org Interactive Fly, Drosophila



Transcriptional Regulation

Maternal effect genes, including oskar, define a shared pathway leading to the provision of two determinants at the posterior pole of the embryo. One determinant is the posterior body patterning morphogen Nanos, and the other directs germ cell formation. Overexpression of oskar causes the shared pathway to be hyperactivated, with excess nanos activity present throughout the embryo and a superabundance of posterior pole cells. In addition, presumptive pole cells appear at a novel anterior position. Formation of these ectopic pole cells is enhanced in nanos mutants, possibly reflecting competition between nanos and the germ cell determinant for a shared and limiting precursor (Smith, 1992).

Targets of Activity

Misexpression of NOS protein at the anterior of the embryo inhibits translation of Bicoid mRNA, thereby suppressing head and thorax development (Gavis, 1994).

In Drosophila embryos, graded activity of the posterior determinant Nanos generates abdominal segmentation by blocking protein expression from maternal transcripts of the Hunchback gene. When active inappropriately at the anterior pole, NOS can also block expression of the anterior determinant Bicoid. Both regulatory interactions are mediated by similar sequences in the 3' untranslated region of each transcript. These NOS response elements (NREs) are both necessary and sufficient to confer nos-dependent regulation, the degree of regulation determined by the number and quality of the elements and the level of NOS in vivo. Based on these and other results, it can be argued that NOS acts as a morphogen, controlling HB expression (and hence abdominal pattern) as a function of its concentration-dependent interaction with the NREs. Thus, it would seem that the requirements for NOS mRNA localization, involving proteins of the posterior group are imposed by the presence of the NRE in BCD and HB mRNAs (Wharton, 1991).

giant is expressed in two broad gradients in precellular embryos, one in anterior regions and the other in posterior regions. GT patterns overlap with protein gradients specified by the gap genes hunchback and knirps. Maternal factors are responsible for initiating gt expression, while gap genes participate in the subsequent refinement of the pattern. The maternal morphogen Bicoid initiates the anterior gt pattern, while nanos, by inhibiting hunchback, plays a role in the posterior pattern (Kraut, 1991).

High local concentrations of NOS protein in the posterior of the embryo are necessary to inhibit translation of the maternal transcript of hunchback in this region, and thus permit expression of genes required for abdomen formation. The restriction of HB protein expression permits the ordered expression of Krüppel, knirps and giant, three genes specifying the abdominal region (Gavis, 1994).

Primitive insects develop segmentation by heterchronic gene activation. Each posterior segment forms independently in a time dependent manner. nanos activity eliminates hunchback from the posterior precellular blastoderm region of primitive insects, and caudal, otherwise repressed by hunchback, can function there at a later stage. caudal proceeds to drive the addition of new segments over time. Thus, a gradient of Caudal would regulate the asynchronous segment development of primitive insects (Rivera-Pomar, 1995).

The closely linked POU domain genes pdm-1 and pdm-2 are first expressed early during cellularization in the presumptive abdomen in a broad domain that soon resolves into two stripes. The broad abdominal domain of pdm-1 protein is lacking in nanos- mutant embryos, and ectopic pdm-1 expression in nanos- embryos leads to a partial restoration of abdominal segmentation (Cockerill, 1993).

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. Following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a 'PGC niche' (Gilboa, 2004).

GSC differentiation is repressed by extrinsic factors, such as Dpp, and also by intrinsic factors. To further test whether PGCs employ the same mechanisms as GSCs to repress differentiation, larval ovaries were examined that were mutant for the translational repressors Nanos (Nos) and Pumilio (Pum), which function within GSCs to repress their differentiation. Indeed, nos mutant LL3 gonads contained many developed cysts. pumilio (pum) mutant gonads also contained cysts, although less so than nos mutants. Gonads that were mutant for both nos and pum did not contain more cysts than gonads that were mutant for nos alone. Because the alleles that were used were very strong, this suggests that nos and pum function together in the repression of PGC differentiation (Gilboa, 2004).

In adult ovaries, the differentiation of cysts requires Bam, and increasing amounts of Bam are present during each subsequent mitotic division. A reporter construct of GFP under control of the bam promoter was used to follow bam expression in the larval cysts. Cysts found in nos ML3 larval gonads also expressed higher amounts of GFP as compared to single PGCs. As in adults, the intensity of GFP labeling corresponds to the developmental state of the cyst. In addition to precocious differentiation, nos mutant germ cells displayed aberrations in the shape of the branched fusome and increased amount of small fusomal material as compared with wild-type. It is concluded that both Nos and Pum, which are required for GSC maintenance, are also required to repress PGC differentiation (Gilboa, 2004).

To further test for a possible partnership between nos and pum in GSC maintenance, the time at which nos or pum mutant germ line clones, generated by the FLP-FRT method, were eliminated from the adult ovary was examined. In wild-type, clones of unmarked GSCs were induced in about 25% of the ovarioles and that percent decreased only slightly during the course of the experiment, probably due to the natural rate of GSC loss. nos and pum mutant GSCs, in contrast, were lost rapidly. GSC loss was observed as early as 4 days after clone induction, and by the 6th or 7th day, most ovarioles did not contain a mutant GSC. The striking similarity in the profiles of nos and pum GSC loss therefore suggests that these genes also function together within GSCs (Gilboa, 2004).

As of the fifth and sixth day after clone induction, it was found that many nos mutant cysts were eliminated from the ovary. These results agree with the death of cysts observed in nos and pum mutants and with the death of nos cysts in pupal ovaries, which may be the cause of the empty ovarioles observed in adult nos females. These results and the previously reported phenotypes of nos and pum suggest that these genes are continually required throughout germ cell life. In the embryo, nos and pum are required for correct migration, transcription, and viability. During larval stages, they are required for the repression of PGC differentiation and, in the adult, for the maintenance and viability of GSCs as well as for the viability of differentiating cysts (Gilboa, 2004).

The targets of Nos and Pum within GSCs remain elusive, and the relationship of these 'intrinsic' GSC maintenance factors to the 'extrinsic' Dpp signal is unclear. To test if Dpp could function partly through Nos, the Nos expression pattern was examined in wild-type and in tkv-mutant GSCs. In wild-type germaria Nos is expressed at intermediate levels in GSCs and their immediate daughters, at very low levels during mitotic divisions of the cyst, and at very high levels in a fraction of the 16-cell cysts. This expression pattern was unchanged in tkv-mutant germ cells. Similar results were obtained for larval PGCs; Nos was expressed at intermediate levels in wild-type and tkv mutant PGCs, at lower levels in cysts undergoing mitosis, and at very high levels in 16-cell cysts. This suggests that Nos expression is independent of Dpp signaling (Gilboa, 2004).

Next, whether nos is required for Dpp function was tested, by analyzing nos mutant PGCs that were overexpressing either Dpp or TkvQD, a constitutively activated form of Tkv. In nos mutant control gonads, fragmented fusomal material as well as branched cysts could be observed. The spherical fusome within nos mutant germ cells remained small or fragmented in nos gonads overexpressing Dpp. Most strikingly, single PGC/GSC like germ cells accumulated in these gonads, and no cysts could be found. Thus, although increased Dpp signaling cannot fully counteract the nos phenotype, it does prevent precocious differentiation of nos mutant PGCs. Similar results were obtained with PGCs expressing TkvQD. In most gonads no cysts could be observed, although occasionally a small branched fusome could be detected, suggesting that Dpp signaling acts directly on PGCs, rather than via a secondary signal. The genetic data show that PGCs that are mutant for nos, can still respond to a Dpp signal, which keeps them in an undifferentiated state (Gilboa, 2004).

During larval stages, PGCs proliferate rather than differentiate. The translational repressors Nos and Pum are required to repress PGCs differentiation during larval stages. It has also been show that the Dpp pathway functions in a similar manner. Both pathways are also required for GSC maintenance. The fact that the spherical fusome remains abnormal in nos mutant gonads even when Dpp is overexpressed may suggest that some of Nos function is downstream of Dpp. However, the Nos expression data and the fact that Dpp signaling can prevent nos mutant PGCs from differentiation are more compatible with the Nos pathway playing a role upstream or in parallel to the Dpp pathway. It remains unclear how these pathways converge within germ cells (Gilboa, 2004).

Germ cells may perceive a Dpp signal from the moment they form at the posterior pole of the embryo until they differentiate to form cysts. Indeed, pMad is present in embryonic pole cells, larval PGCs and adult GSCs. Dpp signaling is not only necessary for GSC maintenance but also required continually through larval stages to actively repress PGC differentiation. Thus, the larval ovary functions in a similar manner to the adult niche with regard to Dpp-mediated repression of differentiation. During the third-larval instar, the adult somatic niche forms, and repression of PGC differentiation may then become limited to the small area of the adult ovary, allowing PGCs outside the confinement of the niche to differentiate (Gilboa, 2004).

Repression of PGC differentiation is required for about 4 days, from the end of embryogenesis to the beginning of pupa formation, whereas GSCs are maintained in the adult for many days. Differences between the 'short-term' and the 'long-term' repression of differentiation may yet be found. However, all the genes tested, dpp, bam, nos, and pum, function similarly in GSCs and PGCs. This similarity suggests that there may not be a clear transition from a 'dividing' PGC to a 'self-renewing' GSC (Gilboa, 2004).

Transport of germ plasm on astral microtubules directs germ cell development in Drosophila

In many organisms, germ cells are segregated from the soma through the inheritance of the specialized germ plasm, which contains mRNAs and proteins that specify germ cell fate and promote germline development. Whereas germ plasm assembly has been well characterized, mechanisms mediating germ plasm inheritance are poorly understood. In the Drosophila embryo, germ plasm is anchored to the posterior cortex, and nuclei that migrate into this region give rise to the germ cell progenitors, or pole cells. How the germ plasm interacts with these nuclei for pole cell induction and is selectively incorporated into the forming pole cells is not known. Live imaging of two conserved germ plasm components, nanos mRNA and Vasa protein, revealed that germ plasm segregation is a dynamic process involving active transport of germ plasm RNA-protein complexes coordinated with nuclear migration (see graphical abstract). Centrosomes accompanying posterior nuclei induce release of germ plasm from the cortex and recruit these components by dynein-dependent transport on centrosome-nucleated microtubules. As nuclei divide, continued transport on astral microtubules partitions germ plasm to daughter nuclei, leading to its segregation into pole cells. Disruption of these transport events prevents incorporation of germ plasm into pole cells and impairs germ cell development. These results indicate that active transport of germ plasm is essential for its inheritance and ensures the production of a discrete population of germ cell progenitors endowed with requisite factors for germline development. Transport on astral microtubules may provide a general mechanism for the segregation of cell fate determinants (Lerit, 2011).

This study has uncovered a dynamic mechanism for germ plasm inheritance involving release of germ plasm RNPs from the posterior cortical actin anchor coordinated with their dynein-dependent transport to centrosomes that are associated with posterior nuclei. Transport of these RNPs occurs primarily, if not exclusively, on astral microtubules throughout the mitotic cycle. The results suggest that directed transport of germ plasm components during pole bud formation ensures the production of a discrete population of germ cell progenitors and partitions factors required for germline development during subsequent divisions. Through this process, germline fate determinants are segregated away from somatic nuclei (Lerit, 2011).

Pole cell formation is highly sensitive to the dosage of germ plasm components, because mutations that reduce the accumulation of germ plasm at the posterior pole result in fewer pole cells. This study found that pole cell formation is similarly reduced when germ plasm transport is disrupted, as it is in Dhc mutants or in mutants that affect centrosome function. Although the molecular mechanism by which germ plasm promotes pole cell formation is unknown, the results suggest that directed transport of germ plasm components toward the small subset of nuclei that are the first to arrive at the posterior pole provides the requisite concentration of one or more factors necessary to impart germline fate and induce pole cell formation. In addition, because the first divisions of the nascent pole cells occur before budding is complete, the persistence of germ plasm transport toward centrosomes during these divisions would ensure that factors required for germline development, such as nos, are maintained within pole buds, segregated to daughter nuclei, and ultimately incorporated into the forming germ cells (Lerit, 2011).

Germ plasm produced ectopically in osk-bcd3′UTR (transgene used to generate embryos with germ plasm containing nos*GFP localized ectopically at the anterior of the embryo in addition to its normal localization at the posterior) and Khc mutant embryos is transported to nearby nuclei, indicating that nuclei are not predetermined to recruit germ plasm. Thus, the release of germ plasm from its actin-based anchor and the onset of germ plasm motility must be tightly coordinated with the arrival of nuclei at the posterior cortex to target germ plasm specifically to these nuclei and prevent the misspecification of cell fate. Egg activation triggers the release of bcd mRNA from the anterior cortex, probably through a generalized activation-dependent restructuring of the cortical actin cytoskeleton. This event does not release nos and Vas, however. Nor is germ plasm release scheduled by an intrinsic timing mechanism, as was shown in this study. Consistent with the observation that nos release is delayed at the anterior in osk-bcd3′UTR embryos, formation of ectopic germ cells at the anterior lags behind pole cell formation at the posterior in these animals. Moreover, centrosomes isolated from nuclei, either pharmacologically or genetically, are sufficient to trigger germ plasm release from the posterior. These data thus support a model whereby centrosomes and/or centrosome-nucleated microtubules associated with migrating nuclei trigger germ plasm release from the cortical anchor (Lerit, 2011).

Astral microtubules provide the tracks along which germ plasm RNPs travel upon their initial release from the cortex. During mitosis in the syncytial embryo, astral microtubules appear to secure the partitioning of germ plasm RNPs to daughter nuclei. The preferential association with astral microtubules may also prevent the dilution of inductive signals during asymmetric division events, when only one aster is proximal to the germ plasm. The apparent specificity for astral microtubules suggests that the RNP-motor complexes may include factors that recognize particular microtubule-associated proteins or modifications that distinguish these microtubules as preferred tracks (Lerit, 2011).

