pumilio

Targets of Activity

The posterior group of maternal genes is required for the development of the abdominal region in the Drosophila embryo. Genetic as well as cytoplasmic transfer experiments have been used to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. Nanos, present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack aspecialized posterior pole plasm and consequently fail to form germ-cell precursors. The products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants (Lehmann, 1991).

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 6^th or 7^th 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 Tkv^QD, 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 Tkv^QD. 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).

Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation

The transition from a Drosophila ovarian germline stem cell (GSC) to its differentiated daughter cell, the cystoblast, is controlled by both niche signals and intrinsic factors. piwi and pumilio (pum) are essential for GSC self-renewal, whereas bag-of-marbles (bam) is required for cystoblast differentiation. This study demonstrate that Piwi and Bam proteins are expressed independently of one another in reciprocal patterns in GSCs and cystoblasts. However, overexpression of either one antagonizes the other in these cells. Furthermore, piwi;bam double mutants phenocopy the bam mutant. This epistasis reflects the niche signaling function of piwi because depleting piwi from niche cells in bam mutant ovaries also phenocopies bam mutants. Thus, bam is epistatic to niche Piwi, but not germline Piwi function. Despite this, bam⁻ ovaries lacking germline Piwi contain approximately 4-fold fewer germ cells than bam⁻ ovaries, consistent with the role of germline Piwi in promoting GSC mitosis by 4-fold. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. It is proposed that niche Piwi maintains GSCs by repressing bam expression in GSCs, which consequently prevents Bam from downregulating Pum/Nos function in repressing the translation of differentiation genes and germline Piwi function in promoting germ cell division (Szakmary, 2005).

This study investigates the regulatory relationships between Piwi, Bam, and Pum, three key regulators of GSC versus cystoblast fates. Among them, Pum and Bam are intrinsic factors, whereas Piwi is expressed both in niche cells as an essential component of niche signaling and in GSCs to promote its division. Pum was originally identified as a maternal effect protein that heterodimerizes with Nanos (Nos) to bind and suppress the translation of its target hunchback mRNA in the posterior of the Drosophila embryo. In addition, Pum and Nos have important germline development zygotic roles, including their cell-autonomous function for GSC maintenance. In contrast to this function of Pum and Nos, Bam is necessary and sufficient in promoting GSC differentiation, even though its molecular activity is not known. bam encodes two protein isoforms: the cytoplasmic (Bam-C) and the fusomal (Bam-F) forms, with Bam-C specifically present in cystoblasts and differentiating cysts but absent in GSCs. Finally, Piwi is the founding member of the evolutionarily conserved Piwi protein family (a.k.a. Argonaute family) involved in stem cell division, RNA interference, transcriptional gene silencing, and other developmental processes. In the Drosophila ovarian germline, Piwi is a nuclear protein that is preferentially expressed in GSCs but is only weakly expressed in cystoblasts and mitotic cysts, consistent with its germline function (Szakmary, 2005).

To investigate the regulatory relationship between piwi and bam, the reciprocal expression pattern was confirmed by double immunofluorescence microscopy of wild-type germaria for Piwi and Bam-C. A fully functional myc-tagged Piwi is expressed at high levels in GSCs and is downregulated in cystoblasts and early mitotic cysts. In contrast, Bam-C is absent from GSCs but accumulates in most cystoblasts and mitotic cystocytes in germarial region 1. The downregulation of Piwi coincides with the zone of Bam-C expression. In a few cases, germ cells were observed expressing both Piwi and Bam-C in cystoblast positions. These cells might represent the transitional stage from GSCs to cystoblasts. At a very low frequency, cystoblast-like cells express low levels of Piwi, but no detectable Bam-C. On the basis of piwi;bam double mutant analysis, these cystoblast-like cells are likely to be undifferentiated or potentially apoptotic. Overall, the reciprocal expression pattern of Piwi and Bam-C proteins supports the opposing functions of piwi and bam genes (Szakmary, 2005).

To determine whether this reciprocal expression pattern is a result of mutually negative regulation toward each other's expression, Bam expression was analyzed in piwi mutants and vice versa. Because piwi mutant ovarioles typically contain germaria that are depleted of germline cells, it is difficult to assay Bam-C expression in them. For this reason, piwi¹ GSC clones were generated with the FLP-DFS (flipase-mediated dominant female sterile) technique. Bam-C is expressed normally in cystoblasts and early mitotic cysts in germaria that contain only piwi¹ germline cells. Moreover, no ectopic Bam expression was detected in GSCs. Therefore, proper Bam-C expression in the adult germline during oogenesis does not require piwi⁺ function in the germline. To address whether piwi expression in apical somatic cells affects bam expression in GSCs, Piwi was eliminated in somatic niche cells. This was achieved by using Yb mutations that eliminate Piwi expression specifically in niche cells. Yb mutants are phenotypically very similar to piwi mutants. However, if examined within the first day of eclosion, Yb mutant germaria still contain germ cells. Bam-C expression is unaffected in adult Yb mutant ovaries, suggesting that there is no specific requirement for YB or Piwi in niche cells for proper Bam-C expression or localization in the germline. Taken together, the above analyses indicate that neither niche nor germline piwi is required for Bam-C expression (Szakmary, 2005).

Whether the absence of Bam affects Piwi expression was examined. A P[myc-piwi] transgene was introduced into a bam null mutant background to monitor Piwi expression. Ovaries were dissected from these females and stained with Myc antibody to monitor the Piwi expression and with Vasa antibodies to label germ cells. Piwi is present in all bam null germline cells. In addition, Piwi is also strongly expressed in apical somatic cells that correspond to terminal filament, cap cells, and inner sheath cells in the wild-type germarium. Therefore, bam⁺ function is dispensable for Piwi expression in the germline and the apical somatic cells (Szakmary, 2005).

Whether piwi or bam negatively regulates the expression of the other was tested. Overexpression of Piwi in apical somatic cells increases the number of GSC-like cells. These ectopic stem cell-like cells fill regions 1 and 2a of the germarium and, thus, displace Bam-C-expressing cells to region 2b. If Piwi and Bam expression are mutually antagonistic, the prediction would be that expanding Bam expression to GSCs would downregulate Piwi expression there during oogenesis. To express Bam-C protein ectopically in GSCs, a heat shock-inducible bam transgene was used that places the bam cDNA under the control of the hsp70 promoter. Flies carrying a P[myc-piwi] and a hs-bam transgene were subjected to heat shock twice daily for 3 days after eclosion. Ovaries were subsequently dissected and stained for Bam-C and Myc to monitor ectopic Bam-C expression and its effects on Piwi expression. As predicted, ectopic expression of Bam in GSCs diminishes Piwi expression specifically in these cells. Interestingly, ectopic Bam expression in both somatic cells and other germline cells within and beyond the germarium has no effect on Piwi expression in these cells. Particularly, Piwi expression in apical somatic cells (i.e., cap cells and inner sheath cells) of the germarium is unaffected by ectopic Bam expression. Thus ectopic Bam expression may specifically downregulate the germline Piwi expression (Szakmary, 2005).

