA binding proteins modulates its targeting of specific mRNAs, and/or the degree to which these different targets are repressed. The expression of Bam correlates with changes in the development role of Mei-P26 but the manner in which Bam alters the composition or activity of the Mei-P26 complex remains unknown. Regardless, the findings that Bam can associate with Mei-P26 and Sxl provide further support for the hypothesis that Bam regulates the translation of specific mRNAs to promote the early steps of differentiation within the Drosophila female germline (Li, 2013).
In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, a mutation was isolated in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. Tut was found to bind preferentially a long isoform of mei-P26 3'UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, this study demonstrates that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Both in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage (Chen, 2014).
Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. This study shows that the CCR4 deadenylase is required for GSC self-renewal; Nos and Pum act through its recruitment onto specific mRNAs. mei-P26 mRNA was identified as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. Fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation (Joly, 2013).
This study provides evidence that the twin gene that encodes the CCR4 deadenylase is essential for GSC self-renewal. GSCs are rapidly lost in twin mutants because they differentiate and cannot self-renew. Clonal analysis shows that twin is required cell autonomously in the GSCs for their self-renewal. Nos and Pum are major factors of GSC self-renewal and are translational repressors. Genetic and protein interactions among twin, nos, and pum indicate that CCR4 acts together with Nos and Pum to promote GSC self-renewal. This identifies the recruitment of the CCR4-NOT deadenylation complex as the molecular mechanism underlying Nos and Pum translational repression in the GSCs. Two mechanisms of action used by Nos/Pum have previously been described in the embryo. First, Nos/Pum represses hb mRNA translation by forming a complex with Brat, which in turn interacts with 4EHP and blocks initiation of translation. Second, Nos/Pum represses cyclin B mRNA translation in the primordial germ cells by recruiting the CCR4-NOT complex through direct interactions between Pum and CAF1 and between Nos and NOT4 (Kadyrova, 2007). Brat is not expressed in GSCs, thus excluding the first mode of Nos/Pum translational repression in these cells. However, Pum, Nos, and CCR4 were found to be present in a complex in GSC-like cells, consistent with the recruitment of the CCR4-NOT complex by Nos/Pum for GSC self-renewal (Joly, 2013).
Interestingly, a mutant form of CCR4 that is inactive for deadenylation is able to partially rescue the lack of CCR4 in GSCs. This is consistent with CCR4 not being the only deadenylase in the complex (Temme, 2010). However, CCR4 does participate in the deadenylation activity of the complex, probably via a structural role. Furthermore, the CCR4-NOT complex has been shown recently to be involved in direct translational repression, in addition to its role in deadenylation (Chekulaeva, 2011; Cooke, 2010). This dual mode of action of CCR4-NOT might also be relevant to GSCs (Joly, 2013).
The miRNA pathway also plays a crucial role in GSC self-renewal. A large body of evidence has shown that an important mechanism of silencing by miRNAs involves deadenylation resulting from the recruitment of CCR4-NOT by GW182 bound to Ago1 (for review, see Braun, 2012). Therefore, the CCR4-NOT complex is also likely to contribute to miRNA-mediated translational repression in the GSCs, thus making this complex a central effector of translational repression in the GSCs (Joly, 2013).
An important result from this study is that mei-P26 mRNA is a major target of Nos/Pum/CCR4 regulation for GSC self-renewal. Nos and Pum are known to be essential players in GSC self-renewal, and many mRNAs are expected to be regulated by this complex. However, to date only one mRNA target of this complex, brat, has been reported. This study has identified another target, mei-P26 mRNA, and has shown that its repression by the Nos/Pum/CCR4 complex has a key role in GSC self-renewal, because the loss of GSCs in the twin mutant is strongly rescued by decreasing mei-P26 gene dosage (Joly, 2013).
Both Brat and Mei-P26 belong to the Trim-NHL family of proteins, which have conserved functions in stem cell lineages from C. elegans to mouse (for review, see Wulczyn, 2010). Proteins within this family are potential E3 ubiquitin ligases and can act by either activating or antagonizing the miRNA pathway, through their association with Ago1 and GW182. In particular, Mei-P26 function switches from activation of the miRNA pathway in the GSCs to inhibition of the pathway in differentiating cysts where Mei-P26 levels are higher. As such, Mei-P26 plays a central role in the control of cell fate in the GSC lineage. The rescue of the twin mutant phenotype of GSC loss by decreasing mei-P26 gene dosage suggests that the levels of Mei-P26 themselves might be important for this switch of its function. This might provide an explanation as to why such a precise regulation of its level is crucial for GSC self-renewal and differentiation (Joly, 2013).