The observed dynein-dependent transport of nos during pole cell formation contrasts with its diffusion-based mode of localization during oogenesis. Given that dynein-dependent transport of bcd mRNA to the oocyte anterior is ongoing during late oogenesis, it is essential that nos be excluded from interaction with the dynein transport machinery. nos may reside in a dynein-associated transport complex that is inactive or incompatible with the various oocyte microtubule subpopulations. Alternatively, the composition of the nos RNP in the oocyte may simply preclude its association with the dynein motor complex. The observed cotransport of nos and Vas in the embryo suggests that nos becomes linked to dynein through its packaging into a complex with Vas and other germ plasm components. Whether germ plasm RNPs are coupled to dynein motors while they are anchored at the posterior or only after their release remains a subject for future investigation. A similar switch between motor-independent and motor-dependent modes of germ plasm mRNA translocation may occur in Xenopus, although the role of motors in Xenopus germ plasm inheritance is not yet clear (Lerit, 2011).

Recent in situ hybridization studies have now identified over 50 mRNAs that are localized at the posterior of the Drosophila embryo and incorporated into pole cells. Further characterization of a subset of these mRNAs showed that they accumulate near posterior nuclei, suggesting that they may be transported similarly to nos. Determining whether the different transcripts are cotransported will require the development of methods to simultaneously visualize multiple RNAs and germ plasm proteins. However, packaging of even subsets of RNAs together into germ plasm RNPs competent for dynein-mediated transport would greatly simplify the problem of partitioning a complex pool of transcripts to pole cells (Lerit, 2011).

Nanos function and histone methylation

In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. A chromatin-based mechanism has been identified that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis (Schaner, 2003).

The C. elegans nanos homologs nos-1 and nos-2 play important overlapping roles in worm primordial germ cell development. Knockdown of both NOS-1 and NOS-2 activities results in sterile animals, a population of which exhibit germ cells that begin proliferation prematurely. Loss of NOS-2 alone can also result in ectopic localization of the primordial germ cells. H3meK4 levels were examined in Z2/Z3 chromatin of embryos from nos-1(gv5);nos-2(RNAi) hermaphrodites. Animals homozygous for the nos-1(gv5) are viable and fertile; RNAi of nos-2 in these animals results in germ cell defects. nos-1 mutant animals were therefore injected with nos-2 dsRNA, and parallel broods were assessed for sterility as adults or stained for H3meK4 in Z2/Z3. In these experiments, 59% of the offspring of the injected animals grew up to be completely sterile adults. An almost identical percentage of parallel brood embryos exhibited inappropriate staining for H3meK4 in one or both of the primordial germ cells, Z2/Z3. The C. elegans NANOS homologs, NOS-1 and NOS-2, therefore play important roles in either establishing and/or maintaining the chromatin-based mode of transcriptional repression in Z2/Z3 (Schaner, 2003).

While the progenitors of the soma and germline are also separated from each other during early embryogenesis in Drosophila, the processes that give rise to the physical segregation of these two distinct cell types in flies are quite different from those in worms. In light of these differences, it was of interest to determine if distinct chromatin states are also established in the somatic and germline nuclei of fly embryos. Staining of early Drosophila embryos with the H3meK4 antibody has revealed that there is little detectable H3meK4 in any nuclei during the rapid synchronous nuclear divisions in the center of the embryo. Similarly, no H3meK4 was detected in the nuclei of newly formed pole cells, or in somatic nuclei when they first migrate to the periphery of the embryo. H3meK4 was first detected in somatic nuclei between nuclear division cycles 12 and 13, after the nuclei around the periphery of the embryo have already undergone several rounds of nuclear division. This change in the methylation status of histone H3 is coincident with, or slightly precedes, the time when transcription is broadly upregulated in the somatic nuclei of syncytial blastoderm embryos. High levels of H3meK4 are maintained through the cellular blastoderm stage, with all somatic nuclei staining with the H3meK4 antibody (Schaner, 2003).

In contrast to the somatic nuclei, little if any H3meK4 was detected in the pole cells (marked with anti-Vasa-specific antibody) of either syncytial blastoderm or cellular blastoderm embryos. Staining of embryos at later stages revealed that H3meK4 could still not be detected in germ cells in early gastrulation stage through midgut invagination. However, once the germ cells traversed the midgut wall and began migrating away from the hindgut toward the somatic gonadal precursor cells, H3meK4-specific signal became readily evident. Therefore, from the time of their formation until stage 9 of embryogenesis, there is little if any H3meK4 in the germ cells' chromatin. H3meK4 becomes readily detectable in the germ cells, however, coincident with the onset of transcription at stage 9/10. The accumulation of H3meK4 in the germ cells, as in the soma, correlates temporally with transcriptional activation in these cells (Schaner, 2003).

Embryos were stained for epitopes corresponding to H4acetylK5, H4acetylK8, H4acetylK12, H3diacetyl, and H3dimeK36. Only the antibodies specific for H3diacetyl and H4acetylK5 exhibited nuclear staining above background levels in early embryonic stages, and the appearance of these epitopes was temporally identical to that observed for H3meK4. In contrast to the H3meK4 modification, however, no differences were observed in the levels of these modifications in pole cell nuclei compared to somatic nuclei. This indicates that a global lack of H3meK4, in combination with the presence of other modifications that correlate with transcriptional competence, is a conserved feature of chromatin structure in transcriptionally inert germ cell nuclei. (Schaner, 2003).

nos function is required for either the establishment or maintenance of transcriptional quiescence in newly formed Drosophila pole cells. In light of results showing a premature appearance of H3meK4 in C. elegans germ cells in the absence of nos activity, an obvious question is whether nos is also required to block this methylation in the early fly germline. To answer this question, fly embryos produced by nos mutant mothers were probed with H3meK4- and Vasa-specific antibodies. H3meK4 can be detected in the pole cells of stage 4 nos- embryos at nuclear division cycle 14. In contrast, H3meK4 is completely absent from pole cell chromatin in wild-type embryos at this stage. Moreover, H3meK4-specific signal was also detected in pole cells in stage 5 embryos that have just initiated gastrulation. It is interesting to note that not all nos- pole cells (27/42) display H3meK4-specific staining. This is consistent with findings that transcription is not activated in every pole cell of nos- blastoderm stage embryos and supports the suggestion that additional factors contribute to the establishment/maintenance of transcriptional quiescence in fly pole cells (Schaner, 2003).

While methylation of lysine 4 of histone H3 is a conserved mark of transcriptionally competent or active chromatin, methylation of lysine 9 of histone H3 (H3meK9) is a highly conserved modification that is enriched in silenced genomic regions, such as either facultative or constitutive heterochromatin. Consequently, it was of interest to determine if the transcriptionally quiescent pole cell nuclei are enriched in H3meK9 as compared to the transcriptionally active somatic nuclei. Drosophila embryos were probed with antibodies against H3meK9 and Vasa to mark the pole cells. In contrast to H3meK4, cleavage stage Drosophila embryonic nuclei exhibited readily detectable H3meK9 even prior to their migration. The modification was also detected in somatic nuclei after nuclear migration, where it appeared enriched in nuclear regions adjacent to the periphery of the embryo that correspond to telomeric and centromeric heterochromatin in the Rabl configuration. These regions, conversely, were observed to lack H3meK4. H3meK9 is also detected in pole cells as soon as they are formed, with the level of H3meK9 antibody staining in the pole cell nuclei being considerably higher than in the somatic nuclei. The observed enrichment of H3meK9 in the pole cell nuclei coincided with loss of the mitosis-specific modification, H3 phospho-Ser10, suggesting that a specific remodeling of the pole cell chromatin accompanies their exit from the cell cycle (Schaner, 2003).

Since depletion of maternal nos activity results in a premature increased accumulation of H3meK4 in early pole cells, whether nos- pole cells exhibit a parallel reduction in H3meK9 was investigated. Indeed, nos- pole cell nuclei do not exhibit the characteristic enrichment for H3meK9-specific signal seen in wild-type embryos. Furthermore, more than 50% of the pole cells examined show considerably reduced staining specific for nuclear H3meK9 compared to the neighboring somatic nuclei. The known dichotomous roles for H3meK4 and H3meK9 in the regulation of transcriptional competence are therefore conserved in pole cells, and their relative abundances in pole cell chromatin are responsive to nos activity (Schaner, 2003).

Identifying the mechanisms that guide the separation of somatic and germ lineages is one of the oldest pursuits in developmental biology. How the totipotent germline is maintained during development has become increasingly relevant to modern science in the search for conserved mechanisms guarding stem cell identity. These data provide new evidence for at least two modes of germline-specific repression that guard the germline during C. elegans embryogenesis, one of which is conserved in Drosophila. In the earliest phase, maternal PIE-1 activity in the germline P blastomeres prevents mRNA production through a mechanism that does not involve substantial, germline-specific alterations in chromatin architecture. After the degradation of PIE-1, however, a second mode involving a dramatic and specific remodeling of chromatin arises in the germ cell precursors, Z2/Z3. Whereas the former mode appears to be unique to C. elegans, the latter mode, which bears the hallmarks of direct transcriptional repression provided by a specific mode of chromatin organization, is a conserved feature of germ cell maintenance (Schaner, 2003).

In both worms and flies, lineage restriction to germ cell fate is marked by a global absence of H3meK4 and a more condensed chromatin structure. In the case of C. elegans, the absence of H3meK4 is the result of a specific depletion; in Drosophila the absence arises from preventing H3meK4 accumulation. This absence is maintained in both organisms until zygotic activation of the genome in germ cells. Premature activation, marked by premature accumulation of H3meK4, results in the loss of the germ cell lineage in both worms and flies. In both species, the lack of H3meK4 occurs in the presence of high levels of other histone modifications that often correlate with an open chromatin configuration, indicating a 'dominance' of H3meK4 as an indicator of transcriptional activity (Schaner, 2003).

The conserved correlation between H3meK4 presence and absence and global activation and silencing, respectively, is striking. The inactive X chromosome in mammals is globally depleted of H3meK4, as are silenced regions of the genome in fission yeast. In Tetrahymena, the germinal micronucleus is transcriptionally silent and H3meK4 is missing. Sexual conjugation causes transformation of the micronucleus into a transcriptionally active somatic macronucleus, which becomes enriched in H3meK4. Thus, even in protozoans, genome-wide diminishment of H3meK4 is a property of the 'germline' and its accumulation accompanies 'somatic' activation (Schaner, 2003).

The lack of H3meK4 in fly pole cells is mirrored by enrichment for H3meK9. This is not observed in Z2/Z3 in C. elegans. The reason for this difference is not understood but may reflect a more substantial role for this modification in genome regulation in Drosophila, which has a more highly repetitive genome than that of C. elegans, and consequently more abundant classically defined heterochromatin. Indeed, in contrast to Drosophila, H3meK9 is only cytologically enriched at telomeres in C. elegans embryos and on the highly condensed, unpaired X chromosome during male meiosis. In both examples, the enrichment for H3meK9 is always mirrored by the absence of H3meK4. (Schaner, 2003).

Posttranscriptional regulation plays a major role in maintaining the embryonic germline in both worms and flies. The conserved classes of proteins involved include the pumilio (Pufs) and nanos families. nanos activities are similarly required for proper migration of the primordial germ cells, maintenance of their mitotic quiescence, proper proliferation of the germline after hatching, and, as shown in this report, the regulation of chromatin organization in Z2/Z3 in both species. In addition, nanos has also been shown to be required to maintain transcriptional quiescence in pole cells. Similar nanos functions may also be required in mammals, where two of three nanos homologs are required for fertility. The phenotype of nanos-3 null mice is consistent with specification of primordial germ cells occurring normally, followed by an inability to maintain germ cell identity during migration. This phenotype is strikingly similar to those observed in nanos(-) worms and flies (Schaner, 2003).

nanos function is essential to maintain embryonic germline repression prior to normal activation of proliferation, presumably through its characterized roles in posttranscriptional regulation. Only a few direct targets of nanos regulation in germ cells have been identified, including maternal cyclin mRNAs. Targets that are the effectors of H3meK4 addition have yet to be identified, but conceivably include an H3 lysine 4-specific methyltransferase. The phenotypes observed upon removal of nanos are also only partially penetrant in both organisms. It is thus likely that other conserved, partially redundant systems exist. A clear candidate is germ cell-less (gcl), which is required in Drosophila to maintain transcriptional quiescence. Preliminary experiments suggest that H3meK4 regulation is also disrupted in gcl mutants. A predicted C. elegans gene with substantial homology to gcl has been identified, but its role in these processes has not as yet been assessed (Schaner, 2003).

It appears clear in flies that nos is required to maintain the repressive state of the germline and that part of this involves preventing accumulation of H3meK4. The pole cells begin life lacking this modification, and Nanos function is required to prevent its addition to pole cell chromatin. It is not as clear-cut in worms, since H3meK4 is initially present and then removed at germline restriction. The detection of H3meK4 in Z2/Z3 of nos-depleted embryos could therefore conceivably reflect a role for nos in either (presumably indirectly) promoting the removal of H3meK4 after PIE-1 and/or preventing the re-addition of H3meK4 before hatching. Given the conservation of most other phenotypes caused by the loss of nos activity in multiple organisms, the latter role for C. elegans NOS proteins is favored (Schaner, 2003).

At approximately the 100-cell stage in C. elegans, PIE-1 levels decrease and this is concurrent with a global loss of a distinct subset of histone modifications, H3meK4 and H4acetylK8, in Z2/Z3. The presence of other histone tail modifications in Z2/Z3 indicates that the loss of H3meK4 and H4acetylK8 epitopes are not due to a general cleavage of histone tails, but rather a specific chromatin-remodeling event. While removal of H4acetylK8 is likely to be performed by a histone deacetylase (HDAC), an activity that demethylates lysines has yet to be identified in any organism. The loss of H3meK4 could therefore represent either a replication-independent or a replication-coupled replacement of histone H3, perhaps by a germline-specific H3 variant. A diminishment of H3meK4 in P4 after gastrulation and prior to its entry into mitosis, which could indicate an S phase-related event, is frequently observed (Schaner, 2003).

