InteractiveFly: GeneBrief

bruno 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Bruno (Bru) is an ovarian and testicular messenger RNA-binding protein that regulates multiple mRNAs involved in female and male gametogenesis and is also active early in embryogenesis. Bruno functions in the translational repression of Oskar mRNA, and Bruno protein interacts physically with Vasa, an RNA helicase that is a positive regulator of Oskar translation. The gene arrest was characterized by Schupbach and Wieschaus (1991) as a female sterile mutation on the second chromosome of Drosophila. Bruno protein, later found to be identical to the protein coded for by arrest, is required for fertility in both sexes. Few germ cells are present in females that are hemizygous for strong alleles and OSK transcript is not detectable (Webster, 1997). Bruno is one of nearly a dozen maternal genes, belonging to the posterior group, which are involved directly or indirectly in assembly of the pole plasm.

Bruno was originally identified in UV cross-linking experiments as an ovarian protein that binds specfic sequences (Bruno response elements or BRE) in the 3' UTR of OSK mRNA (Kim-Ha, 1995). An expression screen based on the binding of Bru to its target sequence was designed in order to identify the gene coding for Bruno. An ovarian complementary DNA expression library was constructed in a plasmid vector and lysates of clones of transformed bacteria were examined for the presence of a protein that would specifically bind BRE+ mRNA. A plasmid encoding the binding activity was purified and used to clone the complete bruno gene (Webser, 1997).

Three distinct segments of OSK mRNA, termed A, B and C regions, contain Bruno-binding sites. The A and B regions are adjacent to one another near the beginning of the 3' UTR, whereas the C region is located downstream of the AB region, close to the polyadenylation site. The Bruno binding sites contain the consensus sequence UU(G/A)U(A.G)U(G/A)U. When this sequence is modified by mutation, Bru fails to bind. Oskar transgenes lacking Bru binding sites produce embryos that display substantial patterning defects. These defects indicate a posteriorization of the embryo and can be attributed to excess or mislocalized osk activity. These results suggest that Bruno normally acts to restrict OSK activity. BRE mutations have no effect on OSK mRNA localization; rather, they affect the level of translation. The posterior group genes cappuccino, spire, mago nashi, staufen and oo18, each of which are required for the localization of OSK mRNA to the posterior pole of the oocyte, are still required for OSK translation when Bruno-mediated translational repression is missing due to deleted BREs (Kim-Ha, 1995).

How does Bruno act to repress translation? It seems likely that although Bru binds OSK mRNA downstream of the protein coding region, it must affect either the initiation or the progression of translation. Two models have been proposed: the first involves the OSK polyadenylation [poly(A)] tail. Changes in poly(A) tail length are associated with changes in translation (see Drosophila Nanos for a discussion of the role of Nanos in Hunchback mRNA polyadenylation). This model predicts that an OSK mRNA mutant lacking BREs should have a longer poly(A) tail than that of wild-type transcript. However there is no obvous difference in tail length between normal and mutant OSK mRNAs. A second model hypothesizes an interaction between the 3' and 5' ends of the OSK message that influences translational initiation. It is possible that such an interaction promotes translational initiation and that Bruno interferes with this interaction.

What does the interaction between Bruno and Vasa protein signify? The fact that Vasa participates in the activation of OSK mRNA and that the requirement for Vasa is independent of the OSK 3'UTR suggests that Vasa does not activate OSK translation simply by relieving Bruno-mediated repression. Vasa may activate translation through interaction with the OSK 5' UTR. An interaction between Vasa and Bruno bound to the OSK 3' UTR might restrict the ability of Vasa to associate with the 5' UTR until after the OSK transcript is appropriately localized (Webster, 1997)

The arrest phenotype (female sterility) indicates that OSK mRNA cannot be the only target of Bruno regulation, since osk is not required for the early stages of oogenesis and the misregulaiton of OSK by mutation of the BREs does not cause defects in gametogenesis. In addition, although osk is expressed only in females, many arrest alleles are also male-sterile because of reduced numbers of sperm bundles and lack of motile sperm (Schupback, 1991 and Castrillon, 1993). Finally, the disrupted segmentation seen in embryos produced from arrestPA62/arrestPD41 transheterozygotes is not a phenotype that can be attributed to the misregulation of Oskar mRNA. Thus, Bruno carries out multiple roles in development; given its role in the repression of OSK mRNA translation, it is expected that Bru regulates the translation of multiple transcripts (Webster, 1997).