Which molecular mechanisms underlie the fine-tuning of Mei-P26 in the GSC lineage? The translational repression of mei-P26 mRNA is not complete in GSCs. This differs from the complete repression by Nos/Pum of cyclin B mRNA in the primordial germ cells, or brat mRNA in the GSCs, and may result from the concomitant activation of mei-P26 by Vasa. Vasa does activate mei-P26 translation, leading to a peak of expression in 8-cell and 16-cell cysts. However, Vasa is expressed in all germ cells, suggesting that it is not the key regulator governing the timing of Mei-P26 peak of expression. It is proposed that translational activation of Mei-P26 by Vasa would be active already in GSCs but counterbalanced by translational repression by Nos/Pum and the CCR4-NOT complex. In cystoblasts, the presence of Bam overcomes Nos/Pum translational repression by decreasing Nos levels, which would thus switch the balance to translational activation by Vasa. This does not lead to a peak of Mei-P26 expression in cystoblasts, but rather to a progressive increase of Mei-P26 levels in proliferating cysts. This progressive accumulation of Mei-P26 could depend on the necessity to build up Vasa-mediated translational activation. However, another possibility could be that a different factor still partially represses mei-P26 translation in cystoblasts and early cysts. A potential candidate is Bam, which has been defined as a translational repressor and has recently been reported to directly repress mei-P26 mRNA translation in the male GSC lineage (Insco, 2012). The Bam expression profile in female germ cells is consistent with this potential role in mei-P26 translational repression, because Bam protein is present from cystoblasts to 8-cell cysts but absent in 16-cell cysts, where Mei-P26 levels are the highest (Joly, 2013).
Recent advances have established the generality of a central role for translational regulations in adult stem cell lineages. Translational repression is required to prevent the synthesis of differentiation factors whose mRNAs are already present in stem cells. In the Drosophila female GSC lineage, recent work has demonstrated that changes in cell fate are driven by different translational regulation programs; associations between translational repressors evolve to trigger stage-specific regulation of mRNA targets. For example, while Nos/Pum maintain female GSCs by repressing a specific set of mRNAs, Pum associates with Brat in cystoblasts to repress a different set. The Trim-NHL proteins appear to be of particular importance in the translational regulations essential for stem cell fate as exemplified by Mei-P26. The fine-tuning of Mei-P26 protein levels by translational repression is essential for GSC self-renewal and implicate CCR4 in this regulation (Joly, 2013).
The functions of Trim-NHL proteins are conserved in many adult stem cell lineages in different organisms, and mutations in the corresponding genes lead to highly proliferative tumors. Elucidating the molecular mechanisms behind their translational control is key to deciphering how these proteins regulate adult stem cell fates (Joly, 2013).
The balance between germ-line stem cell (GSC) self-renewal and differentiation in Drosophila ovaries is mediated by the antagonistic relationship between the Nanos (Nos)-Pumilio translational repressor complex, which promotes GSC self-renewal, and expression of Bam, a key differentiation factor. This study found that Bam and Nos proteins are expressed in reciprocal patterns in young germ cells. Repression of Nos in Bam-expressing cells depends on sequences in the nos 3' UTR, suggesting that Nos is regulated by translational repression. Ectopic Bam causes differentiation of GSCs, and this activity depends on the endogenous nos 3' UTR sequence. Previous evidence showed that Bgcn is an obligate factor for the ability of Bam to drive differentiation, and this study reports that Bam forms a complex with Bgcn, a protein related to the RNA-interacting DExH-box polypeptides. Together, these observations suggest that Bam-Bgcn act together to antagonize Nos expression; thus, derepressing cystoblast-promoting factors. These findings emphasize the importance of translational repression in balancing stem cell self-renewal and differentiation (Li, 2009).
Previous studies show GSC maintenance dually depends on Nos expression to suppress CB differentiation and on transcriptional silencing of bam. Expression of Bam acts as a developmental switch, and is both necessary and sufficient to drive germ cell differentiation. Elucidating the biochemical activity of Bam has been impeded, however, because both the low abundance and lack of recognizable functional domains of the protein. The goals of the experiments presented in this article were to provide new insights into the function of Bam by finding protein partners and the downstream targets of Bam action (Li, 2009).
The translational repressor proteins Pum and Nos are critical GSC maintenance factors and suppress CB differentiation, perhaps by repressing translation of a pool of cystoblast (CB) promoting mRNAs stored in GSCs. The dynamic pattern of Nos accumulation in the germarium suggested the protein disappears as CB differentiation begins. Decreasing levels of Nos expression during CB differentiation are unlikely to reflect changes in the transcription, because a GFP reporter fused to the nos promoter remains active throughout the germarium. Instead, Nos elimination within early cysts is mediated by sequences in the 3' UTR of the transcripts. Substituting a tubulin 3' UTR for the endogenous nos 3' UTR resulted in uniform Nos protein expression throughout the germarium. Further work has narrowed down the region responsible for translational repression of nos in the germarium to the first 100 bases of the 3' UTR of the transcript (Li, 2009).