Nanos downregulates transcription and modulates CTD phosphorylation in the soma of early Drosophila embryos

nanos (nos) specifies posterior development in the Drosophila embryo by repressing the translation of maternal hb mRNA. In addition to this somatic function, nos is required in the germline progenitors, the pole cells, to establish transcriptional quiescence. nos has been shown to be required to keep turned off in the germline of both sexes the Sex-lethal establishment promoter, Sxl-Pe. nos also functions to repress Sxl-Pe activity in the surrounding soma. Sxl-Pe is inappropriately activated in the soma of male embryos from nos mothers, while Sxl-Pe can be repressed in female embryos by ectopic Nos protein. nos appears to play a global role in repressing transcription in the soma since the effects of nos on promoter activity are correlated with changes in the phosphorylation status of the carboxy terminal domain (CTD) repeats of the large RNA polymerase II subunit. Finally, evidence is presented indicating that the suppression of transcription in the soma by Nos protein is important for normal embryonic development (Deshpande, 2005).

During the rapid nuclear division cycles in cleavage stage Drosophila embryos, RNA polymerase II transcription is largely shut down and only a few genes are actively transcribed. RNA polymerase II transcription in somatic nuclei is upregulated soon after they migrate to the periphery of the embryo at stage 9 and by nuclear cycle 10 and 11 many of the key segmentation genes are already actively transcribed. While RNA polymerase II activity is substantially augmented when the nuclei reach the periphery of the embryo in the soma, the opposite occurs in the germline pole cell nuclei. When these nuclei migrate into the posterior pole plasm and pole cells are formed, transcription is shut down rather than activated. Previous studies have implicated the posterior determinants nos and pum in establishing/maintaining transcriptional quiescence in pole cells. In embryos derived from mothers mutant for either nos or pum, RNA polymerase II transcription is not properly downregulated in the pole cells and several genes that are normally active only in somatic nuclei are ectopically expressed (Deshpande, 2005).

Since only nos mRNA localized at the posterior pole is translated, pole cells have the highest levels of Nos. However, translation of the localized message generates a Nos gradient that extends to the center of the embryo. An obvious question is whether this Nos gradient also affects RNA polymerase II activity in somatic nuclei. Indeed, transcription of Sxl-Pe is upregulated in somatic nuclei when Nos protein is removed, and is repressed when Nos protein is ectopically expressed. The role of Nos protein in repressing transcription is not restricted to the sex determination pathway since the activity of other promoters also appears to be increased in the absence of nos function (Deshpande, 2005).

Several mechanisms could potentially explain the ectopic activation of Sxl-Pe in the soma and germline of nos mutant embryos. The most obvious is that this promoter is turned-on by maternal Hb expressed in the absence of nos. However, Sxl-Pe is upregulated in nos embryos even when maternal Hb is eliminated. In addition, ectopic expression of Hb from a transgene lacking NREs seemed to repress rather than activate Sxl-Pe. Another possibility is that the zygotic expression of one or more of the X-linked numerators is elevated in nos embryos, upsetting X chromosome to autosome counting. However, since none of the known numerators has a recognizable NRE in the 3′UTR of its message, it seems unlikely that these genes are direct targets for translational repression by Nos protein. In addition, it is not at all clear why numerator genes (which are transcribed in the zygote) would be subject to translational repression by Nos, while autosomal denominator genes such as deadpan (which turns off Sxl-Pe) would not (Deshpande, 2005).

For this reason, the idea is favored that Sxl-Pe is activated in nosm embryos at least in part because RNA polymerase II activity is upregulated. Support for this idea comes from analysis of CTD phosphorylation. When RNA polymerase is transcriptionally engaged the CTD domain is phosphorylated on serine 2 and 5. In wild-type pole cells, phospho-ser2 cannot be detected, while there is only little phospho-ser5. In contrast, phospho-ser2 is found in nosm pole cells, while the level of phospho-ser5 is increased. nos-dependent alterations in CTD phosphorylation are also evident in the soma. When Nos is absent, the level of ser2 and ser5 CTD phosphorylation is elevated, while both types of CTD phosphorylation are reduced by ectopic Nos protein (Deshpande, 2005).

Additional evidence that nos has a global effect on transcription comes from the finding that nos regulates the methylation of histone H3 in the germline of worms and flies. In both organisms, the methylation of histone H3 on lysine 4 (H3meK4) is upregulated in the soma when zygotic transcription commences in early embryogenesis. In contrast, little or no methylation H3 K4 is observed in the transcriptionally quiescent germline. Inhibition of H3 K4 methylation in germ cells requires nos and H3meK4 is markedly upregulated in nos germ cells. In light of these findings, K4 methylation was examined in the soma of nosm embryos. As might be expected from the effects of nos on CTD phosphorylation, somatic H3meK4 is elevated compared to wild type (Deshpande, 2005).

Since phosphorylation of serines 2 and 5 are correlated with transcription, the nos-dependent alterations in CTD phosphorylation are consistent with the idea that nos has a global impact on RNA polymerase II activity. If this is the case, an important question is whether CTD phosphorylation is the cause or the consequence of nos induced changes in the activity of the transcriptional apparatus. Because actively transcribing RNA polymerase has a hyperphosphorylated CTD domain, any mechanism, which leads to a general increase (or decrease) in transcription, would likely alter the level of CTD phosphorylation. This makes it difficult to distinguish between cause and effect. In contrast, besides being a characteristic feature of elongating polymerase, CTD phosphorylation has been linked to the last steps in the initiation process, promoter clearance and the formation of an elongation competent RNA polymerase complex. Moreover, there is growing evidence that these steps in the transcription cycle are subject to regulation (Deshpande, 2005).

The fact that CTD phosphorylation may be a key control point in the transcriptional cycle raises the possibility that nos exerts its effects on polymerase activity by inhibiting the translation of some factor which promotes CTD phosphorylation. In nos mutants, the level of this factor would increase, leading to a general derepression of transcription. Conversely, the level of this factor would decrease by ectopic Nos, reducing overall transcription (Deshpande, 2005).

While the results clearly show that Sxl-Pe is inappropriately turned on in the soma of male embryos and upregulated in the soma of female embryos in the absence of nos activity, it was initially surprising to find that there is usually not a very pronounced posterior-anterior activation gradient. In fact, the smaller Sxl-Pe0.4 kb promoter is clearly activated not only in the posterior but also in the anterior of nos embryos, while the larger Sxl-Pe3.0, usually shows at most only a very shallow posterior–anterior gradient of β-galactosidase expression. Since the Nos gradient does not extend beyond the midpoint of the embryo, and the repressive effects of Nos on hb mRNA translation are restricted to this posterior domain, one might have expected that the activation of Sxl-Pe in would be tightly restricted to the posterior half of nos mutant embryos. However, proteins of average size would be expected to diffuse (in water or even in cytoplasm) through the volume of a fly embryo over a time scale of minutes, and the establishment of gradients like those seen for Nos or Bcd are likely to require special mechanisms including a localize source of product, as well as the sequestration (e.g. nuclear localization) and degradation of the product. If the Nos target for inhibiting general RNA Pol II activity is translated from a uniformly distributed maternal mRNA and is able to equilibrate through the embryo during the time between the onset of the very rapid nuclear divisions and the formation of the cellular blastoderm, only a shallow gradient of this factor in the soma might be expected at any one time in the presence or absence of Nos. In contrast, in pole cells, where repression of this factor by Nos would presumably be required to impose transcriptional quiescence, the formation of the cell membrane would prevent factor synthesized in the soma from influencing polymerase activity. This would enable Nos in the pole cells to reduce the level of this factor below the threshold required for transcriptional activation (Deshpande, 2005).

As observed in many species, establishing transcriptional quiescence in the newly formed pole cells during early embryogenesis is a critical step in the development of the Drosophila germline. However, it is not immediately obvious what role nos mediated down regulation of polymerase activity would have in the development of the soma. Obviously, hyperactivation of Sxl-Pe in nosm embryos could inappropriately switch on the Sxl autoregulatory feedback loop in males. However, analysis of Sxl accumulation in post-blastoderm stages suggests that only very few nosm 1X/2A embryos actually make the wrong choice in sexual identity. There is also little evidence of a sex-bias in adult progeny of nos hb germline clone mothers. The fact that the Sxl autoregulatory loop is usually not activated in male nosm embryos, which are hemizygous for Sxl, is not altogether surprising. In females that have only a single wild type Sxl gene, activation of the autoregulatory loop is severely compromised by conditions which diminish Sxl-Pe activity. Since the amount of Sxl produced by Sxl-Pe in nosm male embryos is much less than that in wild-type females, the autoregulatory loop should be activated infrequently (Deshpande, 2005).

While nosm males largely escape the effects of activating Sxl-Pe, the increased polymerase activity appears to have other consequences. Previous studies have shown that removal of maternal hb suppresses the posterior defects of nosm embryos. However, it has been shown that only about 40% of the hb+/hb embryos from nos hb mothers survive to adults. Likewise, it was found that only 60% of the embryos produced by hb nos mothers hatch as first instar larva, and that an even lower number survive to the adult stage. Taken together, these experiments argue that nos has important functions in the soma besides blocking translation of maternal hb mRNA. It seems possible that the segmentation/developmental defects evident in progeny of hb nos clone mothers could arise from the upregulation of various patterning genes in the absence of nos activity. It is presumed that in wild-type embryos the activity of the transcriptional apparatus and of target zygote promoters is appropriately adjusted to compensate for the repressive effects exerted by Nos. Because the transcriptional apparatus is hyperactivated in the absence of Nos function, this balance is perturbed and many genes are overexpressed (Deshpande, 2005).

Nos functions indirectly to positively regulates tll and hkb transcription

In Drosophila, the maternal terminal system specifies cell fates at the embryonic poles via the localised stimulation of the Torso receptor tyrosine kinase (RTK). Signalling by the Torso pathway relieves repression mediated by the Capicua and Groucho repressors, allowing the restricted expression of the zygotic terminal gap genes tailless and huckebein. This study reports a novel positive input into tailless and huckebein transcription by maternal posterior group genes, previously implicated in abdomen and pole cell formation. Absence of a subset of posterior group genes, or their overactivation, leads to the spatial reduction or expansion of the tailless and huckebein posterior expression domains, respectively. The terminal and posterior systems converge, and exclusion of Capicua from the termini of posterior group mutants is ineffective, accounting for reduced terminal gap gene expression in these embryos. It is proposed that the terminal and posterior systems function coordinately to alleviate transcriptional silencing by Capicua, and that the posterior system fine-tunes Torso RTK signalling output, ensuring precise spatial domains of tailless and huckebein expression (Cinnamon, 2004).

Terminal gap gene expression must be tightly regulated for the correct specification of terminal cell fates at the nonsegmented poles. Clearly, the Tor pathway plays a key role in driving tll and hkb transcription, given that terminal gap genes are not expressed at the posterior end of terminal group mutants, and as a result terminal structures such as the terminal filzkorper (FK) do not form. In this paper, a novel biological role is unraveled for the maternal posterior system, showing that members of this group, in particular Nos, positively regulate transcription of the zygotic subordinate genes of the terminal system. Torso response elements (TREs) in the tll upstream regulatory region, which are derepressed in cic mutants, also respond to alterations in maternal osk dosage, and the Cic repressor is not excluded from the termini of posterior group mutants. These results are consistent with the posterior system feeding into the Tor signalling pathway, upstream of or at the level of the Cic repressor. It is suggested that the concerted activities of both the terminal and posterior systems, in their spatially overlapping zones of action, generate accurate domains of terminal gap gene expression at the posterior (Cinnamon, 2004).

It was originally proposed that the four maternal systems that pattern the early Drosophila embryo act largely independently of each other. Recent work, however, demonstrated interactions between the Tor pathway and the anterior and D/V systems. For example, tll has been shown to respond to the anterior determinant Bicoid (Bcd) even when Tor signalling is genetically blocked. Indeed, cis-acting DNA elements responsive to these three maternal systems have been found in the tll upstream regulatory region. The current results now link the terminal and posterior systems, previously thought to be independent of each other, in terminal gap gene regulation, reinforcing the idea that maternal systems that pattern the early embryo act in a coordinated manner (Cinnamon, 2004 and references therein).

Why has the positive input, by posterior group genes into terminal patterning, been largely overlooked to date? Classical segmentation studies mostly involved phenotypic analyses at the cuticular level. For this reason, and when taking into account the primary contribution of the terminal system, the delicate input by the posterior group has gone unnoticed. Thus, the unextended FK that develops in posterior group mutant background, which may arise from decreased terminal gap gene expression, had largely been attributed to pleiotropic effects arising from abdominal defects. It has been possible to detect the relatively subtle changes in tll and hkb gene expression patterns only by investigating terminal gap gene regulation at the molecular level. In fact, at least one other molecular study had previously reported reduced terminal gap gene expression in osk mutant embryos (Cinnamon, 2004 and references therein).

One emerging concept is that, for the refinement of the expression levels and spatial extents of RTK signalling targets, it is also imperative to integrate accurately information originating from other, non-RTK sources. In many cases this integration occurs at the level of target gene enhancers, with various effectors of distinct signalling pathways binding to specific DNA elements to regulate transcription. For example, D-Pax2 expression in the cone and pigment cells of the developing eye is regulated by effectors of the EGFR RTK pathway, such as Pointed P2 and Yan, and also by the Notch signalling component Suppressor of Hairless, as well as by the transcription factor Lozenge. The current study shows that terminal gap gene expression requires not only Tor RTK pathway activity but also a contribution from the posterior system. In this instance, inputs from these two maternal coordinate systems are interpreted and linked not at the level of terminal gap gene promoters but at the level of the Cic repressor. Thus, Cic functions as an integrator of multiple regulatory inputs, with both the posterior and terminal systems acting to relieve transcriptional silencing mediated by this repressor (Cinnamon, 2004).