The mutually independent expression of Piwi and Bam does not rule out their regulatory relationship in GSC cell fate, whereas the suppression of piwi in GSCs by ectopic bam expression suggests that these two genes interact antagonistically. To further define the interaction between piwi and bam, females were constructed lacking both piwi and bam function and the double mutants' ovaries were examined. In contrast to the piwi mutant phenotype, in which ovarioles typically contain a germlineless germarium and 2-3 egg chambers, the double mutant ovaries are characterized by 'tumorous' germaria filled with hundreds of undifferentiated germ cells. Moreover, there is no apparent egg chamber development in the double mutant ovary. This phenotype is qualitatively similar to the tumorous phenotype observed in bam mutant ovaries, which can contain up to thousands of undifferentiated germ cells. The piwi;bam double mutant phenotype therefore indicates that bam is epistatic to piwi. Given the opposing functions of piwi and bam, these results suggest that piwi acts upstream of bam to repress its function in promoting GSC differentiation (Szakmary, 2005).

Although the piwi;bam double mutant shows a bam-like phenotype, there is a difference between the defect of the double mutant and that of bam alone. The bam mutant typically contains 300-1000 undifferentiated germ cells, whereas the piwi;bam double mutants contain only 50-300 germ cells. One possible explanation for this difference is the absence of the mitosis-promoting, germline cell autonomous piwi function in the double mutant. The cell autonomous function of piwi in GSCs is to promote mitosis, resulting in a 4-fold increase of mitotic rates. In bam mutants, 'tumorous'germ cells are more mitotic because of the presence of piwi⁺ function, whereas in piwi;bam double mutants, 'tumorous' germ cells are less mitotic because of the absence of piwi⁺ function. Therefore, these analyses suggest that, whereas bam is epistatic to the niche function of piwi, the cell autonomous function of piwi is epistatic to bam (Szakmary, 2005).

To verify the complex epistasis between bam and distinct somatic versus germline functions of Piwi, the effect of specifically removing Piwi protein from either the germline or the somatic niche cells of bam mutants was investigated. The piwi (somatic);bam double mutant was achieved by generating Yb;bam double mutants because Yb specifically eliminates piwi expression in niche cells. The piwi (germline);bam double mutant was achieved by driving transgenic piwi expression specifically in the niche cells of a piwi;bam double mutant background (Szakmary, 2005).

Yb;bam double mutant ovaries display a clear bam phenotype. This phenotype, however, is not as attenuated as in piwi;bam double mutants, but rather appears to be as pronounced as in bam single mutants. This result supports the assumption that the epistasis of bam over piwi reflects the somatic piwi function, and the attenuated bam phenotype of the double mutant reflects the germline cell autonomous piwi function (Szakmary, 2005).

To further verify this hypothesis, the phenotype of piwi (germline);bam double mutants was analyzed. The piwi (germline) mutant was generated with an en-gal4 transgene to drive the expression of piwi^EP to produce specific expression of fully functional Piwi in niche cells. Because piwi^EP is inserted into the piwi locus, it is therefore a piwi mutant allele in the absence of gal4 expression. The en-gal4 piwi¹/piwi^EP transheterozygotes were generated in bam mutant and wild-type backgrounds. The piwi/piwi^EP;bam⁺ ovaries display the expected piwi mutant phenotype. The en-gal4 piwi¹/piwi^EP;bam^Δ86~/TM3 Sb ovaries appear wild-type, aside from a mild reduction in size, and give rise to females capable of laying eggs. This finding directly confirms that Piwi expression in niche cells is sufficient for GSC maintenance, whereas the observed reduction in ovary size may reflect the absence of germline piwi function in promoting GSC mitoses. As expected, the en-gal4 piwi¹/CyO;bam^Δ86/bam^Δ86 flies display typical bam mutant ovarioles. Also as expected, piwi¹/piwi^EP;bam^Δ86/bam^Δ86 and en-gal4 piwi¹/piwi^EP;bam^Δ86/bam^Δ86 ovaries display the phenotypes of piwi (somatic);bam double mutants and the piwi (germline);bam double mutants, respectively. These analyses further verified that bam is epistatic to somatic niche piwi function, yet germline piwi is epistatic to bam function (Szakmary, 2005).

The fact that Piwi expression in somatic cells has a downregulating effect on Bam-C expression in GSCs raises the question of how this signal may be relayed. The reciprocal expression patterns of Piwi and Bam-C in the germline closely resemble those of Pum and Bam-C. Pum maintains GSC self-renewal during oogenesis, whereas Bam promotes GSC differentiation. GSCs are depleted in pum mutants but overproliferate in bam mutants. This raised the possibility that Piwi may exert its functions by acting on Pum. Pum expression was therefore examined in piwi¹ mutants and Piwi expression in pum¹⁶⁸⁸ and pum²⁰⁰³ mutants. The expression of one gene was not detectably altered in the mutant background of the other, suggesting that neither gene regulates the other's expression. However, pum encodes two distinct protein isoforms (156 kDa and 130 kDa). Either isoform is sufficient for maternal function, but both are required for zygotic function, including GSC maintenance. Because pum¹⁶⁸⁸ and pum²⁰⁰³ eliminate the expression of the 156 kDa and 130 kDa Pum isoforms, respectively, these results could suggest that either the 156 kDa or the 130 kDa isoform of Pum alone is sufficient for proper germline Piwi expression. Even if this is the case, the niche expression of piwi is independent of pum because pum is not required somatically to maintain GSCs (Szakmary, 2005).

To more definitively determine the regulatory relationship between Pum and the Piwi-Bam-C pathway, pum,bam double mutants were constructed and analyzed. If somatic Piwi acts through Pum to regulate Bam-C, then pum,bam double mutants should resemble piwi;bam double mutants. This, however, was not the case. In pum^ET1,bam^Δ86/pum^ET9,bam^Δ86 double mutant flies, in which both pum^ET1 and pum^ET9 are null alleles, the majority of germaria were devoid of germ cells. Only a minority of germaria (<10%) contained a number of undifferentiated germ cells with restricted proliferation. This range of defects is indistinguishable from that of the phenotype of typical pum mutant ovaries. These results suggest that pum is epistatic to bam and that the proliferation of germ cells in bam mutants requires Pum function (Szakmary, 2005).