Genetic experiments with bam and pum alleles has suggested that the 2 genes exerted opposite actions on CB differentiation. Nos accumulation declines when Bam is expressed ectopically in several genetic backgrounds, suggesting that Nos accumulation can be linked directly to Bam protein levels and not to signals from somatic cells in the germarium. Data showing that diminished bam or bgcn gene dosage could suppress the germ cell loss phenotype of nos alleles provided additional evidence for the inverse relationship of bam and nos expression. A reduction in bam or bgcn dose may decrease the likelihood that a nos− GSC differentiates precociously, because stem cells are more likely to be maintained in these ovaries. The relevant antagonism could take place within the transient cell identified as the 'precystoblast'. It is also possible that nos− primordial germ cells are more likely to be captured as stem cells during gonadogenesis when bam or bgcn levels were reduced (Li, 2009).
The nos 3' UTR is essential for proper regulation, because the CB differentiation induced by ectopic Bam expression fails when [hs-Bam] flies carry a Nos-tub 3' UTR transgene. Surprisingly, cyst formation proceeds normally in ovaries carrying the Nos-tub 3' UTR transgene even though ectopic expression would be expected to promote GSC self-renewal. One possible explanation is provided by observations that Pum levels also fall as CBs differentiate. Pum levels become limiting even as Nos continues to accumulate from the p[nosP-Nos-tub 3' UTR] transgene. Likewise, redundant pathways may exist to derepress translation of CB-promoting mRNAs, just as multiple pathways appear to exist to silence those mRNAs. If, like Nos-Pum, the miRNA pathway is down-regulated to initiate differentiation, derepression of CB-promoting mRNAs might occur even if Nos expression is maintained during CB and cyst stages. Because ectopic Ago1 expression, but not Nos, produces extra GSCs, miRNAs might be separate and prominent repressors of CB differentiation to maintain GSCs (Li, 2009).
Together, these genetic and biochemical experiments suggest that Bam and Bgcn form a complex that represses nos translation, either directly or indirectly. Mechanistically, it is considered possible that Bam-Bgcn and perhaps other proteins directly repress nos mRNA by binding sequences in the nos 3' UTR. However, it has not been possible to demonstrate a direct physical interaction between Bam-Bgcn and nos mRNA, either from ovary extracts or in vitro. Likewise, attempts to reconstitute Bam-Bgcn-dependent translational repression of the nos 3' UTR in S2 cells failed. These experiments may have failed because S2 cells lack important, but as yet unidentified, cofactors found specifically in germ cells, or because Bam-Bgcn regulate nos translation via an indirect mechanism. For example, it is plausible that Bam-Bgcn promote the expression of the early response target mRNAs, and one or more of these factors could repress nos translation. Alternative mechanisms of action for Bam-Bgcn are unclear as Bam lacks any defined sequence motifs and Bgcn, whereas related throughout the length of the protein to RNA/DNA helicases, lacks the motifs to be a functional helicase (Ohlstein, 2000). Outside the Bam-interacting domains, Bgcn contains a pair of ankyrin repeats that could mediate other protein-protein interactions (Li, 2009).
One potential component of the Bam-Bgcn complex, Mei-P26, was suggested by previous genetic experiments. mei-P26 has been identified as a gene required for early germ cell differentiation and meiosis, and showed that mei-P26 activity depends on the proper dosage of bam. Recently, bam has been reported to require mei-P26 to deplete stem cells, and similarly, mei-P26 required bam to function properly. These observations could imply a close working relationship between bam and mei-P26. However, the interactions and interrelated functions of Bam, Bgcn, and Mei-P26 are likely to be complex. For example, although the phenotypes of bam and bgcn mutations are indistinguishable, the mei-P26 mutant phenotype is distinct. Germ cells lacking mei-P26 apparently form CBs, because they produce Bam-positive cysts with branched fusomes. Given the current results, exploring the functional significance of Bam, Bgcn, and Mei-P26 interactions will be important (Li, 2009).
The view of stem cells that emerges from these studies has several striking elements: (1) that repression mechanisms control many stem cell differentiation circuits, and (2) that translational regulation has an integral role in these decisions. The GSC model highlights an intrinsic capacity to differentiate and the need to apply brakes (Nos-Pum) to retard differentiation. Perhaps this mechanism was advantageous to prevent all gametes from maturing at once in animals that developed with a finite number of germ cells. Of course, differentiation would require a mechanism (Bam-Bgcn) to override the brakes. Within this framework, a stem cell population could arise when a group of stromal cells captured germ cells and produced signals that could repress expression of the factor(s) that would antagonize the brakes. Natural selection would rapidly fix this event, because it would greatly expand the number of gametes produced from individuals by establishing a stem cell as a renewable source of germ cells. This mode of niche evolution might also explain the appearance of stem cell populations in most organs, because it would be expected to enhance fitness by permitting larger body size, lengthening the fecund lifespan and increasing survivability of trauma by providing a mechanism for tissue regeneration. If the mechanisms at work in Drosophila GSCs apply to many stem cells, stem cells should be enriched for many more antidifferentiation genes than true stemness genes (Li, 2009).