Surprisingly, anterior tll and hkb expression is also reduced in posterior group mutants. Similarly, others have reported prolonged bcd expression and head defects in pum mutants. It is speculated that low levels of Osk and Nos, which escape translational repression, similarly regulate terminal gap gene expression via Cic removal at the anterior. In accordance with this, the dismissal of Cic from the anterior pole of posterior group mutants is also ineffective (Cinnamon, 2004).

How does Nos, which has been assigned the role of a translational repressor, positively regulate tll and hkb transcription? The results suggest that Nos does so indirectly, by downregulating the accumulation of the Cic repressor at the termini. The exact mechanism by which the Tor pathway mediates the exclusion of Cic from terminal regions has not been established, but one model argues that phosphorylation of Cic by MAPK causes degradation of the protein, as in the case of Yan. Thus, Nos could be affecting this process in one of several possible ways, at the level or downstream of MAPK. For example, Nos could be facilitating the translocation of phosphorylated MAPK into the nucleus. In posterior group mutants, then, activated MAPK would remain in the cytoplasm rather than enter the nucleus, impeding Cic phosphorylation and degradation. Alternatively, Nos may be modulating MAPK activity, or regulating adaptor proteins that promote Cic phosphorylation by nuclear MAPK. Nos may also be controlling the translation of factors that are involved in the nuclear trafficking (import/export) or degradation of Cic, or perhaps may even be acting on the cic message itself. Future studies will distinguish between these possibilities, and may shed new light on the molecular mechanisms underlying role of Nos in other developmental processes, for example, the establishment/maintenance of transcriptional quiescence in pole cells. The positive input by the posterior group genes is viewed as evolving to modulate terminal pathway activity, merging with other varied modes of Tor regulation to ultimately ensure accurate tll and hkb expression and, consequently, precise cell fate determination (Cinnamon, 2004).

The Tor signal transduction pathway is under multiple tiers of regulation, outside and inside the nucleus. For instance, internalisation and trafficking of the activated Tor receptor to the lysosome for degradation attenuates the signal, as evident by the spatial broadening and temporal prolonging of Tor activation in mutants for hrs, a component of the endosomal recycling machinery (Lloyd. 2002). Yet another level of control is provided by the tyrosine phosphatase corkscrew, which sharpens the gradient of Tor activity. Additionally, multiple cytoplasmic adaptor proteins take part in transducing the Tor signal, conceivably buffering against surplus or deficiency in signalling (Cinnamon, 2004).

In the nucleus, tll and hkb are subjected to silencing by several repressors. Derepression of tll is observed in grainy-head and tramtrack69 (ttk69) mutants, and the proteins encoded by these genes bind tll promoter sequences. Cic and Gro appear to play a leading role in terminal gap gene silencing, given that mutations in cic and gro bring about a significant expansion of the tll and hkb expression domains. Intriguingly, however, tll expression never reaches the middle of the embryo in these mutants. tll is uniformly expressed, albeit weakly, throughout the embryo only when both the developmental corepressors Gro and CtBP are removed concomitantly. This broadened tll expression likely stems from the fact that there is a redundancy in the activities that normally restrict terminal gap gene transcription from inappropriately spreading into the central portion of the embryo; by jointly removing the Gro and CtBP coregulators, activity of the above repressors is compromised. Alternatively, CtBP might be acting in conjunction with a novel, unidentified repressor that prevents tll transcription in the middlemost region of the embryo (Cinnamon, 2004).

So what is the purpose of the input by the posterior group genes into tll and hkb transcription? Quantitative differences in Tor receptor activity have to be eventually interpreted and translated into distinct cell fates at the termini. Strong Tor activation induces both hkb and tll expression, whereas weaker Tor activation only brings about tll expression. It is surmised that the precision endowed by the Tor RTK cascade may not suffice for the complex patterning of the termini, given that mere two-fold fluctuations in Tor signalling result in defective embryonic development. For example, mutants with reduced Tor RTK activity show partial tll expression and the complete loss of hkb. These mutants consequently develop incomplete terminal structures and die at the larval stage. Conversely, overactivation of the Tor pathway leads to anterior expansion of the posterior tll expression domain, perturbing segmentation in central body parts, likely as a result of downregulation of abdominal gap genes by the Tll protein. Thus, the precise spatial confinement of terminal gap gene expression domains requires the coordinated integration of regulatory inputs, coming from two maternal systems and converging on the same effector protein, Cic (Cinnamon, 2004).

Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline

In the Drosophila embryo, Nanos and Pumilio collaborate to repress the translation of hunchback mRNA in the somatic cytoplasm. Both proteins are also required for repression of maternal Cyclin B mRNA in the germline; it has not been clear whether they act directly on Cyclin B mRNA, and if so, whether regulation in the presumptive somatic and germline cytoplasm proceeds by similar or fundamentally different mechanisms. This report shows that Pumilio and Nanos bind to an element in the 3' UTR to repress Cyclin B mRNA. Regulation of Cyclin B and hunchback differ in two significant respects. (1) Pumilio is dispensable for repression of Cyclin B (but not hunchback) if Nanos is tethered via an exogenous RNA-binding domain. Nanos probably acts, at least in part, by recruiting the CCR4-Pop2-NOT deadenylase complex (see twin), interacting directly with the NOT4 subunit. (2) Although Nanos is the sole spatially limiting factor for regulation of hunchback, regulation of Cyclin B requires another Oskar-dependent factor in addition to Nanos. Ectopic repression of Cyclin B in the presumptive somatic cytoplasm causes lethal nuclear division defects. It is suggestd that a requirement for two spatially restricted factors is a mechanism for ensuring that Cyclin B regulation is strictly limited to the germline (Kadyrova, 2007).

Thus Nos and Pum directly regulate maternal CycB mRNA, binding to an NRE in its 3' UTR. Differences in the spacing and arrangement of protein-binding sites within the hb and CycB NREs appear to account for the regulation of hb but not CycB by Brat. For regulation of CycB, the main function of Pum is to recruit Nos, a role that can be bypassed by tethering Nos via an exogenous RNA-binding domain. CycB-bound Nos is then likely to act, at least in part, by recruiting a deadenylase complex, interacting with its NOT4 subunit. Regulation of CycB is limited to the PGCs to avoid the deleterious consequences of repression in the presumptive somatic cytoplasm. The requirement for both Nos plus at least one additional germline-restricted factor may be part of a mechanism to ensure that CycB regulation is strictly limited to the PGCs (Kadyrova, 2007).

The co-crystal structure of human Pum bound to a fragment of the hb NRE shows that a single Pum RBD directly contacts eight nucleotides of the RNA. However, Puf proteins bind with essentially wild-type affinity to many mutant sites, suggesting that all eight nucleotides are not rigidly specified. How, then, do Puf proteins recognize specific mRNA targets in vivo (Kadyrova, 2007)?

Part of the answer appears to be that, within functional NREs, more than eight nucleotides are recognized, at least by Drosophila Pum. Mutations that simultaneously disrupt Pum binding in vitro and regulation in vivo are spread over 20 nts of the hb NRE and 18 nts of the CycB NRE. These extended Pum mutational 'footprints' are too large to be accounted for by binding of a single RBD; it is suggested that two or more Pum RBDs bind each NRE, an idea supported by the detection of two RNA-protein complexes in gel mobility shift experiments using both the CycB and hb NREs. This model disagrees with earlier experiments that suggested only a single Pum RBD binds to the hb NRE. Further biochemical and structural studies will be required to resolve the issue (Kadyrova, 2007).

The distribution of Pum- and Nos-binding sites within the CycB and hb NREs is different. In the former, the Nos binding site lies 5' to the Pum-binding site(s), whereas in the latter, the Nos-binding site is flanked by nucleotides recognized by Pum. It is assumed that the different arrangement of Nos- and Pum-binding sites is responsible for the assembly of Pum-NRE-Nos complexes with different topographies, such that Brat is recruited to hb but not to CycB. Further definition of each RNP structure will ultimately be required to understand the combinatorial assembly of different repressor complexes on each NRE (Kadyrova, 2007).

In addition to the NRE, Pum also binds with high affinity to at least two other sites in the CycB 3' UTR; however, binding to these sites does not mediate translational repression in the PGCs, perhaps because neither supports recruitment of Nos. These sites may simply bind Pum fortuitously, or they may mediate Nos-independent regulation at other stages of development. Pum has been suggested to destabilize bcd mRNA at the anterior of the embryo in a Nos-independent manner. Another Nos-independent function of Pum is the repression of CycB translation throughout the prospective somatic cytoplasm during the early syncitial nuclear cleavages. These processes might be mediated by elements in Fragments A and F of the 3' UTR, that bind Pum but not Nos (Kadyrova, 2007).

A general framework has been provided for understanding how Puf proteins act to control either the translation or stability of target mRNAs (Goldstrohm, 2006). The yeast Puf protein MPT5 interacts directly with Pop2, one of the catalytically active subunits of a large deadenylase complex. Subsequent deadenylation could either silence the mRNA or cause its degradation, depending on other signals in the transcript or the composition of the deadenylase complex (or both). The Puf-Pop2 interaction is conserved across species (including Drosophila), supporting the idea that the mechanism uncovered for MPT5 might generally be applicable to Puf proteins (Kadyrova, 2007).

In this context, it is surprising that Pum is dispensable if Nanos is tethered to CycB via MS2 CP. It is suggested that yeast Puf proteins both recognize target mRNAs and recruit the deadenylase, but that in the Drosophila germline these functions are partitioned, with Pum primarily responsible for target mRNA recognition and Nos primarily responsible for effector recruitment. This model has the attraction of attributing an important role to Nos, which is essential for Puf-mediated regulation in Drosophila, and probably other metazoans as well. What, then, might be the role of the conserved interaction between Pum and Pop2? One possibility is that it acts cooperatively with Nos to recruit the deadenylase; unlike CycB, other mRNA targets (e.g. hb) might require recruitment by both Nos and Pum to ensure efficient deadenylation. Another possibility is that it plays an essential role for mRNAs regulated by Pum but not Nos (Kadyrova, 2007).

Oscillations in CycB activity underlie normal cell cycle progression. During the early embryonic syncitial nuclear cleavages, degradation in the vicinity of the nuclei is thought to deplete CycB locally. Recent work has shown that Pum can inappropriately repress de novo translation of CycB mRNA during the initial nuclear cleavages if not antagonized by the PNG kinase, resulting in mitotic failure. This early Pum-dependent repression is thought to be Nos-independent, as it occurs efficiently in the anterior, where Nos activity is undetectable (Kadyrova, 2007).

The results show that if CycB is inappropriately subjected to Pum+Nos-dependent repression via the hb NRE, CycB is locally depleted, resulting in mitotic failure during nuclear division cycles 10-13. Since it is thought to be the case during the early cycles (1-7), de novo synthesis of CycB apparently is required to counteract the local degradation that probably occurs during M phase of each cycle. The CycB NRE must therefore be precisely tuned to repress translation only in the PGCs and not in the presumptive somatic cytoplasm (Kadyrova, 2007).

Osk is the limiting factor for assembly of pole plasm in the embryo; the results suggest that it stimulates the accumulation or activity of at least one factor in addition to Nos that is required for repression of CycB in the PGCs. The existence of a co-factor is inferred from the finding that ectopic Nos can repress CycB in the somatic cytoplasm only in the presence of ectopic Osk. Regulation of CycB may depend on more than one germline-restricted factor to ensure that potentially deleterious repression does not occur in the somatic cytoplasm (Kadyrova, 2007).

A germline Nos co-factor might act in a variety of ways. It could bind to the CycB NRE adjacent to Pum and Nos, substituting functionally for Brat, which is recruited to the Pum-hb NRE-Nos complex. The 50 nt CycB NRE is inactivated by a truncation at both ends that leaves the Pum- and Nos-binding sites intact, consistent with the idea that another factor binds to the element. Another possibility is that the co-factor is a germline-specific component of the adenylation/deadenylation machinery, as is the case for the GLD-2 cytoplasmic poly(A)-polymerase in C. elegans. Distinguishing among these ideas awaits identification of the cofactor (Kadyrova, 2007).

Spatial regulation of nanos is required for its function in dendrite morphogenesis

Spatial control of mRNA translation can generate cellular asymmetries and functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis. Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation. Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole. Abolishment of nos localization prevents abdominal development, whereas translational derepression of unlocalized nos mRNA suppresses head/thorax development, emphasizing the importance of spatial regulation of nos mRNA. Loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons, suggesting that nos also might be regulated in these larval sensory neurons. This study shows that localization and translational control of nos mRNA are essential for da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons but suggests a different transport mechanism. Targeting of nos mRNA to the processes of da neurons may reflect a local requirement for Nos protein in dendritic translational control (Brechbiel, 2008).

Translational activation of nos at the posterior pole is tightly coupled to translational repression of unlocalized nos mRNA to prevent accumulation of Nos in the anterior of the embryo, where Nos suppresses anterior development. Because nos localization during oogenesis is inefficient, this linkage is essential to silence nos mRNA that remains distributed throughout the bulk cytoplasm. Translational repression of nos mRNA is mediated by a structural motif, the translational control element (TCE), within the nos 3'UTR. TCE function requires the formation of two stem loops, designated as II and III, that have temporally distinct activities. Whereas stem-loop III mediates repression of nos during oogenesis, through its interaction with Glorund (Glo), stem loop II is responsible for repression of nos in the early embryo, through its interaction with a different repressor, Smaug (Smg) (Brechbiel, 2008).