The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche

Maintenance of adult stem cells is largely dependent on the balance between their self-renewal and differentiation. The Drosophila ovarian germline stem cells (GSCs) provide a powerful in vivo system for studying stem cell fate regulation. It has been shown that maintaining the GSC population involves both genetic and epigenetic mechanisms. Although the role of epigenetic regulation in this process is evident, the underlying mechanisms remain to be further explored. This study found that Enoki mushroom (Enok), a Drosophila putative MYST family histone acetyltransferase controls GSC maintenance in the ovary at multiple levels. Removal or knockdown of Enok in the germline causes a GSC maintenance defect. Further studies show that the cell-autonomous role of Enok in maintaining GSCs is not dependent on the BMP/Bam pathway. Interestingly, molecular studies reveal an ectopic expression of Bruno, an RNA binding protein, in the GSCs and their differentiating daughter cells elicited by the germline Enok deficiency. Misexpression of Bruno in GSCs and their immediate descendants results in a GSC loss that can be exacerbated by incorporating one copy of enok mutant allele. These data suggest a role for Bruno in Enok-controlled GSC maintenance. In addition, it was observed that Enok is required for maintaining GSCs non-autonomously, functioning in cap cells. Compromised expression of enok in the niche (cap) cells (CpC) impairs the niche maintenance and BMP signal output, thereby causing defective GSC maintenance. This is the first demonstration that the niche size control requires an epigenetic mechanism. Taken together, studies in this paper provide new insights into the GSC fate regulation (Xin, 2013).

As a Drosophila putative histone acetyltransferase of the MYST family, Enok has been shown to be essential for neuroblast proliferation in the mushroom body (Scott, 2001). This paper presents evidence that Enok is required intrinsically and extrinsically for maintaining GSCs in the ovary. In the case of intrinsic mechanisms, Bruno was identified as an intermediate factor for Enok- controlled GSC maintenance. Molecular and genetic studies revealed that enok mutations in the germline lead to ectopic expression of Bruno in the GSCs, thereby inducing GSC loss probably via promoting cell differentiation. Meanwhile, Enok was also shown a having a non-cell autonomous role in controlling GSC self-renewal throughregulating the niche maintenance and niche-derived BMP signaling output. Thus, this study unraveled a novel regulatory mechanism governing the GSC maintenance mediated by a putative epigenetic regulator in Drosophila. Since Moz and Qkf, the mammalian homologs of Enok, are involved in controlling self-renewal of adult stem cells such as hematopoietic and neural stem cells, the new findings in this paper will help to address how the adult stem cell fate regulation occurs in higher organisms (Xin, 2013).

Numerous studies have shown that GSC maintenance in the Drosophila ovary depends on at least three intrinsic machineries: the BMP/Bam pathway, the Nos/Pum complex and the miRNA pathway. The present study observed that Enok in the germline controls GSC self-renewal independently of BMP/Bam pathway. In the meantime, it was found that loss of enok function does not intrinsically alter the expression pattern of either Nos or Pum in the GSCs, and that enok displays no genetic interactions with either nos or pum in GSCs maintenance. Hence, the results exclude the possibility that the Nos/Pum complex is implicated in Enok-controlled GSC maintenance. Intriguingly, the molecular studies identified Bruno as a potential target of Enok involved in the GSC maintenance. Further genetic analyses suggest that increased expression of Bruno in the GSCs mutant for enok contributes to the GSC loss. bruno encodes an RNA-Recognition-Motifs-containing RNA binding protein which targets a number of mRNAs for their translational repression in the ovary and early embryo. Early on, Bruno was shown to function in patterning the embryo along the AP and DV axis by regulating the translation of oskar and gurken mRNA during late oogenesis. Later, it was reported that Bruno plays a pivotal role in CB differentiation and germline cyst formation at early oogenesis via targeting the Sex-lethal (Sxl) gene. This study has defined a novel function for Bruno in mediating the intrinsic requirements of Enok for maintaining GSCs (Xin, 2013).