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).
Vasa (Vas) is a DEAD-box RNA-binding protein required in Drosophila at several steps of oogenesis and for primordial germ cell (PGC) specification. Vas associates with eukaryotic initiation factor 5B (eIF5B), and this interaction has been implicated in translational activation of gurken mRNA in the oocyte. Vas is expressed in all ovarian germline cells, and aspects of the vas-null phenotype suggest a function in regulating the balance between germline stem cells (GSCs) and their fate-restricted descendants. A biochemical approach was used to recover Vas-associated mRNAs. >mei-P26, whose product represses microRNA activity and promotes GSC differentiation, was identified. vas and mei-P26 mutants interact, and mei-P26 translation is substantially reduced in vas mutant cells. In vitro, Vas protein bind specifically to a (U)-rich motif in the mei-P26 3' untranslated region (UTR), and Vas-dependent regulation of GFP-mei-P26 transgenes in vivo was dependent on the same (U)-rich 3' UTR domain. The ability of Vas to activate mei-P26 expression in vivo was abrogated by a mutation that greatly reduces its interaction with eIF5B. Taken together, these data support the conclusion that Vas promotes germ cell differentiation by directly activating mei-P26 translation in early-stage committed cells (Liu, 2009).
This study demonstrated that Vas regulates mei-P26 expression in vivo, and that a (U)-rich element in the mei-P26 3' UTR interacts with Vas in vitro and is required for Vas-mediated regulation in vivo. DEAD-box helicases such as Vas were not believed previously to be sequence-specific nucleic acid-binding proteins. This study has shown however, that Vas demonstrates specific binding, but that this requires domains that are distinct from the motifs shared by all DEAD-box proteins. Vas contains nine RGG repeats located between amino acids 17 and 165, within the region implicated in binding specificity that lies outside of the canonical DEAD-box segment. While the region N-terminal to the common DEAD-box motifs is highly variable in the sequences of Vas orthologs from different species, the presence of RGG repeats within that region is conserved; for example, zebrafish Vas contains nine such motifs, and human Vas (DDX4) contains four. Two RGG repeats are present in mammalian fragile X mental retardation protein (FMRP), and have been shown to specifically recognize a G quartet structure in semaphorin 3F RNA, indicating that this motif can discriminate among target RNAs. RGG repeats are often present in proteins that contain other RNA-binding domains; for example, Vas contains a DEAD-box signature, while FMRP contains two hnRNPK homology (KH) domains; thus it has been proposed that they serve an auxiliary role in RNA binding. It is suggested that the RGG repeats of Vas play such a role by conferring specificity to its association with RNA. RGG motifs are also targets for arginine methyltransferases, and arginine methylation has been linked to modulating the RNA-binding activity of heterogeneous nuclear RNP (hnRNP) A1. It is tempting to speculate that the RNA-binding activity of Vas might be similarly modulated, perhaps through the activity of the arginine methyltransferase Capsuléen, which like Vas is essential for germ cell specification (Liu, 2009).
A model has been proposed for Vas-mediated translational activation whereby Vas recruits eIF5B to target mRNAs. This study suggests that Vas can itself discriminate among potential mRNA targets, although it cannot be excluded that Vas may be recruited to other target mRNAs through indirect associations involving partner RNA-binding proteins. Precedents for regulation of translation at the step of subunit joining exist in several systems. For example, in early erythroid precursor cells, the mRNA encoding 15-lipoxygenase (r15-LOX) is translationally silenced at this step of translation initiation, dependent on a cytidine-rich 3' UTR element termed DICE (differentiation control element) and on two RNA-binding proteins: hnRNP K and hnRNP E1. Phosphorylation of a specific tyrosine residue of hnRNP K by c-Src reduces its affinity for DICE, thus activating translation. Another example is provided by the ASH1 mRNA in Saccharomyces cerevisiae, which is translationally repressed before localizing to the bud cortex by Puf6p, which binds the RNA and blocks subunit joining through an interaction with eIF5B. As for r15-LOX, repression is alleviated by phosphorylation of the RNA-binding protein. Vas differs from these other translational regulators in that it positively regulates its targets; like hnRNP K and Puf6p, however, post-translational modification of Vas has also been linked to a reduction of its activity (Liu, 2009).