Replacement of the nos 3'UTR by α-tubulin 3'UTR sequences (nos-tub3'UTR) abolishes nos localization and translational repression, leading to unrestricted synthesis of Nos and defects in anterior development. GAL4 mediated overexpression of a UAS-nos-tub3'UTR transgene in class IV da neurons also is deleterious, causing decreased branching complexity. This overexpression phenotype is ameliorated by reinsertion of the nos TCE. The observation that both loss and overexpression of nos cause similar defects indicates that although nos is required for dendrite morphogenesis, the level of Nos protein must be carefully modulated in da neurons. Moreover, the ability of the TCE to suppress the toxicity of nos mRNA overexpression in da neurons suggests that it may normally function to control Nos levels in the PNS. Attempts were therefore made to determine whether endogenous nos is regulated by the TCE in da neurons (Brechbiel, 2008).

Ectopic expression studies have identified several additional somatic cell types in which the TCE can repress translation, including neuroendocrine cells and the dorsal pouch epithelium. However, TCE function in the dorsal pouch does not depend on the Glo or Smg binding sites but requires a distinct sequence motif with homology to the Bearded (Brd) box. Mutation of the Brd box-like motif does not abrogate the ability of the TCE to suppress excess nos activity in da neurons. Consequently, to determine whether endogenous nos mRNA might be regulated by the TCE, da neurons were analyzed in glo and smg mutant larvae (Brechbiel, 2008).

Larvae mutant for glo or smg survive until third instar stage, permitting examination of the effect of eliminating either repressor on dendrite morphology of da neurons. Compared to wild-type class IV da neurons, glo mutant larvae show a significant decrease in the number of higher order dendritic branches as reflected by a decreased number of terminal dendritic processes. Because glo mutant larvae exhibit additional defects, glo function was disrupted specifically in class IV da neurons either by using GAL4477 to express a UAS-gloRNAi transgene or by using the MARCM method to generate mosaic animals. In both cases glo mutant da neurons show decreased branching complexity. Mutation of smg or GAL4477-mediated overexpression of a UAS-smg transgene also causes loss of high-order branches. Larvae doubly mutant for glo and smg do not show a more severe phenotype than larvae mutant for either gene alone, suggesting that each repressor contributes independently. Thus, defects due to loss or overexpression of the repressors are consistent with defects caused by loss or overexpression of nos. Due to the inadequacy of anti-Nos antibodies, changes in Nos protein levels could not be monitored in glo and smg mutant da neurons. However, when combined with the analysis of Glo and Smg binding site mutations, these results strongly support a role for glo and smg in regulation of nos for dendrite morphogenesis (Brechbiel, 2008).

In the oocyte, Glo binds specifically to the distal double-stranded helix of TCE stem-loop III (the Glo Recognition Helix or GRH. In the embryo Smg interacts with nos TCE stem loop II via nucleotides within the loop designated as the Smg Recognition Element (SRE). A second SRE located downstream of the TCE in the nos 3'UTR appears to act redundantly. To determine whether the defects observed in glo and smg mutant da neurons are due to loss of TCE-mediated repression, whether mutation of the nos GRH or SREs produces a similar phenotype was tested. Mutations that disrupt both SREs (SREs-), the binding site for Glo (GRH-), or the SREs and GRH (SREs-GRH-) together were introduced into the gnos transgene. The resulting gnosSREs-, gnosGRH-, and gnosSREs-GRH- transgenes all produce mRNAs that show wild-type localization in the early embryo but whose translation is not restricted to the posterior pole. When compared to larvae expressing the wild-type gnos transgene, branching complexity is significantly reduced in da neurons of larvae expressing gnosSREs-, gnosGRH-, and gnosSREs-GRH- transgenes. Moreover, each of these transgenes behaves similarly to the gnos-tub3'UTR transgene, which lacks the entire nos 3'UTR, indicating that mutation of the GRH and/or SREs is sufficient to disrupt nos regulation in the PNS. Together, these results show that TCE-mediated regulation of nos in da neurons is essential for dendrite morphogenesis. Furthermore, the finding that the same phenotype is produced by either eliminating the repressors or mutating their binding sites provides strong evidence that this regulation is mediated by Glo and Smg (Brechbiel, 2008).

In many cell types protein synthesis is spatially regulated through the transport of translationally silent mRNAs and activation of these mRNAs at the target destination. Linkage of translation and localization serves not only to prevent premature accumulation of nos during transit to the oocyte posterior but also to silence the large pool of nos that remains unlocalized due to inefficient posterior localization. It cannot yet be distinguish whether localization of nos in da neurons is similarly inefficient or whether translational repression of nos serves primarily to repress translation during transport. However, the deleterious effect on dendrite morphogenesis caused by mutations that disrupt TCE function show that, as for maternally synthesized nos mRNA, localization alone is not sufficient to modulate its activity (Brechbiel, 2008).

It is concluded that nos plays an important role in dendrite morphogenesis, and this study shows that nos function in da neurons requires spatial regulation of nos mRNA. Cis-acting sequences and two cognate factors that control nos mRNA localization and/or translation in the oocyte and early embryo are redeployed during larval stages to regulate localization and translation of nos in da neurons. Localization of nos mRNA to the processes of class IV da neurons is essential for dendritic branching. Movement of RNA particles in neurons of intact animals was shown to take place, and analysis of nos mRNA particle movement suggests that nos localization occurs by different mechanisms depending on cellular context. Taken together, these results support a role for Nos as a local regulator of translation in the PNS (Brechbiel, 2008).

In the early embryo Nos functions in a complex with the RNA-binding protein Pumilio (Pum) to repress hunchback mRNA translation, thereby promoting abdominal development. Whereas Pum is produced throughout the embryo, restriction of Nos synthesis to the posterior limits the spatial domain of the repressor complex. Mutations in nos and pum produce similar defects in dendrite morphogenesis, suggesting that Nos and Pum also act together to repress translation in da neurons. Thus, spatial regulation of nos may serve a similar function in the PNS as it does in the early embryo, by restricting the activity of the Nos/Pum repressor complex to dendrites (Brechbiel, 2008).

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).

Protein Interactions

Nanos interaction with Cup

Nanos (Nos) is a translational regulator that governs abdominal segmentation of the Drosophila embryo in collaboration with Pumilio (Pum). In the embryo, the mode of Nos and Pum action is clear: they form a ternary complex with critical sequences in the 3'UTR of Hunchback mRNA to regulate its translation. Nos also regulates germ cell development and survival in the ovary. While this aspect of its biological activity appears to be evolutionarily conserved, the mode of Nos action in this process is not yet well understood. In this report it is shown that Nos interacts with Cup, which is required for normal development of the ovarian germline cells. nos and cup also interact genetically: reducing the level of cup activity specifically suppresses the oogenesis defects associated with the nosRC allele. This allele encodes a very low level of mRNA and protein that, evidently, is just below the threshold for normal ovarian Nos function. Taken together, these findings are consistent with the idea that Nos and Cup interact to promote normal development of the ovarian germline. They further suggest that Nos and Pum are likely to collaborate during oogenesis, as they do during embryogenesis (Verrotti, 2000).

To identify proteins that interact with Nos, a yeast two-hybrid screen was performed using full-length Nos fused to the GAL4 DNA-binding domain as the bait. Two of the interactors proved to be fragments of Cup, a novel cytoplasmic protein of unknown biochemical function that is required for normal oogenesis (Keyes, 1997). In the ovaries of cup mutant females, egg chamber maturation arrests between stages 5 and 14, and the nurse cells have aberrant nuclear morphology. However, all of the extant cup alleles encode detectable protein, and thus the null phenotype may be stronger. The interaction with Nos appears to be specific, since Cup fails to interact with a variety of other baits in yeast. In particular, Cup does not interact with the RNA-binding domain of Pum (Verrotti, 2000).

To test the interaction between Nos and Cup in vitro, the minimum region of Cup required for interaction in yeast was defined by deletion analysis. Residues 593-963 constituted the smallest fragment of Cup tested that interacted with Nos in yeast. A GST fusion protein bearing these Cup residues was prepared in bacteria and was incubated with embryonic extracts from either wild-type or transgenic flies that produced a Myc epitope-tagged Nos that was fully functional and rescued the defects in otherwise nos- embryos and ovaries. Approximately 10% of the Nos-Myc from the extract is retained by GST-Cup under the reaction conditions, whereas a negligible amount of Nos-Myc is retained in a control reaction with GST. In summary, Nos appears to interact specifically with Cup in yeast and in vitro (Verrotti, 2000).

Which portion of Nos mediates the interaction with Cup? Nos contains two regions -- a well-conserved C-terminal Zn2+-binding domain that mediates the interaction with Pum and HB mRNA, and a poorly conserved N-terminal region. The N-terminal region of Nos mediates interaction with Cup. No biochemical function has previously been ascribed to this portion of Nos, which is very poorly conserved even among closely related Dipteran homologs. Further deletion analysis of the N-terminal region reveals that it contains at least two redundant sub-domains that can interact with Cup (Verrotti, 2000).

The function of the N-terminal region of Nos in vivo is not clear. A recent analysis of 60 nos- alleles reveals that mis-sense mutations that eliminate both ovarian and embryonic function alter residues in the conserved C-terminal domain, consistent with the idea that this domain is required for function in both tissues (Arrizabalaga, 1999). In contrast, no such mis-sense mutations are found in the coding region for the poorly conserved N-terminal domain. Microinjection of mRNAs encoding deletion derivatives of Nos into nos- embryos suggests that no single part of the N-terminal region is essential for regulation of HB mRNA. No comparable analysis of Nos residues required for ovarian function has been reported (Verrotti, 2000).

To identify residues of Nos essential for its activity in the ovary, nos transgenes were prepared that encode deletion derivatives. No part of the N-terminal region of Nos is essential for Nos to function in either the embryo or the ovary. Maternal expression from a single transgene encoding each deletion derivative rescues the ovarian morphology and egg-laying defects associated with nosRC. Each deletion derivative also rescues the abdominal segmentation defects associated with nosBN either completely (a full complement of 8 abdominal segments) or nearly completely (6-8 abdominal segments). One derivative, deltaG, appears to have somewhat less activity than the others; however, expression from two maternal copies of the deltaG transgene completely rescues abdominal segmentation in 100% of embryos. Thus, it is concluded that the N-terminal region of Nos contains no unique sequence that is essential for its activity in either the embryo or the ovary. This latter observation is consistent with the finding that interaction with Cup is mediated by redundant elements in the N-terminal region of Nos (Verrotti, 2000).

To determine whether the interaction between Nos and Cup is functionally significant, it was asked whether lowering the level of Cup modified any of the ovarian or embryonic phenotypes associated with altered Nos function. In one case, a strong genetic interaction was observed: introduction of a single cup allele substantially suppresses the oogenesis defects in hemizygous nosRC mutant females. Cystoblasts that give rise to the germline components of the egg chamber do not develop normally in nosRC mutant ovaries, and germline stem cells that give rise to cystoblasts are not maintained. As a result, nosRC mutant ovaries contain only rare mature egg chambers. In contrast, in cup-/+; nosRC/Df(nos) ovaries, many of the egg chambers appear normal and mature into oocytes that are fertilized and oviposited. (The resulting embryos develop no abdominal segments, presumably because they lack sufficient Nos activity to repress HB translation.) The cup-/+; nosRC /Df(nos) females lay eggs for at least 3 weeks, suggesting that germline stem cells are maintained and function normally. Thus, reducing the level of Cup appears to specifically suppress the oogenesis defects associated with the nosRC allele, but not the embryonic defects. Nine different cup alleles tested suppress the defects associated with nosRC, suggesting that it is simply a reduction of Cup activity that suppresses the oogenesis phenotype. In contrast, the genetic interaction appears to be specific to the RC allele; the nosRD mutant encodes an unstable protein bearing a substitution at one of the conserved Cys residues in the C-terminal domain. This allele exhibits oogenesis defects similar to nosRC, but these defects are not ameliorated by lowering the level of Cup, presumably because the level of active Nos protein is insufficient. In addition, reducing the level of Cup has no effect on the oogenesis defects associated with two different allelic combinations of pum. Thus, reduction of Cup activity does not appear to globally suppress oogenesis defects resulting from alterations in Nos or Pum activity, but specifically suppresses the defects associated with nosRC (Verrotti, 2000).

The genetic interaction between nos and cup suggests that expression of the protein encoded by each gene coincides, and previous reports support this idea (Keyes, 1997). However, it was of interest to visualize the distributions of Nos and Cup simultaneously to determine whether the spatiotemporal distribution of the proteins is consistent with the observed genetic interaction. Using anti-Nos antibodies, Nos protein could not be reliably detected in the germarium. Therefore, the localization of Cup and Myc-tagged Nos was examined in the transgenic flies that carry a fully functional nos+ transgene altered to encode a Myc epitope tag at the C terminus of the protein. Cup is present throughout the cytoplasm of all the germ cells in the germarium, the terminal region of the ovary that contains the stem cells, cystoblasts and most immature egg chambers. In contrast, the Nos-Myc distribution is not uniform. It is present in the germline stem cells and cystoblasts in region 1 of the germarium, falls beneath the level of detection during the early cystoblast cleavages in regions 1 and 2, rises to relatively high levels in the germline cysts in region 2, and falls to somewhat lower levels in the maturing cysts of region 3. The significant finding is that Nos and Cup co-localize to the cytoplasm of the stem cells, the cystoblasts and the cysts, consistent with the genetic interaction described above (Verrotti, 2000).