In summary, these results show that somatic niche Piwi function antagonizes Bam-C, which in turn antagonizes Pum and germline Piwi. The niche function of Piwi in downregulating BAM function appears to converge with the Dpp signaling pathway that is also required for GSC maintenance. This is based on three observations. (1) the expression of dpp does not require piwi. Therefore, Dpp is not a downstream signal of piwi. (2) dpp overexpression does not rescue piwi mutant defects. Therefore, Dpp and niche Piwi are functionally parallel. (3) The dpp signaling pathway directly represses bam transcription. Likewise, piwi niche signaling also downregulates bam expression because bam is epistatic over piwi and because overexpression of Piwi in germarial somatic cells causes overproliferation of GSCs and displaces Bam-C expression beyond region 1 and 2a of the germarium. Taken together, these results indicate that these two signaling pathways must converge at some point to regulate Bam function. The convergence point could be in niche cells, where piwi directly affects Dpp signal production by aiding in its modifications, stability, and/or secretion. Alternatively, it could be in GSCs, where Piwi suppresses a Dpp agonist(s) or perhaps even the Bam/Bgcn complex. This scenario would require that Piwi produce an intercellular signal independent of Dpp. At present, these two possibilities cannot be distinguished (Szakmary, 2005).

How does Bam function as a converging target in promoting GSC differentiation? It has been suggested that benign gonial cell neoplasm (Bgcn) is an obligatory partner for Bam-C as a differentiation factor. Bgcn is expressed in GSCs, but not in somatic cells. This may explain why ectopic bam expression only downregulates Piwi in GSCs, but not in somatic cells (Szakmary, 2005).

How is Pum involved in the Piwi-Bam pathway? Piwi and Pum do not affect one another's expression, yet pum is clearly epistatic to bam. This precludes the possibility that Piwi exerts its effect on Bam-C via Pum. The epistasis of pum to bam is best explained by ascribing a translational repressing function of Pum/Nos in the germline toward mRNAs that promote differentiation. This repression is released by Bam/Bgcn. In GSCs, Bam-C is itself transcriptionally silenced; therefore, Pum and Nos are active in suppressing differentiation. In cystoblasts, Bam/Bgcn are expressed, thereby antagonizing Pum/Nos function. This allows differentiation-promoting mRNAs to be translated. Bgcn is related to the DexH-box family of RNA-dependent helicases. Recently, it has been suggested that the majority of RNA helicases function by displacing proteins from RNA strands rather than by unwinding RNA. It is therefore conceivable that the Bam/Bgcn complex displaces Pum/Nos from their target RNAs (Szakmary, 2005).

A model is proposed for switching between self-renewal and differentiation of GSCs in the Drosophila germarium. The niche cells signal to GSCs by secreting Dpp/Bmp and possibly other proteins. The Dpp signal is received by GSCs through its receptors Punt and Thick Veins (TKV), and it is transduced by pMad to silence bam transcription in these cells. This is achieved via the direct binding of Smads to a discrete silencing element in the bam gene. Piwi in niche cells has an essential and cooperative functional involvement in this signal. Piwi and Dpp signaling pathways converge at some point upstream of bam, in either niche cells or GSCs. The absence of Bam allows Pum and Nos to be active, which suppresses the translation of differentiation genes, thus maintaining the stem cell fate. In the cystoblast and differentiating germline cysts, the Dpp signal is no longer effective, thereby relieving the transcriptional repression of bam. The Bam/Bgcn complexes then repress Pum/Nos function, allowing these cells to differentiate. Therefore, Pum/Nos can be considered a switch between self-renewal and differentiation, whereas niche signaling through Bam/Bgcn regulates this switch at a single cell level (Szakmary, 2005).

Gene circuitry controlling a stem cell niche

Many stem cell populations interact with stromal cells via signaling pathways, and understanding these interactions is key for understanding stem cell biology. In Drosophila, germline stem cell (GSC) maintenance requires regulation of several genes, including dpp, piwi, pumilio, and bam. GSCs also maintain continuous contact with cap cells that probably secrete the signaling ligands necessary for controlling expression of these genes. For example, dpp signaling acts by silencing transcription of the differentiation factor, bam, in GSCs. Despite numerous studies, it is not clear what roles piwi, primarily a cap cell factor, and pumilio, a germ cell factor, play in maintaining GSC function. With molecular and genetic experiments, it is shown that piwi maintains GSCs by silencing bam. In contrast, pumilio is not required for bam silencing, indicating that pumilio maintains GSC fate by a mechanism not dependent on bam transcription. Surprisingly, it was found that germ cells can differentiate without bam if they also lack pumilio. These findings suggest a molecular pathway for GSC maintenance. dpp- and piwi-dependent signaling act synergistically in GSCs to silence bam, whereas pumilio represses translation of differentiation-promoting mRNAs. In cystoblasts, accumulating Bam protein antagonizes pumilio, permitting the translation of cystoblast-promoting transcripts (Chen, 2005).

dpp-dependent silencing of bam transcription defines a key -- probably the primary -- mechanism for maintaining GSCs. By repressing bam transcription in the germ cells attached to cap cells, dpp signaling prevents these cells from forming cystoblasts and assigns them as GSCs. It is speculated that all GSC maintenance genes might act by repressing bam transcription and this prediction was tested for piwi and pumilio (Chen, 2005).

Two genetic observations suggested that piwi might negatively regulate bam expression: (1) bam was epistatic to piwi in double mutants, indicating that the piwi GSC-loss phenotype required an active bam gene; (2) Bam coexpression suppressed the formation of extra GSCs induced when piwi was overexpressed. Thus, piwi-dependent GSC formation depends on maintaining low levels of bam expression (Chen, 2005).

Both overexpression and loss-of-function phenotypes could be explained if piwi, like dpp signaling, were necessary to silence bam transcription in GSCs. This possibility was tested by scoring the expression of bam transcriptional reporters in piwi mutant GSCs. Because piwi inactivation causes GSC loss, P{bamP-GFP} reporters were assayed in piwi bgcn (benign gonial cell neoplasm) double mutant flies that preserve GSCs. GSCs lacking bgcn were GFP negative, but GSCs that lacked both piwi and bgcn were GFP positive. Thus, like dpp signaling, piwi⁺ was necessary to silence bam transcription in GSCs (Chen, 2005).

piwi⁺ action in somatic, but not germline, cells is critical for GSC maintenance, and, therefore, piwi must act indirectly to repress bam transcription. A putative piwi target (or targets) in GSCs must integrate with dpp signaling because previous work has established that the Mad:Medea binding site in bam is a sufficient silencer element. Two recent findings drew attention to the E3-ligase Dsmurf as a candidate for a germ cell piwi target: (1) Dsmurf inactivation produces extra GSCs, just as does ectopic piwi expression and (2) Dsmurf suppresses dpp signaling by targeting phosphorylated Mad for degradation (Chen, 2005).

If piwi silences bam transcription by repressing Dsmurf in GSCs, then GSCs might be restored in piwi mutants if Dsmurf were simultaneously removed. Therefore ovaries of piwi Dsmurf double mutant females were examined and it was found that most germaria contained supernumerary GSCs and a continuous supply of egg chambers. It was verified that piwi Dsmurf GSC-like cells behaved as GSCs by noting that they did not express BamC protein. In 62/80 double mutant germaria, no cells expressing BamC were detected, whereas BamC-positive germ cells were detected in 18/80 germaria. In those cases, the most apical cells, in the GSC position, were BamC negative (Chen, 2005).