Several phenotypes manifested in vas-null ovaries point toward a function for Vas in restricting cell fate during cystocyte divisions, and these could result from reduced mei-P26 expression. The relationship between Mei-P26 and Vas may be more complex, however, as both are linked to small RNA metabolism. Mei-P26 binds to AGO1, an RNase that is a core component of the RNA-induced silencing complex (RISC) that is involved in miRNA-mediated translational repression and RNA degradation pathways. Both Mei-P26 and AGO1 have been implicated in regulating GSC fate; Mei-P26 restricts growth and proliferation and promotes differentiation, while AGO1 does the reverse. Piwi, a key component of rasiRNA and piwi-interacting RNA (piRNA) pathways, has been implicated in stem cell self-renewal. Vas is associated with Piwi, Aubergine, and other components of the rasiRNA pathway, and has itself been linked to retrotransposon silencing. Therefore, Vas appears to be involved in both the AGO1 pathway, through its regulation of Mei-P26, and the rasiRNA pathway, potentially making it a key regulator of processes mediated by small RNAs in GSCs and early-stage committed cells (Liu, 2009).
Drosophila neuroblasts and ovarian stem cells are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. This study shows that the Trim-NHL protein Mei-P26 restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. Argonaute-1, a component of the RISC complex, was identified as a common binding partner of Brat and Mei-P26, and it was shown that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages (Neumuller, 2008).
The data suggest that brat and mei-P26 might act in a similar manner to control proliferation in stem cell lineages. In both mutants, cells that normally stop self renewal increase ribosome biogenesis, grow abnormally large and fail to exit the cell cycle leading to the formation of a tumour. The general upregulation of microRNAs in mei-P26 mutants leaves several possibilities for how these proteins might regulate the microRNA pathway. The presence of a RING finger in Mei-P26 suggests a role in protein degradation. The high amounts of AGO1 detected in Mei-P26 immunoprecipitates make it unlikely that AGO1 itself is degraded by Mei-P26. However, another member of the RISC complex might be a degradation target of Mei-P26. Equally likely, Mei-P26 could prevent the incorporation or increase the turnover of microRNAs in the RISC complex (Neumuller, 2008).
Many human tumours contain cancer stem cells that drive tumour growth and metastasis. Although the similarities between Drosophila tumours and human cancer are limited, brat mutant brains and mei-P26 mutant ovaries (as well as the other mutant conditions causing stem cell tumours provide an invertebrate model for stem-cell-derived tumour formation. In mei-P26 mutants, tumours originate from cystocytes, the transit-amplifying pool of the ovarian stem cell lineage. In mei-P26 mutants, these cells re-gain the ability to self-renew: after bam overexpression—which leads to premature differentiation of stem cells—the germ line is depleted in a wild-type but not in a mei-P26 mutant background. Thus, mei-P26 tumours arise from growth defects in the transit-amplifying compartment of the ovarian stem cell lineage—a mechanism that could occur in human tumours as well (Neumuller, 2008).
The data establish Trim-NHL proteins as regulators of stem cell proliferation. Vertebrate members of this family exist and are downregulated in human cancer cell lines suggesting that their tumour-suppressor function might be conserved in vertebrates as well (Neumuller, 2008).
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-P261 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. Tested were 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, in order to further assess the role of mei-P26 in germline differentiation. A null mutation at the bam locus was found to act as a dominant enhancer of mei-P26 in both males and females. Interestingly, meiotic exchange in mei-P261; bamDelta86/+ females is also severely decreased in comparison to mei-P261 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).
mei-P26-induced defects in female germline differentiation appear to occur during the early mitotic divisions in cyst formation, and hypomorphic alleles of mei-P26, such as mei-P261, produce egg chambers with abnormal numbers of nurse cells. Cysts with an increased number of nurse cells may be due to an additional round of mitosis occurring in some or all of the cystocytes. Alternatively, a defect in follicle cell packaging of normal cysts may result in chambers containing an excess of cells. However, egg chambers containing two oocytes, which would be expected if two 16-cell cysts were packaged together, were not found. Similarly, cysts with too few nurse cells could also result from packaging of cysts that have abnormally broken their intercellular connections to form smaller clusters of cells. Cysts containing 7 nurse cells and an oocyte may also result from the cystocytes undergoing only three divisions (Page, 2000).
Allelic combinations of mei-P26 that alter the number of nurse cells also reduce the number of normal eggs produced, but they do not cause complete sterility. In addition to the defects in nurse cell number, a tumorous ovary phenotype is more frequent in severe alleles. In females homo- or hemi-zygous for these mutations, the egg chambers become filled with hundreds of small cells, and no oocyte develops. This phenotype is similar to that exhibited by mutants in the bam and benign gonial cell neoplasm (bgcn) genes (Page, 2000).