The data reported above support the idea that Cup interacts with the N-terminal region of Nos and thereby lowers its activity, perhaps titrating it away from regulatory targets. This follows from the observed physical interaction and the genetic interaction between nosRC and cup. However, nosRC, which bears a mutation in the splice donor of intron 1 in the pre-mRNA, has been described as a null allele, and no mature mRNA is detectable by either in situ hybridization or Northern blot. How then can a physical interaction between Cup and Nos account for the genetic interaction between cup and nosRC? To address this question, it was asked whether nosRC actually encodes a very low level of functional protein. Semi-quantitative RT-PCR was used to determine whether nosRC flies contain low levels of mRNA. Using primers that flank intron 1, a low level of NOS mRNA was detected in extracts prepared from whole flies. Since the major site of transcription in adult females is the ovary, it is assumed that most of this mRNA is derived from the rudimentary nosRC ovaries. Two major cDNA species were detected by ethidium bromide staining of the PCR product following electrophoresis. To further characterize these cDNAs, the PCR products were subcloned and six individual clones were sequenced. The six clones appear to represent mRNAs generated by processing from cryptic splice sites; the open reading frame is preserved in three different clones, and one of these encodes a Nos derivative that is four amino acids larger than wild type. This cDNA clone plausibly represents the mRNA species that gives rise to the nosRC-encoded protein (Verrotti, 2000).

In an attempt to detect protein encoded by nosRC directly, transgenic flies were prepared bearing a nosRC-myc gene that encodes an epitope-tagged protein that is otherwise identical to the nos+-myc gene described above. Using Western blots, a very low level of nearly full-length protein was detectable in nosRC-myc ovaries from five different transgenic lines. Comparison with dilutions of extracts prepared from nos+-myc transgenic ovaries suggests that the level of protein encoded by nosRC is in the order of 1%-2% of wild type. By crossing the nos+-myc and nosRC-myc transgenes into cup-/+ backgrounds and comparing the level of Nos protein in ovarian extracts, it was found that reducing the level of Cup does not significantly affect the level of protein encoded by either transgene. Thus, low levels of Cup do not appear to suppress the nosRC ovarian phenotype by stabilizing Nos. It is concluded that, in the presence of reduced levels of Cup, 1%-2% of the wild-type level of Nos is sufficient to promote normal maintenance of the germline stem cells and differentiation of the cysts (Verrotti, 2000).

It is concluded that reducing the level of functional Cup suppresses the oogenesis defects in hemizygous nosRC ovaries. This finding suggests that Cup acts to inhibit the residual protein encoded by nosRC and prevent it from acting on potential regulatory targets. The identities of such targets are not currently known. In addition, the sequence of Cup sheds no light on its function. A search of the current database reveals no significant homologies to proteins of known function, neither does it possess recognizable motifs using programs such as Prosite, although Keyes (1997) has suggested that Cup may be a microtubule-associated protein. A fragment of human sequence bears high homology to a part of the fly protein, and thus it seems likely that one or more of the Cup functions are evolutionarily conserved. Further analysis of the significance of the Nos-Cup interaction awaits definition of the biochemical activities of Cup and the identification of Nos-regulated genes in the ovary (Verrotti, 2000).

While physiological levels of Cup are capable of inhibiting the low level of Nos activity in hemizygous nosRC flies, it is not understood what role the Cup-Nos interaction plays in the ovaries of wild-type flies. Over-expression of Nos is deleterious in many different tissues -- the embryo, the eye imaginal disc and the male germline -- suggesting that Nos is a potent regulator of gene expression. Consistent with this idea, it has been found that extremely low levels of Nos, in the order of a few percent of the wild-type amount, suffice for biological activity in the ovary. Thus, it seems possible that the interaction with Cup helps restrict Nos activity, which otherwise might interfere with normal ovarian development. Alternatively, Nos and Cup may act together to govern some aspect of germ cell development. Cup appears to play a role in early germline development, since cup and ovarian tumor interact genetically in the ovary, leading to over-proliferation of germline cells (Keyes, 1997; Verrotti, 2000).

Nanos cooperates with Pumilio to regulate Hunchback mRNA

The Drosophila Nanos protein is a localized repressor of Hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. One equivalent of zinc is bound with high affinity by each of the CCHC motifs. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. Full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the Hunchback mRNA target sites for Nanos-dependent translational repression are found to disrupt translational repression in vivo, but have little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target Hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex (Curtis, 1997).

Translational regulation of Hunchback mRNA is essential for posterior patterning of the Drosophila embryo. This regulation is mediated by sequences in the 3'-untranslated region of HB mRNA (the Nanos response elements or NREs), as well as two trans-acting factors -- Nanos and Pumilio. Pum binds to a pair of 32-nucleotide sequences (named Nanos response elements -- NREs) in the 3'-UTR of maternal HB mRNA in order to repress HB translation in the posterior of the embryo. This translational repression is essential for normal abdominal segmentation. The RNA-binding domain of Pum is structurally similar to that of another translational regulator, FBF (fem-3 mRNA-binding factor) found in C. elegans (Zhang, 1997). The minimal RNA-binding domain of each protein consists of eight imperfect repeats plus flanking residues. These structural similarities define a conserved 'Puf' motif (Pum and FBF) that is found in proteins from diverse organisms from yeast to humans. However, the RNA partner of no other Puf domain protein has been identified, nor is it clear whether other Puf proteins regulate translation or some other aspect of RNA metabolism. Thus, Pumilio recognizes the NREs via a conserved binding motif. The mechanism of Nanos action has not been clear. In this report protein-protein and protein-RNA interaction assays in yeast and in vitro were used to show that Nanos forms a ternary complex with the RNA-binding domain of Pumilio and the NRE. Mutant forms of the NRE, Nos, and Pum that do not regulate HB mRNA normally in embryos do not assemble normally into a ternary complex. In particular, recruitment of Nos is dependent on bases in the center of the NRE, on the carboxy-terminal Cys/His domain of Nos, and on residues in the eighth repeat of the Pum RNA-binding domain. These residues differ in a closely related human protein that also binds to the NRE but cannot recruit Drosophila Nos. Taken together, these findings suggest models for how Nos and Pum collaboratively target HB mRNA. More generally, they suggest that Pum-like proteins from other species may also act by recruiting cofactors to regulate translation (Sonoda, 1999).

In one model, Nos simultaneously makes specific contacts with Pum and nucleotides 17-20 of the NRE. On their own, neither the Nos-Pum nor the Nos-NRE contacts are strong enough to recruit Nos to hb mRNA (at least in the presence of competitor proteins and RNAs), because binary complexes with Nos are not detectable. In another model, unbound Pum cannot interact with Nos, but binding to the NRE induces a conformational change in Pum, which subsequently recruits Nos via protein-protein contacts. In this model, nucleotides 17-20 of the NRE interact with Pum to induce the conformational change without affecting its affinity for the RNA, and nonspecific interactions between Nos and other portions of the RNA help stabilize the complex. Either model is consistent with the nonspecific RNA-binding activity reported for the carboxy-terminal portion of Nos in vitro and the RNA-Nos cross-link found in this study. Further structural and biochemical experiments will be required to distinguish between these (or alternative) models (Sonoda, 1999 and references therein).

The mechanism by which the ternary complex blocks translation is not yet clear. mRNAs subject to Nos- and Pum-dependent repression are deadenylated in vivo. In addition, Nos and Pum have been shown to regulate internal ribosome entry site (IRES)-dependent translation in imaginal disc cells, suggesting that their regulatory target lies downstream of cap recognition and scanning. It is assumed that some surface of the ternary complex, formed jointly by Nos and Pum, targets a component of the polyadenylation or translation machinery. This surface appears to be altered in the Pum680 mutant protein, which binds the NRE normally but is defective in regulating HB translation in the embryo. The Pum680 mutant recruits Nos into a ternary complex normally and thus apparently is defective in a subsequent step of the repression reaction. The RNA-binding domain of Pum therefore appears to have at least three different functions in regulating HB: recognizing the NRE, recruiting Nos, and acting as a corepressor (with Nos) to block translation (Sonoda, 1999 and references therein).

In the experiments reported in this study, focus was placed on discrete regions of both Nos (the carboxy-terminal 97 amino acids) and Pum (the minimal RNA-binding domain), which play an essential role in formation of the ternary complex. However, other regions of Nos are known to be required for its function in repressing translation in the embryo. In addition, residues elsewhere in Pum play an unknown role in augmenting the intrinsic translational repression activity of the RNA-binding domain. Thus, the ternary complex formed by the 157-kD, full-length Pum protein may be stabilized by auxillary protein-protein or protein-RNA interactions in addition to those that mediate recruitment of the carboxy-terminal domain of Nos by the RNA-binding (or Puf domain) of Pum. The results suggest that Puf domain proteins generally may act by recruiting cofactors to specific RNA binding sites. Cofactor specificity may be mediated, at least in part, by the eighth repeat of the Puf domain. Although Puf domain proteins have been described in organisms from yeast to humans, for only one protein other than Drosophila Pum, C. elegans FBF, is the relevant RNA regulatory target known. FBF regulates the sperm/oocyte switch in the hermaphrodite germ line by governing the translation of fem-3 mRNA (Zhang, 1997). The FBF RNA-binding domain interacts with one of the C. elegans Nos homologs (Kraemer, 1999). Further experiments will be required to determine whether the Pum/fly Nos complex and the FBF/worm Nos complex function in a similar manner (Sonoda, 1999 and references therein).

Translation regulation plays an essential role in the differentiation and development of animal cells. One well-studied case is the control of Hunchback mRNA during early Drosophila embryogenesis by the trans-acting factors Pumilio, Nanos, and Brain Tumor. This study reports a crystal structure of the critical region of Pumilio, the Puf domain, that organizes a multivalent repression complex on the 3' untranslated region of Hunchback mRNA. The similarity between Pum RBD and that of another translation regulator FBF, which binds to the 3'UTR of fem-3 mRNA in C. elegans, defines a Puf (Pum and FBF) domain, which is conserved in organisms as diverse as plants, yeast, and humans. The Puf domain is characterized by eight imperfect repeats of ~36 amino acids (Puf repeats), followed by a C-terminal extension. All eight repeats appear to be required for proper folding of the Puf domain, since limited proteolysis fails to yield stable smaller fragments. The Puf domain is thus amongst the largest sequence-specific RNA binding motifs to be discovered; the RRM, the KH domain (70 residues), and the dsRBD (65 residues), are much smaller. The PUF domain structure reveals an extended, rainbow shaped molecule, with tandem helical repeats that bear unexpected resemblance to the armadillo repeats in beta-catenin and the HEAT repeats in protein phosphatase 2A. Based on the structure and genetic experiments, putative interaction surfaces for Hunchback mRNA and the cofactors Nanos and Brain Tumor are identified. This analysis suggests that similar features in helical repeat proteins are used to bind extended peptides and RNA (Edwards, 2001).

Two lines of evidence from mutagenesis studies support the idea that the Pum concave surface binds RNA. (1) A gene encoding the 322 residue minimal Pum RBD was randomly mutagenized and variants were isolated that bind normally to the wild-type NRE in yeast. Collectively, these variants bear substitutions at 61 residues, 55 of which map to the structure; the remaining six are in the putative 9th repeat, not in this crystal structure. Of these, only 3 (presumably silent) substitutions fall on the solvent exposed concave surface, with the remaining 52 lying elsewhere. The relative paucity of substitutions within the inner surface is consistent with this being the area that contacts the RNA. (2) Based on the structure, single substitutions were introduced in solvent-exposed residues along the inner surface in five of the eight Puf domains and RNA binding activity was tested in yeast. Each of these mutants is inactive. Thus, the concentration of positive charge and the distribution of both silent and inactivating substitutions together suggest that the RNA interacts with the inner concave surface (Edwards, 2001).

It is proposed that HB mRNA binds to this inner surface in an extended single-stranded conformation. Algorithms that predict RNA structure suggest the NRE does not adopt a stable secondary or tertiary structure. The minimal NRE for high affinity Pum binding consists of nucleotides 3-27, which bracket specific contacts with nucleotides 9, 11-13, and 21-24. The length of this minimal NRE, in an extended single-stranded conformation (112 Å), agrees approximately with the contour length (90 Å) of the concave surface of the Puf domain. It is noteworthy that beta-catenin also has the highest concentration of positive charge within its concave surface (or groove), which is the proposed binding site for segments of cadherins, APC, and members of the LEF-1/TCF family of transcription factors. A recent crystal structure of a beta-catenin/TCF complex shows the TCF segment tethered along the positively charged groove. In the case of karyopherin-alpha, the concave surface is the binding site for the NLS peptide. Taken together, the binding of ligands to concave surfaces is a recurring theme in helical repeat proteins. The Pum Puf domain shows that this type of extended surface can be used to bind RNA, as well as peptides (Edwards, 2001).

Repression of HB mRNA depends not only on Pum, but also on the recruitment of Nanos and Brat to form a quaternary complex. Previous work suggested that Nos is recruited via residues in Puf repeat 8. These residues map to the extra long loop between helices H1 and H2 in repeat 8, that is the main protrusion from an otherwise relatively smooth outer Pum surface. Two different insertions into this loop have no effect on Pum-RNA binding but eliminate recruitment of Nos. To further define the Nos interaction surface, the collection of Pum mutants that bind normally to RNA was tested for Nos recruitment in yeast. Of the 61 substitutions distributed throughout the domain, only two abrogate interaction with Nos. One is a substitution in the putative ninth Puf repeat that is not represented in this structure, while the other changes the solvent exposed phenylalanine on the H1/H2 loop to a serine (F1367S). Thus, the Pum surface that interacts with Nos appears to be limited to a small region that includes the eighth repeat and the C-terminal tail. If this tail indeed does fold into a ninth Puf repeat, then the Pum-Nos interface would span a length of ~15-20 Å on the outer convex surface. It is tempting to think that the C-terminal tail may only fold when Pum binds to the RNA, thereby explaining why Nos is only recruited to the Pum/NRE binary complex and not to Pum alone. The insertions into the long flexible loop in repeat 8 may modify its conformation such that F1367 is no longer exposed for interaction with Nos. The proposed Phe-Nos interaction is reminiscent of the way in which a solvent exposed phenylalanine on the receptor CD4 interacts with the HIV gp120 glycoprotein (Edwards, 2001).