Dpp signaling and piwi act as GSC maintenance factors by repressing bam transcription. pumilio (pum) is a component of an evolutionarily conserved mechanism of translational control and is also essential for ovarian GSCs. The expression of P{bamP-GFP} reporter was examined in pum mutant germ cells to determine if bam transcriptional silencing also depends on pum⁺. In contrast to piwi, the reporter was properly silenced in pum bam GSCs. For example, GSCs in 84.6% of pum^MSC bam^BG/pum²⁰⁰³ bam^Δ86 germaria were GFP negative. Because pum mutant germ cells are unstable, it was suspected that the few GFP-positive cells in the GSC position had either differentiated or were dying (Chen, 2005).

GSCs required (1) dpp⁺ and piwi⁺ signaling to repress cystoblast (CB) differentiation by silencing bam transcription and (2) pum⁺ to repress CB differentiation by a mechanism that is independent of bam silencing. Because previous work has shown that Pum forms a translational repressor complex with Nanos, it was reasoned that pum⁺ might maintain GSCs by repressing translation of CB-promoting mRNAs. One candidate target mRNA is bam itself, but, because dpp-dependent transcriptional silencing of bam fully accounts for the absence of bam from GSCs, it is unlikely that Pum sustains GSCs by repressing bam translation (Chen, 2005).

The Pum:Nos repressor complex probably blocks translation of other unidentified target mRNAs that are essential for CB differentiation. Cystoblast formation would then depend on relieving this block, and, because bam is both necessary and sufficient to induce CB differentiation, bam might antagonize or bypass translational repression. The phenotypes of double mutants can distinguish between these possibilities. If bam bypasses translational repression, pum bam germ cells would not form CBs and would resemble bam mutant gonads. If, however, bam antagonizes Pum/Nos-mediated translational repression, pum bam germ cells might differentiate (Chen, 2005).

Ovaries formed in various pum and bam genotypes were compared with several alleles of each gene. Double mutant ovaries produced a complex phenotype that was distinct from either single mutant. Staining nuclei with DNA dyes revealed a mixture of apparently undifferentiated cells and overtly polyploid cells. Indeed, in many cases the polyploid chromosomes were also thick and expanded like nurse cell chromosomes. Most remarkably, these pseudo-nurse cells were occasionally organized within an epithelial layer of follicle cells, like a cyst, although these cysts never contained a full complement of 16 cystocytes. Cells with hallmarks of post-CB differentiation occurred only in the pum bam double mutant ovaries, where they were seen in over half the ovarioles scored (see Table S2) (Chen, 2005).

The appearance of pseudo-nurse cells and even cysts suggested that double mutant germ cells had formed functional cystoblasts, remarkably bypassing the requirement for bam⁺ expression. To verify that pum bam germ cells were undergoing differentiation, the double mutant cells were examined with several additional markers of differentiation (Chen, 2005).

Orb is expressed in all germ cells, but its levels increase dramatically in the cystocytes of developing cysts. Orb protein levels remain at very low levels in bam mutant cells. Double mutant cells, however, expressed Orb at levels seen in differentiating cysts and well above the levels in bam cells. Orb accumulation revealed that many of the pum bam germ cells that did not yet have obvious pseudo-nurse cell chromosomes had, in fact, progressed well beyond the "pre-CB" stage of bam cells. Pseudo-nurse cells also had high levels of Orb expression, similar to accumulation seen in developing nurse cells (Chen, 2005).

Ring canal formation is a distinctive feature of germ cell cysts, and the multiple cell pum bam cysts contained ring canals. The incidence of these pum bam cysts was modest but reproducible in all double-mutant combinations, including those containing null alleles of bam and very strong or null alleles of pum. It is suspected that the infrequent appearance of multi-nurse cell cysts is due to a second requirement for bam⁺ to drive cystocyte divisions during cyst formation. This requirement would not have been recognized previously because bam mutations arrested cells as 'pre-CBs' (Chen, 2005).

Although they are not normal, the appearance of these cysts is a striking manifestation that CBs lacking bam could differentiate as long as they also lacked pum. Combined with previous studies showing that ectopic bam expression is sufficient to direct GSC differentiation, the pum bam phenotype strongly suggests that bam acts as a CB-promoting factor by antagonizing, rather than bypassing, pum action. The data suggests further that dpp signaling, which directly regulates bam expression, does not control pum⁺ expression. A similar conclusion has been reached about the relationship between dpp signaling and nanos expression on the basis of studies of primordial germ cell differentiation (Chen, 2005).

A unifying model is proposed to explain the gene circuitry of GSC and CB fate within the GSC niche. The results suggest that Drosophila ovarian GSCs are retained as stem cells because Pum:Nos complexes repress translation of a pool of mRNAs that induce CB differentiation (Chen, 2005).

In wild-type GSCs that contact cap cells, Pum:Nos translational repression remains active because dpp signaling from stromal cells silences bam transcription and thus blocks the formation of Bam:Bgcn complexes that would antagonize Pum:Nos translational repression. Expression of piwi in stromal cells contributes a key, but unknown, signal that stabilizes or strengthens the Dpp response and bam transcriptional silencing (Chen, 2005).

After the GSC divides, the strength of Dpp signaling falls to levels that can no longer efficiently silence bam transcription in the cell displaced to the posterior and away from cap cells. This could be due to declining Dpp levels or diminished piwi-dependent signals that lead to reduced phospho-Mad levels. As bam transcription increases, Bam:Bgcn complexes antagonize Pum:Nos action and cause derepression of CB-promoting mRNAs, initiating the events of CB differentiation. Because pumilio and nanos are evolutionarily conserved proteins, it will be important to determine if a similar 'multiple-negative' circuitry is at work in mammalian stem cell niches (Chen, 2005).

Genome-wide identification of mRNAs associated with the translational regulator Pumilio

Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. Pumilio is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. Transgenic flies were generated expressing affinity-tagged Pumilio under the control of an ovary-specific promoter, and Pumilio from whole adult flies and embryos and associated mRNAs were analyzed by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. A characteristic sequence motif was identified in the 3'-untranslated regions of mRNAs associated with Pumilio, and the sufficiency of this motif for interaction with Pumilio was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by Pumilio and uncover evolutionary features of this conserved family of RNA-binding proteins (Gerber, 2006).