The most severe mei-P26 allele affects germline differentiation in both males and females. In mei-P26mfs1, ovaries consist entirely of tumorous egg chambers and, in males carrying this allele, spermatid differentiation progresses only to the point of producing elongated spermatid bundles, and mature spermatozoa are not produced. The mei-P26mfs1 allele thus bears some similarities to mutants in the bam and bgcn genes, both of which cause a tumorous phenotype in ovaries and arrest of spermatogenesis. However, spermatid differentiation is arrested at an earlier stage in bam and bgcn mutants. Ovary morphology differs somewhat, in that mei-P26 mutant ovarioles consist of a series of defined chambers, while bam and bgcn ovarioles often appear as distended germaria with few distinct chambers. Nevertheless, the interaction of mei-P26 with bam suggests that the similarities in the phenotypes in these mutants are not coincidental (Page, 2000).
Certain mutations in the Sex-lethal (Sxl), ovarian tumor (otu), and ovo genes also cause sterility in females due to the formation of tumorous egg chambers. Interestingly, mild to severe defects in meiotic recombination frequencies are observed in females bearing heteroallelic combinations of female sterile alleles for these loci and in females in which the effects of Sxl mutations have been partially suppressed using genetic modifiers. These results further suggest that the processes that control exchange position and determine germline cyst formation may be coordinately controlled (Page, 2000).
The structure of the MEI-P26 protein suggests a few possibilities for the role of mei-P26. mei-P26 is predicted to encode a member of the RBCC family of proteins, which contain, in their N-terminal regions, a RING finger motif followed by one or two copies of a second cysteine-rich motif called the B-box and a coiled coil region. The RING finger and B-box motifs are believed to mediate physical interactions with other proteins. RBCC proteins are only a subset of the large number of known RING finger proteins, which have diverse roles in oncogenesis, transcriptional regulation, signal transduction, and development (Page, 2000).
Several RBCC proteins, such as PML and the TIF1 family, are known to regulate transcription by binding to nuclear hormone receptors as coactivators or corepressors. While assembling factors for transcriptional regulation is one role for RBCC proteins, certain other RBCC proteins appear to function in capacities such as signal transduction, or by forming ribonucleoprotein complexes (SS-A/Ro). Although a growing number of RBCC proteins have been identified, only a handful also contain NHL repeats, named after the proteins NCL-1, HT2A, and LIN-41. The NHL repeat has been shown to be involved in protein-protein interactions in the RBCC proteins HT2A and BERP (Page, 2000).
The RBCC-NHL proteins are mostly of unknown function or have not been extensively characterized at a molecular level. RBCC-NHL proteins, which contain both RBCC and NHL domains, include at least two potential protein-protein interaction motifs, so a strong possibility is that these also may participate in the formation of multiprotein complexes. One possible candidate for a MEI-P26 partner protein is Bam (Page, 2000).
A genetic interaction between mei-P26 and bam has been demonstrated. Heterozygosity for a null mutant in bam enhances the phenotype of mei-P26, causing sterility in males, an increase in tumor formation in females, and a decrease in meiotic exchange. On the basis of the characterization of the bam and mei-P26 gene products, the nature of the interaction between these two genes is open to speculation (Page, 2000).
(1) MEI-P26 may act as a transcriptional or translational regulator that controls bam expression. A variety of ovarian defects like those observed in mei-P26, including tumorous chambers and cysts with abnormal numbers of nurse cells, are also seen in mutants for the Drosophila Rbp9 gene. The Rbp9 gene has also been shown to encode an RNA binding protein that binds specifically to the bam transcript and may act to regulate Bam expression in the germarium. Similarly, misregulation of bam expression in the encore (enc) mutant may underlie the effect of enc on nurse cell number. However, since heterozygosity for bam exacerbates the meiotic phenotype of mei-P26, this model predicts that the meiotic defects are due to the misregulation of bam, rather than through other genes possibly regulated by mei-P26 (Page, 2000).
(2) mei-P26 could be required for the proper localization or function of Bam. The product of the bam gene is expressed in the cytoplasm of cystoblasts and early germline cysts in females, where it is required for cystoblast differentiation. Bam protein also associates with the fusome, a large organelle, comprised mostly of cytoskeletal and vacuolar components, which is present in early germline cysts. According to this model, MEI-P26 may physically interact with Bam, possibly through the RBCC, NHL, or other motifs in the MEI-P26 protein. Alternatively, this regulation may be indirect, requiring other proteins. For example, MEI-P26 may regulate the bgcn gene product, which is required for Bam function. Again, this suggests that the effects on meiotic exchange are mediated by bam (Page, 2000).