Development of the Drosophila abdomen requires repression of maternal Hunchback (HB) mRNA translation in the posterior of the embryo. This regulation involves at least four components: nanos response elements within the HB 3' untranslated region and the activities of Pumilio (Pum), Nanos (Nos), and Brain tumor. To study this regulation, an RNA injection assay was developed that faithfully recapitulates the regulation of the endogenous HB message. Previous studies have suggested that Nos and Pum can regulate translation by directing poly(A) removal. RNAs that lack a poly(A) tail and cannot be polyadenylated and RNAs that contain translational activating sequences in place of the poly(A) tail are still repressed in the posterior. These data demonstrate that the poly(A) tail is not required for regulation and suggest that Nos and Pum can regulate HB translation by two mechanisms: removal of the poly(A) tail and a poly(A)-independent pathway that directly affects translation (Chagnovich, 2001).

To determine whether injected RNAs require the same factors for regulation as the endogenous maternal HB transcript, the effect of nos, pum, and NRE mutations on the translation of the HFH mRNAs was studied. The test mRNA (HFH) contains the maternal HB 5' UTR, the F-Luc coding region, and the maternal HB 3' UTR. HFH transcripts injected into nos or pum mutant embryos are translated equally well in the anterior versus the posterior. This finding is consistent with the requirement for Nos and Pum in regulating HB translation in vivo. Further, HFH transcripts containing mutant NREs in which the six guanosines have been changed to uracil (GU) show no significant difference in translation between the anterior and posterior of the embryo, regardless of the genetic background. This NRE mutation disrupts Pum binding to the NRE in vitro and eliminates translational repression of maternal HB mRNA in vivo. Thus factors that are required for the regulation of the endogenous maternal HB transcript also are required for the regulation of the injected transcripts (Chagnovich, 2001).

HFH reporter mRNAs were synthesized that contained the Drosophila histone H1 3' terminal stem loop (HSL) in place of the poly(A) tail (HFH HSL). To determine whether the NRE represses translation of HSL-containing mRNAs by directing removal of the HSL, just as it directs removal of the poly(A) tail, radiolabeled, m7GpppG-capped HB 3' UTRs containing either a poly(A) tail or the HSL were injected into WT embryos. Consistent with previous findings, RNAs containing a poly(A) tail maintain the poly(A) tail in the anterior, but the poly(A) tail is rapidly removed in the posterior. However, when RNAs containing the HSL are injected, the HSL is maintained in the anterior and posterior of the embryo, demonstrating that the NRE complex is not directing removal of HSL. Next tested was whether the differences in translation of the HSL-containing reporters were caused by differential stability of the mRNAs. In these experiments, the levels of HSL or poly(A)+ reporter RNAs compared with a poly(A)+ control RNA containing a mutant NRE were examined. The relative levels of the reporter mRNAs are not significantly altered in the anterior versus the posterior for either the polyadenylated or the HSL-containing mRNAs. Together these data demonstrate that translational regulation of HB does not require removal of the poly(A) tail. It is concluded that NRE-directed repression can be independent of the poly(A) tail (Chagnovich, 2001).

Nanos cooperates with Pumilio to regulate Bicoid mRNA

Bicoid is a key determinant of anterior Drosophila development. The prototypical Puf protein Pumilio temporally regulates bicoid (bcd) mRNA translation via evolutionarily conserved Nanos response elements (NRE) in its 3'UTR. Disruption of Pumilio-bcd mRNA interaction by either Pumilio or bcd NRE mutations causes delayed bcd mRNA deadenylation and stabilization, resulting in protracted Bicoid protein expression during embryogenesis. Phenotypically, embryos from transgenic mothers that harbor bcd NRE mutations exhibit dominant anterior patterning defects and similar head defects have been discovered in embryos from pum minus mothers. Hence, Pumilio is required for normal anterior development. Since bcd mRNA resides outside the posterior gradient of Nanos, the canonical partner of Pumilio, the data suggest that Pumilio can recruit different partners to specifically regulate distinct mRNAs (Gamberi, 2002).

To identify sequences regulating bcd mRNA expression, focus was placed on the perfect bipartite NRE sequence GUUGU-N5-AUUGUA (A box-N5-B >box) in the 3'UTR of bcd, starting 50 nucleotides downstream of the bcd translational stop codon. This bcd motif was noticed previously, but its role in normal development was unclear because it resides outside the Nanos embryonic domain. The hb 3'UTR contains two NRE motifs, while bcd has one NRE and an additional B box at position +79 (termed 1 1/2 NREs). By aligning the bcd and hb 3'UTRs from all available species, it was found that the bcd motifs are closer to the second hb NRE. Moreover, the 1 1/2 NREs was absolutely conserved in the bcd 3'UTR of eight fly species that diverged more than 60 million years ago, underscoring functional constraint. Thus, the role the NREs play in bcd expression and their contribution to normal embryonic development were analyzed (Gamberi, 2002).

Pumilio temporally regulates bcd mRNA expression: its mutation causes delayed deadenylation and stabilization of the bcd message, resulting in protracted Bicoid protein expression. Disruption of this molecular control perturbs normal Drosophila head development (Gamberi, 2002).

While affected Bicoid targets are presently only speculative, it was fortuitously noticed that late zygotic hb expression (hbzyg) was increased in Northern blots of bcd NRE mutants, hinting at one potential affected molecule. This is consistent with the pum- data. A second target candidate arises from the defective mouth hook (mh) base present in both bcd NRE mutant transgenics and pum- embryos. This alteration, which is suggestive of a maxillary segment defect, similarly occurs when orthodenticle (otd) is expressed ectopically. Interestingly, Bicoid activates otd transcription and resulting Orthodenticle has the same DNA-binding specificity as Bicoid. Hence, the prolonged Bicoid expression in mutant bcd NRE transgenics and pum- embryos may interfere with normal head development through a complex pattern of interactions (Gamberi, 2002).

Pum was originally characterized as a posterior group gene: Pumilio and Nanos cooperate to repress maternal hb (hbmat) in the posterior of the embryo, allowing abdominal patterning. However, ubiquitous expression of Pumilio in excess of hb implies it could possess additional function(s) elsewhere. This study demonstrates that Pumilio also participates in Drosophila anterior embryonic patterning. pum embryos exhibit head defects. The Pumilio anterior function is mediated via bcd post-transcriptional expression, since similar anterior abnormalities occur when Pumilio's presumptive bcd mRNA-binding site is mutated (Gamberi, 2002).

It was asked if bcd NRE regulation required the Pumilio canonical partner Nanos. When bcd mRNA is injected posteriorly or Nanos is expressed anteriorly by genetic means, Pumilio and Nanos can affect bcd expression because all factors co-exist. In each case, large Nanos amounts are present and head morphogenesis is inhibited (Gamberi, 2002).

A major Nanos role in normal head formation seems unusual because Nanos and bcd mRNA reside at opposite ends of the embryo. Surprisingly Nanos does influence bcd expression and subsequent anterior development to some degree. This suggests undetectable Nanos amounts may regulate bcd mRNA in the anterior. Analogously, a contribution of low Nanos levels in oogenesis has been reported (Gamberi, 2002).

bcd mRNA might encounter low Nanos levels via the NRE-dependent back-up mechanism postulated to repress it when it escapes localization, diffuses posteriorly and intercepts the Nanos gradient. Alternatively, sufficient Nanos moieties might diffuse anteriorly, analogous to when enough Bicoid molecules exist in the posterior of the embryo to elicit hairy stripe 7 expression or to cooperate with Caudal in knirps activation. In a different scenario, nos mRNA translational repression throughout the embryo may be leaky, yielding low basal Nanos levels everywhere, including the anterior. How a Pumilio-bcd mRNA complex can recruit enough Nanos for action and whether this involves additional (anterior?) factors to modulate Nanos activity are questions for future studies (Gamberi, 2002).

nos- severe head involution defects occur at a significantly lower frequency than in pum- cuticles (4% versus 81%; null versus presumptive null), raising the intriguing possibility that an additional partner(s) for Pumilio exists at the anterior that affects bcd NRE function independently of Nanos. Consistently, the sequence between the A and B boxes of the bcd and hb NREs diverges at two of the four nucleotide positions known for hb recruitment of Nanos. While Pumilio and Nanos are usually thought of as functioning in concert, they have only partially overlapping roles in the Drosophila germline and may function independently in oogenesis. The alternate Pumilio partner for bcd might be an anterior Nanos paralog (although only one nos gene was found in the fly genome) or a distinct moiety. Interestingly, S. cerevisiae has five Puf proteins involved in mRNA metabolism but no Nanos homologs, suggesting some Puf proteins can function with novel partners (Gamberi, 2002).

Molecular data indicate the bcd NREs act temporally, repressing translation in a deadenylation dependent way. Mutating either Pumilio or the bcd NREs results in protracted Bicoid expression. Presently, it cannot be distinguish if the bcd NREs primarily constitute a translational control element with mRNA deadenylation and instability accompanying specific repression or a regulated instability element whose downstream effects are seen at the protein level. Interestingly, in addition to detecting specific Pumilio-dependent bcd NRE regulation, a second effect of pum- mutation was noticed: stabilization of maternal mRNAs devoid of NREs. While it is unclear whether this effect is direct, it may reflect a novel Pumilio function in general NRE-independent mRNA turnover (Gamberi, 2002).

Complementary phenotypic analyses of bcd NRE mutant transgenes has revealed that prolonged Bicoid expression interferes with maxillary segment determination, which may affect head involution by altering the intersegmental contacts required for appropriate head morphogenetic movements. Incomplete overlap between the highly penetrant mouth hook defect and the partially penetrant head involution defect might reflect the complexity of fly head development, which is subjected to redundancy and fail-safe mechanisms (Gamberi, 2002).

The conservation of the bcd and hb NREs, their Pumilio association, and their ability to direct translational regulation imply functional similarity between these elements. However, the hb regulatory system operates on a uniformly distributed mRNA to repress its expression in the embryonic posterior where Nanos is most concentrated. By contrast, bcd mRNA is spatially restricted to the anterior via localization, which conceivably impacts NRE action and predicts underlying functional differences between bcd and hb NREs (Gamberi, 2002).

The novel Pumilio role in anterior development documented here raises the exciting possibility that the prototypical Puf protein Pumilio operates more generally than previously thought, regulating multiple physiological pathways in different Drosophila embryonic locales. Furthermore, since Pumilio is also expressed in the adult fly and pum- flies exhibit additional uncharacterized phenotypes, Pumilio may function in mRNA metabolism throughout the life of the fly (Gamberi, 2002).

To date, NREs have been identified in three mRNA species: hb, bcd and cyclin B. For each, NRE organization differs: hb and bcd contain two and 1 1/2 copies, respectively, of the basic (A box-N5-B box) NRE motif, while cyclin B contains one NRE motif with a larger spacer. Furthermore, hbmat and hbzyg mRNA have identical NREs, but hbzyg mRNA seems relatively insensitive to regulation by Pumilio/Nanos. Differences among NREs combined with distinct distributions of NRE-containing mRNAs and their known effectors underlie a potential combinatorial model of NRE recognition in which a common factor (Pumilio) associates with the mRNA target sequence and subsequently recruits different (sets of) factors (e.g. Nanos, Brat for hbmat mRNA) to regulate ultimately and specifically unique target expression. How Pumilio functions on different NRE-containing mRNAs, what factor combinations are employed in distinct situations and whether Nanos homologs are involved in every case are experimental questions begging to be answered (Gamberi, 2002).

Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance

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-tub3′-UTR transgene. Surprisingly, cyst formation proceeds normally in ovaries carrying the Nos-tub3′-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).

Direct inhibition of Pumilo activity by Bam and Bgcn in Drosophila germ line stem cell differentiation

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. This study shows that Bam and Bgcn inhibit Pum function through direct binding. A ternary complex involving Bam, Bgcn, and Pum has been identified in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells (Kim, 2010).

Two important intrinsic factors, Bam and Bgcn, play critical roles in stem cell differentiation. Loss-of-function mutations in either Bam or Bgcn cause stem cell differentiation to arrest. Conversely, ectopic expression of Bam in stem cells overrides stem cell self-renewal capabilities and promotes differentiation. Genetic analyses have shown that Bam and Bgcn require each other for function. Bgcn is present in stem cells as well as cystoblasts and early mitotic cysts, whereas Bam is not expressed in stem cells but is expressed in cystoblasts and early mitotic cysts. Bam silencing in stem cells is governed by the BMP2/4 homolog Decapentaplegic signal emanating from the niche cells (Kim, 2010 and references therein).

In addition to the extrinsic factors emanating from niche cells, stem cell maintenance requires intrinsic stem cell factors. Pumilio (Pum) and Nanos (Nos) are such intrinsic factors. Pum is an RNA-binding protein with a C-terminal Puf (Pum and Fem3-binding factor) domain, which binds the Nanos response element (NRE) sequences at the 3'-untranslated region of its target mRNAs. Binding of the Puf domain to NRE recruits Nos to this complex, resulting in the repression of the translation of the target mRNAs. Because Pum and Nos are required for repression of differentiation in germ line stem cells, it is conceivable that this complex targets a suite of genes that are required for differentiation, although the identities of these genes are unknown (Kim, 2010 and references therein).