More that 1,000 distinct Pum-associated mRNAs were identified, many of which encode functionally related proteins and contain characteristic 3' UTR sequence elements sufficient for interaction with Pum. This finding represents a tremendous increase in potential mRNA targets that may be subject to translational or other posttranscriptional regulation by Pum, and highlights the potential importance of posttranscriptional regulation in multicellular organisms. The roles of Pum in embryonic development, stem cell biology, and the function of the nervous system were discovered by classical forward-genetic approaches; although these approaches uncovered essential functions of Pum and identified several important target mRNAs, the genomic analysis points to many hitherto unrecognized targets mRNAs whose products may be involved in processes less readily accessed by classical genetic approaches. However, the assay is unlikely to exclusively and completely uncover target mRNAs that are associated with Pum in vivo: nontarget mRNAs may associate with Pum and true mRNA targets may dissociate during the affinity-isolation procedure. Moreover, the assay does not reveal whether Pum interacts directly with its target mRNA or indirectly via another protein. Further biochemical and functional experiments are required to verify and dissect regulation of particular mRNAs by Pum. Nevertheless, the identification of a sequence motif for Pum and the striking functional links among mRNA targets strongly suggest an underlying biological role for many of the interactions identified (Gerber, 2006).

Not only was the number of mRNAs associated with Pum remarkably large, but the protein products of these mRNAs shared functional links, including function in the anterior–posterior patterning system, most cyclins, and most subunits of the vacuolar H-ATPase. These functional links add to the growing evidence for an extensive posttranscriptional regulatory system and support recent models for functionally related posttranscriptional modules organized by RBPs. Perhaps Pum, in concert with other proteins, coordinates the temporal or spatial pattern of translation of a large set of mRNAs. For instance, Pum may help ensure that maternally derived mRNAs, which are stored in the unfertilized egg, are translated at the correct developmental stage. Translational repression of maternally derived mRNAs before fertilization is an important mechanism to control the onset of expression of anterior–posterior patterning genes. Another role of Pum is to regulate mitosis of migrating pole cells by inhibition of cycB expression. The finding that most cyclin mRNAs were associated with Pum introduces the possibility of a more general role for Pum in the coordination of the cell-cycle, although cyclins can also have non-cell-cycle-related functions. Regulation of cyclins by Pum may also have a role in sustaining proliferation of stem cells, a proposed ancestral function of Puf-family proteins (Gerber, 2006).

In this work, a consensus RNA-binding element was defined by a genome-wide unbiased search for common sequence motifs among mRNAs selected by a biochemical procedure. The 8-nt core motif [UGUA(A/C/U)AUA] defined in this study is remarkably similar to the sequences in and surrounding box B of the hb NRE, and it resembles core motifs bound by diverse Puf family proteins. It is also in agreement with a crystal structure of human PumHD in complex with hb RNA, which revealed that each of the eight repeats comprising the PumHD interacts with one of eight bases in the bound RNA and suggested that RNA recognition is highly modular. Interestingly, the three amino acid residues in each repeat that directly interact with one RNA base are conserved between Pum and yeast Puf3p paralleling the almost identical core sequences bound by these proteins. Other Puf proteins that bear the same critical amino acid residues for RNA base contacts may bind to highly similar RNA consensus sequences (Gerber, 2006).

The definition of core motifs allows a search for additional potential mRNA targets that were not identified in the affinity isolation procedure. About 10% of all genes in Drosophila contain a computationally defined 8-nt core motif in their 3' UTR; a search for GO terms overrepresented among these 1431 annotated genes found that an unexpectedly large fraction encode proteins involved in morphogenesis or organ development (243 genes, neurogenesis, transcriptional regulation, or proteins that are localized to membranes, in particular the plasma membrane. Interestingly, many of the mRNAs that have the putative Pum binding site but were not enriched in these assays encode proteins with neuronal functions; e.g., Complexin (Cpx; CG32490), which bears a cluster of 10 core motifs in its 3' UTR. Because TAP-Pum was specifically expressed in the ovaries of flies, neuron-specific mRNA targets would not have been accessible to TAP-Pum in vivo and therefore were not expected to be identified. It will be important to extend this analysis to other tissues and organs including the nervous system by the use of tissue-specific drivers available in Drosophila. In addition to identification of tissue-specific potential mRNA targets of Pum, these experiments will also allow the determination of whether and to what extent exchange of Pum-associated mRNAs occurs after cell lysis (Gerber, 2006).

Systematic identification of mRNAs associated with homologous RBPs in various species provides a basis for considering their evolution. Large sets of target mRNAs can now be compared with respect to their structural and functional commonalties and differences. In the case of Pum, conservation of amino acid residues in the PumHDs between homologous Puf proteins correlates with the identified core motifs in 3' UTR of mRNA targets. However, the proteins encoded by the mRNA targets appeared not to be particularly conserved. This discordance suggests that acquisition or loss of RBP-binding motifs in UTRs of genes may provide a surprisingly fluid evolutionary mechanism to modify posttranscriptional regulatory connections (Gerber, 2006).

Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline; Pumilio is dispensable for repression of Cyclin B if Nanos is tethered via an exogenous RNA-binding domain

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, 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 suggested 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).

Protein and RNA Interactions

Posterior patterning in Drosophila embryos is governed by Nanos inhibition of the translation of maternal transcripts of the hunchback gene. nanos response elements (NREs) are sites in HB mRNA that mediate this inhibition. Two proteins present in embryonic extracts, neither one NOS, bind specifically to the NRE in vitro. Binding in vitro correlates with NRE function in vivo. One of the NRE-binding factors is encoded by pumilio, a gene that, like nos, is essential for abdominal segmentation. This suggests that pum acts by recognizing the NRE and then recruiting nos. Presumably, the resulting complex inhibits some component of the translation machinery (Murata, 1995).

Nanos protein promotes abdominal structures in Drosophila embryos by repressing the translation of maternal hunchback mRNA in the posterior. To study the mechanism of nanos-mediated translational repression, the mechanism by which maternal Hunchback mRNA is translationally activated was examined. In the oocyte from wild-type females, where no HB translation is detected, the mRNA has a poly(A) tail length of approximately 30 nucleotides. However, concomitant with translation of the mRNA at between 0.5 and 1.5 hours after egg deposition, the poly(A) tail is elongated to approximately 70 nt. In the absence of nanos activity, the poly(A) tail of Hunchback mRNA is elongated to approximately 100 nt concomitant with its translation, suggesting that cytoplasmic polyadenylation directs activation. However, in the presence of nanos the length of the Hunchback mRNA poly(A) tail is reduced via the nanos response element present in the HB 3'UTR. To determine if nanos activity represses translation by altering the polyadenylation state of Hunchback mRNA, various in vitro transcribed RNAs were injected into Drosophila embryos and changes in polyadenylation were determined. nanos activity reduces the polyadenylation status of injected Hunchback RNAs by accelerating their deadenylation. Pumilio activity, which is necessary to repress the translation of Hunchback mRNA, is also needed to alter polyadenylation. An examination of translation indicates a strong correlation between poly(A) shortening and suppression of translation. These data indicate that nanos and pumilio determine posterior morphology by promoting the de-adenylation of maternal Hunchback mRNA, thereby repressing its translation (Wreden, 1997).