The evidence presented in this study does not allow a determination of the relative positions of bam and mei-P26 in a pathway. Therefore, (3) bam may be required for mei-P26 function, which in turn would be required for proper germline cyst development and meiotic recombination. In this third model, MEI-P26 may physically interact with Bam and/or other proteins in the cytoplasm, possibly as a component of the fusome, from which MEI-P26 may facilitate normal germline development and meiotic exchange. Alternatively, MEI-P26 might be indirectly controlled by Bam as a downstream effector. While the relationship between mei-P26 and bam has not been fully elucidated, these models are intriguing, as they all suggest a role for bam in a pathway ensuring proper meiotic exchange (Page, 2000).
mei-P26 appears to behave as expected for a female meiotic precondition mutant. This group of Drosophila mutants presents a phenotype in which the total frequency of meiotic exchange is often reduced, although to differing levels, and residual exchanges are abnormally distributed in a polar fashion, with reduced frequencies in the distal parts of the chromosome arms. In mei-P26, exchange is decreased overall and the distribution is polar. In more severe alleles, recombination is more severely affected, and E0 is increased. To provide a first step toward explaining the various components of the mei-P26 phenotype and the connection with germline differentiation, the following rather speculative model for precondition mutants in general is proposed (Page, 2000).
In many organisms, telomeres have been proposed as sites responsible for initiating at least part of the pairing interactions between homologous chromosomes. Evidence for the clustering of telomeres during meiotic prophase has been gathered through cytological studies in many species. In these studies, chromosomes in meiotic prophase were observed to form what has been described as a 'bouquet' configuration, where the telomeres are positioned together in a small portion of the nuclear volume. The telomeres are often clustered near a region of the nuclear envelope adjacent to the position of the cytoplasmic centrosome. It is thought that the clustering of telomeres may facilitate homolog pairing in meiosis (Page, 2000).
The existence of a bouquet configuration has been demonstrated recently in Saccharomyces cerevisiae, and telomeres are responsible for a delay in meiotic progression observed in haploid yeast strains that are disomic for a single chromosome pair. Furthermore, the delay associated with the presence of telomeres requires an intact NDJ1 gene. In a wild-type background, deletions of NDJ1 interfere with synapsis and alter the distribution of recombination events. Ndj1p localizes to telomeres and is believed to be necessary for telomeric pairing. Defects in synapsis and a decrease in the frequency of recombination are observed in the presence of mutations in yeast KAR3, which encodes a kinesin-like protein. This observation suggests that Kar3p may be involved in chromosome movement that is necessary for proper homolog alignment during meiotic prophase (Page, 2000).
This clustering of telomeres has not been demonstrated in Drosophila oocytes, but the observations by electron microscopy may not have detected transient telomere clustering at the leptotene or zygotene stage of meiosis. Thus, perhaps the association of telomeres is important for homologous chromosome pairing in Drosophila. The mechanism by which telomere clustering occurs may be similar to that in other organisms. In particular, the movement of the chromosomes may require associations with the cytoskeleton, resulting in the clustering of telomeres near centrosomes. Within germline cysts, the fusome acts to orient the spindles in the cystocyte divisions by interacting with one centrosome at each mitosis. Although the fusome begins to break down after the cessation of the mitotic divisions, remnants of this structure may remain in the oocyte during meiotic prophase (germarium regions 2 and 3). Therefore, the fusome may position a determinant, possibly in the form of a cytoskeletal element, necessary for telomere clustering at a point on or near the nuclear membrane. Perhaps Bam, as a component of the fusome, is indirectly necessary for telomere clustering by marking the site of clustering on the nuclear membrane. The polar recombination defect observed in mei-P26 could therefore be the indirect result of abnormal telomeric clustering due to a disruption of Bam function (Page, 2000).
Since proximal euchromatic regions may rely on the pairing of centric heterochromatin, and perhaps not require telomeric clustering, disruption of telomeric clustering may primarily impact the frequency of exchange in the distal regions of the chromosomes. Thus, the specific disruption of telomeric interactions may result in a distribution of residual exchanges like that seen in recombination precondition mutants. Further work will be necessary to determine whether the meiotic defects in precondition mutants are the result of abnormalities in telomeric clustering (Page, 2000).