Genetic epistasis analysis of double mutants of Bam and Pum indicated that Bam antagonizes Pum function to promote differentiation of stem cells. For the differentiating cystoblasts to begin differentiation, the Pum/Nos activity must be inhibited in the cystoblast. This study explored the possibility that the Bam-Bgcn complex may inhibit Pum-Nos activity at the protein level and discovered a direct interaction between Bam and Pum. Notably, the Bam-Pum interaction is greatly increased in the presence of Bgcn, and this interaction allows for the formation of a strong ternary complex involving Bam, Bgcn, and Pum. Consistent with this physical interaction, Bam inhibits Pum activity in a heterologous reporter assay, which monitors the activity of Pum. On the other hand, no ternary interaction between Bam, Bgcn, and Nos was detected, suggesting that Bam and Bgcn specifically target Pum directly to negatively regulate Pum/Nos activity and promote stem cell differentiation (Kim, 2010).

Previous genetic analysis suggested that Bam and Bgcn form a complex because they require each other for function. Therefore this study utilized diverse assays to probe the biochemical relevance of these genetic results. Surprisingly, both the fragment complementation analysi (FCA) and the yeast two-hybrid assay failed to detect any interaction between Bam and Bgcn. However, the two assays detected a strong Bam-Bgcn-Pum complex. In contrast, the co-immunoprecipitation assay detected direct Bam-Bgcn interaction without Pum involvement, which is in accord with other recent reports. The inability to detect direct Bam-Bgcn interaction by the FCA and the yeast two-hybrid assay may indicate that Bam-Bgcn interaction is weak in vivo (Kim, 2010).

Both yeast two-hybrid and FCA showed that there is a weak interaction between Bam and Pum. Particularly, the interaction revealed by FCA appears authentic because the Bam-Pum interaction brought the N- and C-terminal fragments of fluorescent reporter mKG (monomeric Kusabira-Green) into the cytoplasm, reflecting the cytoplasmic localization of Bam and Pum. In contrast, the control interaction of the p65 and p55 subunits of NF-kappaB occurred in the nucleus. Importantly, both the FCA and yeast tri-hybrid assay detected a strong ternary interaction involving Bam, Bgcn, and Pum, suggesting that weak interaction between Bam and Pum is greatly enhanced by the presence of Bgcn, through additional Bam-Bgcn interaction (Kim, 2010).

The ternary interaction involving Bam and Bgcn is mediated by the N terminus of Pum, which lacks the C-terminal Puf region. Consistent with this, the Puf region fails to form a ternary complex formation with Bam and Bgcn. It is known that the Puf domain mediates both Nanos response element (NRE) binding and Nos binding of Pum. The binding of Bam and Bgcn to the N-terminal region of Pum appears not to interfere with the binding of Nos to the Puf region, because Bam immunoprecipitates contained Bgcn, Pum, and Nos. Neither Bam nor Bgcn binds to Nos, and a ternary complex involving Bam, Bgcn, and Nos was not observed. Therefore, these results indicate that Pum can recruit both Bam/Bgcn and Nos in distinct sites and thus can account for the fact that Bam precipitates contain Bgcn, Pum, and Nos (Kim, 2010).

Using a luciferase reporter system involving the NRE sequence at the 3'-untranslated region, the relevance of Bam/Bgcn binding to Pum activity was addressed in heterologous cells. Expression of Pum repressed luciferase expression, which requires an intact NRE sequence. Bam was able to abrogate this repression by Pum, suggesting that a weak interaction between Bam and Pum is sufficient for Bam inhibition of Pum activity in this assay. The Bam inhibition of Pum function appears to require Bam binding to Pum, because Bam does not bind to Puf and failed to abrogate Puf-dependent repression. Bgcn failed to interact with Pum or affect Pum repression of the reporter gene expression. These results yield insight into the role of Bgcn in vivo and suggest that Bgcn may be confined to facilitating Bam binding to Pum under physiological conditions where Bam protein levels may not be sufficient for the binding and inhibition of Pum (Kim, 2010).

In conclusion, following stem cell division, one daughter cell moves away from the niche cells and begins to initiate differentiation as a cystoblast. For the cystoblast to begin differentiation, Pum/Nos activity must be inhibited in the cystoblast and early dividing germ cells. One possible mechanism for this inhibition is the decrease of Pum and Nos at the protein level. In fact, these levels are gradually reduced in the cystoblasts and immediate early dividing cysts; however, not all Pum and Nos protein disappears. Thus, other mechanisms must exist to inhibit Pum/Nos activity in the differentiating cells. These data suggest that Bam and Bgcn present in the cystoblast cells play such a role by binding and inhibiting Pum directly at the protein level (see Model depicting Bam/Bgcn binding and inhibition of Pum/Nos activity). This notion is consistent with findings that ectopic Bam expression in stem cells triggers stem cell differentiation, which might occur because of direct Bam/Bgcn inhibition of Pum/Nos activity (Kim, 2010).

The CCR4 Deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal

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).

Distinct modes of recruitment of the CCR4-NOT complex by Drosophila and vertebrate Nanos. EMBO J 35(9):974-90. PubMed ID: 26968986

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non-conserved N-terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4-NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4-NOT deadenylase complex (see Drosophila Twin) in invertebrates. This study identified an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4-NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C-terminal domains of NOT1-3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N-terminal regions of Nanos proteins recruit CCR4-NOT to assemble analogous repressive complexes (Raisch, 2016).

Post-transcriptional mRNA regulation plays an essential role in embryonic development. This regulation is mediated by RNA-binding proteins that control the spatial and temporal expression of target mRNAs through the recruitment of effector complexes. The RNA-binding proteins of the Nanos family are conserved post-transcriptional mRNA regulators that play essential roles in embryonic germline specification, germline stem cell maintenance, and neuronal homeostasis in Drosophila melanogaster (Dm) and a wide range of other. The Dm Nanos protein is also required for posterior pattern formation in the embryo (Raisch, 2016).

Three Nanos paralogs (Nanos1-3) exist in vertebrates and various invertebrate species, whereas there is only one family member in Dm and other insects. This protein family is defined by a highly conserved CCHC-type zinc-finger (ZnF) domain and divergent N- and C-terminal unstructured regions of variable lengths. The ZnF domain mediates binding to RNA and to Pumilio, a conserved Nanos partner that confers mRNA target specificity. The unstructured regions are required for interaction with effector complexes, which include the CCR4-NOT deadenylase complex embryo (Raisch, 2016).

The CCR4-NOT complex catalyzes the removal of mRNA poly(A) tails and consequently represses translation. In addition, dead-enylation by the CCR4-NOT complex is coupled to decapping and 5'-to-3' exonucleolytic degradation by XRN1 and can therefore lead to full mRNA degradation in some cellular contexts. Furthermore, the CCR4-NOT complex can also repress translation independently of deadenylation (Raisch, 2016).

The CCR4-NOT complex consists of several independent modules that dock with NOT1, a central scaffold subunit (Temme, 2014). NOT1 consists of independently folded α-helical domains that provide binding sites for the individual modules. A central domain of NOT1, structurally related to the middle domain of eIF4G (the NOT1 MIF4G domain), provides a binding site for the catalytic module, which comprises two deadenylases, namely CAF1 (or its paralog POP2) and CCR4a (or its paralog CCR4b) (Raisch, 2016).

The C-terminal region of NOT1 contains the NOT1 superfamily homology domain (SHD) and assembles with NOT2-NOT3 heterodimers to form the NOT module. The NOT module provides binding sites for RNA-binding proteins, such as vertebrate Nanos and Dm Bicaudal-C, which recruit the CCR4-NOT complex to their mRNA targets (Raisch, 2016).

The three vertebrate Nanos paralogs contain a 17-amino acid NOT1-interacting motif (NIM) that binds directly to the NOT1 SHD domain. Although the NIM is conserved in vertebrate Nanos, Nanos proteins of insects and worms do not have a detectable NIM. Nevertheless, Dm Nanos has been reported to interact with NOT4 through its unstructured N-terminus. However, because NOT4 is not stably associated with the CCR4-NOT complex in metazoans, it has remained unclear whether Dm Nanos recruits the CCR4-NOT complex to mRNA targets directly or rather relies on its interaction with additional partners, such as Pumilio (PUM) and Brain tumor (BRAT), to exert its repressive function (Raisch, 2016).

This study shows that although Dm Nanos does not contain a NIM, it interacts directly with the CCR4-NOT complex using an extended region that is termed the Nanos effector domain (NED). The NED overlaps with a region previously shown to contribute to Nanos function in Dm embryos. The crystal structure of a central region of the NED (termed the NOT module binding region, NBR) bound to the NOT module revealed a bipartite interface that contacts both the NOT1 SHD and the NOT3 NOT-box domains. The binding site for the Dm NBR on NOT1 does not overlap with the vertebrate NIM-binding site. These results indicate that Nanos proteins have maintained the ability to interact with the CCR4-NOT complex using divergent motifs in disordered protein regions (Raisch, 2016).

This study has shown that Dm Nanos recruits the CCR4-NOT complex directly through a Nanos effector domain (NED) that is conserved in the Drosophila species. Similar to the vertebrate Nanos NIM, the Dm NED is necessary and sufficient to repress translation in the absence of mRNA degradation and to promote degradation of bound mRNAs. Thus, the NED and the NIM are the main determinants for the repressive activity of Nanos in Dm and vertebrates, respectively. Although the NED and the NIM are functionally analogous, they do not share sequence similarities, demonstrating that the absence of sequence conservation is not an indicator of functional irrelevance, in particular, when disordered, low complexity protein regions are involved. Such regions often mediate their function by interacting with binding partners using short linear motifs (SLiMs). SLiMs can evolve rapidly due to the lack of constraints to maintain a protein fold and thus enable the evolution of distinct binding modes in orthologous proteins, especially in cases where these proteins are in a competitive scenario or even under positive selection. In this way, these proteins can maintain the ability to interact with the same partners using different binding modes (Raisch, 2016).

The Dm NED and the vertebrate NIM use different modes to mediate the recruitment of the CCR4-NOT complex to Nanos mRNA targets. The NIM is a short 17-residue motif present in the N-terminal disordered region of vertebrate Nanos proteins, which binds to the NOT1 SHD domain. In contrast to vertebrates, flies have only a single Nanos protein but with an extended NED. The Dm NED is 187 amino acids in length and contains multiple and redundant binding sites for the CCR4-NOT complex. These multiple binding sites may increase the affinity of Dm Nanos for the CCR4-NOT complex through avidity effects. Redundancy may also confer a competitive advantage to Dm Nanos over other RNA-binding proteins that compete for recruitment of the CCR4-NOT complex (Raisch, 2016).

Interestingly, redundancy to recruit the CCR4-NOT complex is not only observed within the Nanos protein but also in the context of Nanos repressive complexes. Indeed, Nanos cooperates with PUM to bind and repress natural mRNA targets, and PUM also has the ability to recruit the CCR4-NOT complex independently of Nanos. This may partially obscure the effects of deletion or mutations in the Nanos NED. However, PUM does not act as a general substitute for Nanos because mutations in the Nanos ZnF domain that prevent mRNA binding caused strong developmental defects despite the presence of endogenous PUM. Thus, PUM probably acts both additively and alternatively with the Nanos NED, resulting in distinct modes of engaging the CCR4-NOT complex. In their various combinations, the different binding modes can thus lead to a highly specific and tunable repression of mRNA targets in a cell context-dependent manner (Raisch, 2016).

A large number of RNA-associated proteins have been shown to recruit the CCR4-NOT complex to their mRNA targets to repress translation and/or to promote mRNA degradation. In addition to Nanos, these proteins include the GW182 proteins, which are involved in miRNA-mediated gene silencing in animals, and the Dm proteins CUP, Bicaudal-C, Smaug, and PUM. Additional examples from vertebrates are Roquin and tristetraprolin (TTP), a protein required for the degradation of mRNAs containing AU-rich elements (ARE-mediated mRNA decay) (Raisch, 2016).

For the recruitment of the CCR4-NOT complex, most of these proteins rely on short linear motifs (SLiMs) embedded in peptide regions of predicted disorder. However, a detailed characterization of their interaction with the CCR4-NOT complex on a molecular level is only available for TTP, GW182, and vertebrate and Dm Nanos. For TTP and vertebrate and Dm Nanos, the motifs adopt α-helical conformations that possibly form only upon binding. Specificity results from aromatic and hydrophobic side chains that insert primarily into pockets on the surface of the NOT1 domains that consist of HEAT-like repeats. By contrast, GW182 peptides likely bind to the CCR4-NOT complex in an extended conformation and insert tryptophan residues into tandem hydrophobic pockets exposed at the surface of the NOT9 subunit (also known as CAF40) of the CCR4-NOT complex and probably into additional pockets in NOT1 that remain to be identified (Raisch, 2016).

Similar to GW182 proteins, the Nanos NBR not only contacts NOT1 but also binds to NOT3, providing the first detailed insight into how an mRNA-binding protein recruits the CCR4-NOT complex by contacting two of its subunits simultaneously. Interestingly, the surface on NOT1 that is contacted by Dm Nanos partially overlaps with the binding surface for NOT4 as observed in the Saccharomyces cerevisiae (Sc) complex. This would suggest that Dm Nanos competes with NOT4 for binding to NOT1, providing additional opportunities for the regulation of gene expression. However, it is not known whether the NOT4 binding mode is conserved between Dm and Sc because NOT4 does not co-purify with the CCR4-NOT complex in metazoans and the Sc NOT4 sequences that bind NOT1 are not well conserved in metazoans (Raisch, 2016).

Together with previous studies (Fabian, 2013; Bhandari, 2014; Chen, 2014; Mathys, 2014), these results reveal that the recruitment of the CCR4-NOT complex is mediated by highly diverse sequence motifs and distinct binding modes. It is speculated that these motifs represent a combinatorial code that is read by the CCR4-NOT complex to funnel the effects of diverse RNA-binding proteins into a common repressive pathway, which results in the removal of the mRNA poly(A) tail, translational repression, and, depending on the cellular context, full degradation of the mRNA (Raisch, 2016).

nanos: Biological Overview | Evolutionary Homologs | mRNA localization and post-transcriptional regulation | Developmental Biology | Effects of Mutation | References

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