Posterior patterning in the Drosophila embryo requires the action of Nanos and Pumilio, which collaborate to regulate the translation of maternal Hunchback mRNA. Pum recognizes sites in the 3' UTR of HB mRNA. The RNA-binding domain of Pum has been defined and residues essential for translational repression are shown to be embedded within this domain. Nos and Pum can repress cap-independent translation from an internal ribosome entry site (IRES) in vivo, suggesting that they act downstream of the initial steps of normal, cap-dependent translation (Wharton, 1998).

Translation of Hunchback(mat) (HB[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the HB[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of Pum to the NRE is thought to target HB(mat) mRNA for repression. The RNA-binding domain of Pum is an evolutionarily conserved, 334 amino acid region at the carboxy terminus of the approximately 158 kDa Pum protein. This contiguous region of Pum retains the RNA binding specificity of full length Pum protein. Proteins with sequences homologous to the Pum RNA binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the Pum RNA binding domain in other far flung species suggests that the domain is an ancient protein motif, and conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila Pum (Zamore, 1997).

Translational repression of Hunchback mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The Pumilio protein is thought to bind the NREs and thereby repress HB translation. The RNA-binding domain of Pum defines an evolutionarily conserved family of RNA-binding proteins: the Pum-Homology Domain (Pum-HD) proteins, which have been identified in yeast, plants, and animals. The Pum RNA-binding domain [the Drosophila PUM-HD (DmPUM-HD)] has been shown to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of Pum might recognize one NRE. The RNA-binding affinity and stoichiometry of the DmPUM-HD has been analyzed and it is found that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). No cooperative interactions is detected between DmPUM-HD monomers bound at adjacent sites. These results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on an estimate of the intraembryonic concentration of PUM (>40 nM), it is proposed that in vivo nearly all NREs are occupied by a PUM monomer (Zamore, 1999).

The Ovarian tumor protein (also acting upstream of Sex-lethal) is required for the correct distribution of the Pumilio and Oskar mRNAs, while the Bic-D, K10 and Staufen mRNAs are localised in wild type fashion in otu mutants. These observations refer to the early PUM mRNA distribution in nurse cells. A region of homology exists between the carboxy-terminal part of the OTU protein and the mammalian microtubule associated proteins. The more severe the mutation in this region of homology, the more disturbed mRNA distribution is observed in otu mutants (Tirronen, 1995).

Recruitment of Nanos to hunchback mRNA by Pumilio

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).

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).

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

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

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

Structure of Pumilio reveals similarity between RNA and peptide binding motifs

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).

The surface that interacts with Brat appears to be limited to repeats 7, 8, and 9, based on analysis of the collection of Pum mutants that bind normally both to the NRE and to Nos. Five single mutants and one double mutant bearing substitutions in this region of the protein do not interact with Brat. The mutations in repeats 7 and 8 map to the loops, between helices H1 and H2, that are exposed on the convex surface. The Brat binding site is localized immediately adjacent to the Nos binding site on the outer Pum surface, raising the possibility of cooperative interactions between the two cofactors. The close proximity of the sites may explain why Brat is only recruited once Nos has joined the Pum/NRE complex (Edwards, 2001).

The Pum/Nos partnership extends beyond the regulation of HB mRNA to the correct development of the germline. In addition to HB mRNA, Pum and Nos jointly repress translation of maternal cyclinB mRNA in the germline precursor cells. Although the sequences required for this regulation have not yet been defined, Pum binds to an element in the cyclinB 3'UTR that is similar in sequence to the NRE. While the Pum/cyclinB RNA complex can recruit Nos, the resulting ternary complex does not bind Brat efficiently. This suggests a structural difference between Pum/Nos bound to the hb NRE versus the cyclinB RNA, allowing the former to recruit Brat and the latter to recruit a different cofactor present in the germ line. It is noteworthy that much of the Pum outer surface is 'empty' or 'unspecified', and it may be this portion of the molecule that interacts differently with Nos (and other cofactors) when bound to cyclin B mRNA. To understand the basis of this geometric difference will require cocrystallization of Pum/Nos with different RNA sequences. While the allosteric effects of closely related DNA sites on the conformation of transcription factors are well documented, this issue is largely unexplored with RNA binding proteins (Edwards, 2001).

Pum joins a family of helical repeat proteins that includes beta-catenin and karyopherin-alpha with Arm repeats, pp2A with HEAT repeats, karyopherin-beta (also called importin-beta) with a mixture of HEAT and Arm repeats, and protein phosphatase 5 with tetratricopeptide repeats. A broader definition of the family would include proteins with a repeating alpha/beta substructure, such as ribonuclease inhibitor with leucine-rich-repeats. All of these family members are characterized by an extended surface that until now had been thought to be ideally suited for protein-protein interactions. The Pum structure shows that the same kind of surface can also be used to recognize RNA. It is curious that several members of the family, including beta-catenin, karyopherin-alpha, and karyopherin-beta, (and I-kappaB) are involved in movements in and out the cell nucleus. It is tempting to speculate that Puf domains may have roles beyond regulation of mRNA translation, perhaps involving the trafficking of RNA out of the nucleus in some species (Edwards, 2001).

Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo

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).

An anterior function for the Drosophila posterior determinant Pumilio

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).

An intricate combination of spatial and temporal controls orchestrate expression of a gene hierarchy resulting in appropriate embryonic patterning. For bcd, initial spatial restriction in the embryo is provided by anterior localization of translationally silent bcd mRNA. The RNA is then translationally deployed over a short period, resulting in a pulse of Bicoid. This latter process of temporal control of localized bcd mRNA expression is regulated by the evolutionarily conserved bcd NRE to ensure proper head development. Either NRE or Pumilio mutation causes protracted bcd translation. Resulting Bicoid found later in development would have prolonged access to its downstream targets and/or novel access to inappropriate targets from which it is temporally segregated in wild type. Either could interfere with anterior development, ultimately causing head defects (Gamberi, 2002).

While affected Bicoid targets are presently only speculative, it was fortuitously noticed that late zygotic hb expression (hb^zyg) 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 (hb^mat) 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 (Olivas, 2000) 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, hb^mat and hb^zyg mRNA have identical NREs, but hb^zyg 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 hb^mat 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).

Model of the Brain tumor-Pumilio translation repressor complex

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

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

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

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

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

The translational repressor Pumilio regulates presynaptic morphology and controls postsynaptic accumulation of translation factor eIF-4E

Translational repression by Drosophila Pumilio (Pum) protein controls posterior patterning during embryonic development. Pum is an important mediator of synaptic growth and plasticity at the neuromuscular junction (NMJ). Pum is localized to the postsynaptic side of the NMJ in third instar larvae and is also expressed in larval neurons. Neuronal Pum regulates synaptic growth. In its absence, NMJ boutons are larger and fewer in number, while Pum overexpression increases bouton number and decreases bouton size. Postsynaptic Pum negatively regulates expression of the translation factor eIF-4E at the NMJ, and Pum binds selectively to the 3'UTR of eIF-4E mRNA. The GluRIIa glutamate receptor is upregulated in pum mutants. These results, together with genetic epistasis studies, suggest that postsynaptic Pum modulates synaptic function via direct control of eIF-4E expression (Menon, 2004).