Search PubMed for articles about Mei-p26
Braun, J. E., Huntzinger, E. and Izaurralde, E. (2012). A molecular link between miRISCs and deadenylases provides new insight into the mechanism of gene silencing by microRNAs. Cold Spring Harb Perspect Biol 4. PubMed ID: 23209154
Chau, J., Kulnane, L. S. and Salz, H. K. (2012). Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis. Proc Natl Acad Sci U S A 109: 9465-9470. PubMed ID: 22645327
Chekulaeva, M., Mathys, H., Zipprich, J. T., Attig, J., Colic, M., Parker, R. and Filipowicz, W. (2011). miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs. Nat Struct Mol Biol 18: 1218-1226. PubMed ID: 21984184
Chen, D., Wu, C., Zhao, S., Geng, Q., Gao, Y., Li, X., Zhang, Y. and Wang, Z. (2014). Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila. PLoS Genet 10: e1004797. PubMed ID: 25412508
Cooke, A., Prigge, A. and Wickens, M. (2010). Translational repression by deadenylases. J Biol Chem 285: 28506-28513. PubMed ID: 20634287
Hammell, C. M., Lubin, I., Boag, P. R., Blackwell, T. K. and Ambros, V. (2009). nhl-2 Modulates microRNA activity in Caenorhabditis elegans. Cell 136: 926-938. PubMed ID: 19269369
Insco, M. L., Leon, A., Tam, C. H., McKearin, D. M. and Fuller, M. T. (2009). Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage. Proc Natl Acad Sci U S A 106: 22311-22316. PubMed ID: 20018708
Insco, M. L., Bailey, A. S., Kim, J., Olivares, G. H., Wapinski, O. L., Tam, C. H. and Fuller, M. T. (2012). A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage. Cell Stem Cell 11: 689-700. PubMed ID: 23122292
Joly, W., Chartier, A., Rojas-Rios, P., Busseau, I. and Simonelig, M. (2013). The CCR4 Deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal. Stem Cell Reports 1: 411-424. PubMed ID: 24286029
Kedde, M., van Kouwenhove, M., Zwart, W., Oude Vrielink, J. A., Elkon, R. and Agami, R. (2010). A Pumilio-induced RNA structure switch in p27-3' UTR controls miR-221 and miR-222 accessibility. Nat Cell Biol 12: 1014-1020. PubMed ID: 20818387
Kimble, J. and Crittenden, S. L. (2007). Controls of germline stem cells, entry into meiosis, and the sperm/oocyte decision in Caenorhabditis elegans. Annu Rev Cell Dev Biol 23: 405-433. PubMed ID: 17506698
Knoblich, J. A. (2008). Mechanisms of asymmetric stem cell division. Cell 132: 583-597. PubMed ID: 18295577
Kadyrova, L. Y., Habara, Y., Lee, T. H. and Wharton, R. P. (2007). Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline. Development 134(8): 1519-27. PubMed ID: 17360772
Li, Y., Minor, N. T., Park, J. K., McKearin, D. M. and Maines, J. Z. (2009). Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance. Proc Natl Acad Sci U S A 106: 9304-9309. PubMed ID: 19470484
Li, Y., Maines, J. Z., Tastan, O. Y., McKearin, D. M. and Buszczak, M. (2012). Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling. Development 139: 1547-1556. PubMed ID: 22438571
Li, Y., Zhang, Q., Carreira-Rosario, A., Maines, J. Z., McKearin, D. M. and Buszczak, M. (2013). Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary. PLoS One 8: e58301. PubMed ID: 23526974
Liu, N., Han, H. and Lasko, P. (2009). Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3' UTR. Genes Dev 23: 2742-2752. PubMed ID: 19952109
Neumuller, R. A., Betschinger, J., Fischer, A., Bushati, N., Poernbacher, I., Mechtler, K., Cohen, S. M. and Knoblich, J. A. (2008). Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature 454: 241-245. PubMed ID: 18528333
Page, S. L., et al. (2000). Genetic studies of mei-P26 reveal a link between the processes that control germ cell proliferation in both sexes and those that control meiotic exchange in Drosophila. Genetics 155: 1757-1772. PubMed Citation: 10924476
Rybak, A., Fuchs, H., Hadian, K., Smirnova, L., Wulczyn, E. A., Michel, G., Nitsch, R., Krappmann, D. and Wulczyn, F. G. (2009). The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2. Nat Cell Biol 11: 1411-1420. PubMed ID: 19898466
Schwamborn, J. C., Berezikov, E. and Knoblich, J. A. (2009). The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. Cell 136: 913-925. PubMed Citation: 19269368
Shen, R., Weng, C., Yu, J. and Xie, T. (2009). eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary. Proc Natl Acad Sci U S A 106: 11623-11628. PubMed ID: 19556547
Temme, C., Zhang, L., Kremmer, E., Ihling, C., Chartier, A., Sinz, A., Simonelig, M. and Wahle, E. (2010). Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation. RNA 16: 1356-1370. PubMed ID: 20504953
Wulczyn, F. G., Cuevas, E., Franzoni, E. and Rybak, A. (2010). MiRNA need a TRIM regulation of miRNA activity by Trim-NHL proteins. Adv Exp Med Biol 700: 85-105. PubMed ID: 21627033
Xia, L., Jia, S., Huang, S., Wang, H., Zhu, Y., Mu, Y., Kan, L., Zheng, W., Wu, D., Li, X., Sun, Q., Meng, A. and Chen, D. (2010). The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response. Cell 143: 978-990. PubMed ID: 21145463
date revised: 25 March 2015
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.