Pum's postsynaptic localization at the NMJ suggests that it acts on mRNAs located in the synaptic region of muscle fibers. It could repress local translation of synaptic mRNAs, and this would be consistent with its roles during early development. However, the data are compatible with models in which Pum regulates mRNA stability, localization, or transport at synapses (Menon, 2004).

Conceptually, local translation does not seem necessary for synaptic regulation in the Drosophila neuromuscular system, since NMJs are often close to muscle nuclei and the strengths of individual synapses within an NMJ branch are not known to be separately controlled. However, evidence has been provided that local translation does occur at the larval NMJ. It has been suggested that this regulation provides a mechanism to allow rapid assembly of postsynaptic elements under conditions where NMJs need to be strengthened within a short time period (Menon, 2004).

Increasing larval motility produces a rapid increase in synaptic strength at the NMJ and causes the eventual addition of new boutons. These changes are associated with the appearance of eIF-4E aggregates at NMJs. PolyA binding protein (PABP) spots are also seen at NMJs, and these appear to colocalize with the eIF-4E aggregates. Aggregate numbers can be elevated genetically by overexpressing eIF-4E in muscles or altering PABP expression. Polysome profiles have been identified in transmission electron micrographs of the subsynaptic reticulum (SSR), and eIF-4E/PABP aggregates are hypothesized to colocalize with polysomes and thus label sites of postsynaptic translation. However, this has not been directly demonstrated (Menon, 2004).

The cap binding protein eIF-4E is the limiting factor for translation initiation in many systems. Thus, the appearance of an eIF-4E aggregate might indicate that sufficient local eIF-4E has accumulated to allow efficient translation at that site. GluRIIa mRNA is localized to the NMJ region, and GluRIIa receptor-containing puncta increase in number and intensity under conditions that induce appearance of translational aggregates. This accumulation of GluRIIa may be a consequence of local translation of synaptic GluRIIa mRNA (Menon, 2004).

Pum represses eIF-4E accumulation at synapses as shown by staining LOF mutants with anti-eIF-4E antibody. In pum larvae in which movement had been induced, large increases (5- to 12-fold) were seen in the number of synaptic eIF-4E NMJ aggregates relative to two wild-type reference strains. The amount of eIF-4E in aggregates at each NMJ (evaluated by quantitating aggregate areas and fluorescence intensities) was also elevated by 5- to 12-fold. These phenotypes were fully rescued by restoring Pum expression in muscles. Genetic manipulations, such as altering the levels of PABP or eIF-4E or introducing dunce mutations, have been reported to produce increases of <3-fold in the number of eIF-4E aggregates. This suggests that eIF-4E expression may be maximally derepressed when Pum is absent (Menon, 2004).

Pum is required for repression of eIF-4E accumulation at the NMJ in less motile larvae (those maintained in liquid slurry food), because pum mutants have large numbers of eIF-4E aggregates both before and after transfer to solid media and consequent induction of movement. In contrast, manipulation of PABP levels increased eIF-4E aggregate number only after induction of movement. This implies that Pum is upstream of PABP in the control of eIF-4E expression. It is interesting to speculate that the motility-induced appearance of eIF-4E aggregates in wild-type larvae might be due to partial inactivation of Pum in response to motor activity (Menon, 2004).

Having observed this effect on eIF-4E aggregates in pum mutants, Pum binding in vitro to the eIF-4E 3′UTR was examined and eif-4E was found to contains at least one high-affinity site. A 51 nt subfragment was defined that binds selectively to Pum; binding was shown to be effectively competed by wild-type hb NRE RNAs but not by mutant NREs that do not bind Pum with high affinity. These results indicate that the accumulation of eIF-4E observed at the NMJ in pum mutants could be caused by an increase in the translational efficiency and/or stability of eIF-4E mRNA when it is not bound to Pum. If Pum does repress eIF-4E synthesis, such repression acts selectively on eIF-4E mRNA in the postsynaptic region, since eIF-4E protein does not accumulate elsewhere in the muscles of pum mutants. This is consistent with the fact that Pum protein in muscles is restricted to the NMJ region (Menon, 2004).

eIF-4E overexpression in muscles produces an increase in the number of boutons, perhaps due to the recruitment of new active zones to sites of local translation. If Pum represses eIF-4E synthesis at the NMJ, one might expect that simultaneous elevation of muscle Pum levels would suppress the increase in Ib bouton number produced by crossing UAS-eIF-4E to a muscle-specific driver. This is in fact the case (Menon, 2004).

New and brighter GluRIIA puncta appear in response to increases in larval motor activity or to genetic manipulations of eIF-4E or pabp. These changes in receptor expression do not lead to an increase in postsynaptic responsiveness to transmitter, as measured by mEJP amplitude. They do, however, increase evoked transmitter release. The frequency of spontaneous transmitter release (mEJP frequency) also increases, and additional active zones accumulate at each NMJ. These results suggest that the additional GluRIIa puncta recruit new active zones through a retrograde signaling mechanism and that the new active zones have a normal density of functional receptors. The same effects are observed when GluRIIa is overexpressed in muscles (Menon, 2004).

To examine the downstream effects caused by removal of Pum and the consequent increase in eIF-4E aggregates, pum mutant larvae were stained for GluRIIa. A dramatic increase was observed in the number and intensity of receptor puncta. This result suggests that synaptic GluRIIa mRNA may be translated more efficiently or is more stable at NMJs lacking pum function. When examined by electrophysiology, pum mutant NMJs display elevated mEJP frequencies, suggesting that the extra GluRIIa puncta seen in these larvae may also define additional active zones (Menon, 2004).

An increase was seen in the number of Is boutons in pum mutants, and this phenotype is rescued by postsynaptic Pum expression. Since Is boutons are particularly rich in GluRIIa puncta in pum mutants, one might have expected that new active zones that mediate evoked responses would exist at these supernumerary boutons. However, perhaps postsynaptic defects caused by dysregulation of other, as yet undefined, Pum mRNA targets impair the ability of the new GluRII puncta to increase the evoked response (Menon, 2004).

The morphologies of the Ib and Is NMJs are both regulated by Pum, but in opposite directions and from opposite sides of the synapse. Ib boutons are decreased in number in pum LOF mutants, and this phenotype is rescued by restoring Pum in neurons; Is boutons are increased in number, and this phenotype is rescued by postsynaptic Pum (Menon, 2004).

The divergent Ib NMJ phenotypes produced by loss and overexpression of Pum in neurons suggest that Pum has an instructional role in controlling the growth and morphology of these presynaptic terminals. Loss of pum function and overexpression of Pum also have divergent effects on the morphologies of dendrites. In larval peripheral sensory neurons, Pum overexpression produces a reduction in higher-order dendritic branches, while loss of pum function causes an increase in the length of dendritic spikes. The morphological changes observed in presynaptic terminals and dendrites when pum function is reduced or elevated suggest that it might directly or indirectly repress translation of mRNAs encoding cytoskeletal components (Menon, 2004).

A gene encoding a cytoskeletal protein has been characterized whose mutant phenotypes parallel those of pum. DVAP-33A LOF mutations and DVAP-33A neuronal overexpression produce phenotypes like the pum LOF and neuronal overexpression phenotypes describe in this study. DVAP-33A mutations affect the structure of the synaptic microtubule cytoskeleton (Pennetta, 2002), and microtubules are altered in a similar manner in the NMJs of pum LOF mutants. These findings do not suggest that DVAP-33A is a target of Pum repression but may indicate that it is required for Pum-regulated presynaptic functions (Menon, 2004).

Electrophysiological studies have suggested that ion channels might be targets of Pum regulation. The hypomorphic pum^bem mutation does not produce changes in basal synaptic transmission at the NMJ, but persistent facilitation in motor neurons is prematurely induced by repetitive nerve stimulation. Facilitation is also produced by neuronal overexpression of the para Na⁺ channel or by LOF mutations in the Hyperkinetic K⁺ channel gene, so dysregulation of these or other channels could explain the pum^bem phenotype (Menon, 2004).

It is likely that phenotypes caused by loss of neuronal Pum are complex, arising from the combined derepression of several different targets. Expression of Pum itself might be regulated by environmental conditions, since it has been found that pum mRNA levels increase in the adult Drosophila brain under conditions favoring long-term memory formation (Menon, 2004).

In summary, Pum-mediated posttranscriptional regulation is likely to be important for synaptic morphology and function in both larvae and adults. Pum has distinct roles on the presynaptic and postsynaptic sides of the larval NMJ. Synaptic regulatory pathways involving Pum might be conserved in mammals, since mammalian Pum proteins are very similar in sequence to fly Pum and are expressed in the brain (Menon, 2004).

Regulation of neuronal excitability through Pumilio-dependent control of a sodium channel gene

Dynamic changes in synaptic connectivity and strength that occur during both embryonic development and learning have the tendency to destabilize neural circuits. To overcome this, neurons have developed a diversity of homeostatic mechanisms to maintain firing within physiologically defined limits. In this study, activity-dependent control of mRNA for a specific voltage-gated Na⁺ channel [encoded by paralytic (para)] is shown to contribute to the regulation of membrane excitability in Drosophila motoneurons. Quantification of para mRNA, by real-time reverse-transcription PCR, shows that levels are significantly decreased in CNSs in which synaptic excitation is elevated, whereas, conversely, they are significantly increased when synaptic vesicle release is blocked. Quantification of mRNA encoding the translational repressor pumilio (pum) reveals a reciprocal regulation to that seen for para. Pumilio is sufficient to influence para mRNA. Thus, para mRNA is significantly elevated in a loss-of-function allele of pum (pum^bemused), whereas expression of a full-length pum transgene is sufficient to reduce para mRNA. In the absence of pum, increased synaptic excitation fails to reduce para mRNA, showing that Pum is also necessary for activity-dependent regulation of para mRNA. Analysis of voltage-gated Na⁺ current (I_Na) mediated by para in two identified motoneurons (termed aCC and RP2) reveals that removal of pum is sufficient to increase one of two separable I_Na components (persistent I_Na), whereas overexpression of a pum transgene is sufficient to suppress both components (transient and persistent). It is shown, through use of anemone toxin (ATX II), that alteration in persistent I_Na is sufficient to regulate membrane excitability in these two motoneurons (Mee, 2004).

Analysis of para mRNA shows an NRE-like sequence located in the 5'-UTR indicative that this mRNA might be subject to Pum-dependent translational repression. However, the presence of a cis-regulatory region homologous to the hb NRE motif is, by itself, insufficient evidence to implicate translational repression. To show this beyond doubt, a number of criteria must first be satisfied. These include a demonstration of specific binding of Pum to mRNA containing the cis-regulatory motif. Although gel-shift assays have been used to show an interaction between Pum and the 3'-UTR of hb, strong interaction between Pum and the para NRE-like sequence has not yet been demonstrated. This lack of binding requires that alternate mechanisms for the observed effect of Pum must be considered. One such mechanism could be that Pum is a transcriptional regulator of para, although there is no evidence from previous work to indicate that Pum has any other activities in addition to that of a translational repressor. The observation of altered para mRNA after manipulation of pum is consistent with both transcriptional and translational mechanisms. This is because Pum-dependent translational repression of hb may also increase the rate of degradation of hb mRNA by removal of the poly(A) tail. Of course, Pum could regulate para mRNA through interaction with an intermediate factor. Clearly, additional studies are required to establish the true nature of this regulatory mechanism (Mee, 2004).

Voltage-gated Na⁺ channels are major determinants of neuronal excitability and as such have been identified as a convergent locus for intracellular regulation through PKA- and PKC-dependent mechanisms. In a majority of neurons examined, phosphorylation mediates a reduction in maximal conductance in I_Na that, in Drosophila, has been shown to be sufficient to downregulate membrane excitability in vivo. Analysis of I_Na in aCC/RP2 shows two distinct components, a fast transient current that inactivates rapidly and a smaller persistent current that slowly inactivates. Although the transient current is suited to the initiation of single spikes, its function is compromised if membrane potentials remain depolarized. Motoneurons, including those in Drosophila, produce plateau potentials that amplify and sustain their motor output. Persistent sodium and calcium currents are principal contributing conductances that underlie these plateau potentials. In the light of this, it is satisfying that regulation of I_Na(p) in a Pum-dependent manner is observed. However, it is intriguing that, in the absence of pum (Pum^bem), or when para is upregulated [Tp(1;2)r^+75c], only I_Na(p) is increased, whereas I_Na(t) seemingly remains unchanged. This is even more puzzling when one considers that overexpression of pum is sufficient to downregulate both current components. That these two current components show differential regulation is suggestive that they may arise from different splice variants of para. Para is a highly complex gene with the capacity to produce multiple splice variants. It is quite probable that these isoforms will, through differing kinetics and regulation, contribute to neuronal signaling in unique ways. Indeed, alternative splicing of exons a and i within the first intracellular loop is sufficient to alter I_Na(t), and it is therefore not inconceivable that other isoforms will preferentially affect I_Na(p) (Mee, 2004).

In mammals, the kinetics of I_Na are also reported to be influenced by subunit composition. For example, the presence of the Na_v1.6 Na⁺ channel subunit, in rat Purkinje neurons, confers a greater degree of persistent Na⁺ current than in its absence. Thus, the increase in I_Na(p) that is observed in aCC/RP2 may be accounted for by additional regulatory mechanisms that alter the predominant spli