Misexpression of Bruno in the germline causes a derepression of PGC differentiation in the gonads from the late third instar larvae. This precocious differentiation phenotype further suggests that bruno gain-of-function in the enok mutants promotes GSC differentiation, thereby eliciting a stem cell loss (Xin, 2013).

To better understand how enok mutation-induced ectopic expression of Bruno promotes the GSC differentiation, it is necessary to identify the potential mRNA target(s) of this RNA-binding protein in the GSCs and their immediate descendants that may function as the differentiation-inhibiting factor in this context. Of all known target genes of Bruno, only Sxl is dynamically expressed in early germ cells including GSCs and CBs, and essential for the GSC/CB fate switch. Preliminary data show that the expression pattern of Sxl remains unchanged in the mutant GSC or CB clones homozygous for the enok allele, ruling out a possible role of Sxl in Enok/Bruno-mediated differentiation control process. Given that the Bruno Response Element (BRE) consensus sequences located in the 3'UTR of the target mRNAs is important for Bruno binding, target candidates from the ovarian mRNAs that contain putative BRE sequences will be sought, based on bioinformatics approaches. However, it is noteworthy that Bruno can also regulate the expression of its target mRNA in a BRE-independent manner. Thus, high-throughput screens such as microarray analysis for differentially expressed genes in the enok mutant ovaries may give more clues for unraveling the mystery (Xin, 2013).

It has been shown that mammalian Moz can acetylate histones H3 and H4 at a number of specific lysine residues. In particular, this MYST family histone acetyltransferase is required for H3K9 acetylation at Hox gene clusters, thus for correct body segment patterning in mice. As the Drosophila homolog of Moz, Enok possesses a conserved MYST histone acetyltransferase (HAT) domain, as well as two PHD fingers and a shared N-terminal domain. Previous studies showed that a point mutation in the MYST HAT domain of Enok causes an arrest in neuroblast proliferation of mushroom body as a null allele (Scott, 2001). Combined with the observation in this paper that the same mutation (enok²) gives defective GSC maintenance phenotype, it is proposed that the HAT activity is implicated in Enok's function during the indicated developmental processes. To further test this scenario, studies will attempt to determine whether the expression of Bruno in the early germ cells could be under the epigenetic control of Enok by examining a possible binding of Enok to bruno gene using chromatin immunoprecipitation (ChIP). In this case, high-throughput screens based on a combination of ChIP-seq and microarray analysis may lead to identification of more target genes of Enok that could mediate the GSC fate regulation controlled by this putative epigenetic factor (Xin, 2013).

The GSC niche plays a key role in controlling GSC self-renewal in the ovary. Although the niche regulation itself is less understood, recent studies showed that systemic factors such as insulin signaling control the niche size, and consequently GSC maintenance at adulthood. Specifically, systemic insulin-like signals maintain the cap cell (CpC) population via modulating Notch signaling. The present study provides the first evidence that the niche maintenance also requires a putative epigenetic factor, and that decrease in the CpC number induced by enok knockdown in the niche is attributable to impaired Notch signaling. Thus, identification and functional characterization of the targets of Enok in controlling the niche size would provide more insights towards understanding how the niche is maintained. Given that insulin signaling is required for controlling the normal decline of both CpCs and GSCs in the aging process, and that epigenetic regulation is important for aging stem cells in mammals, it is assumed that Enok-mediated niche maintenance via Notch signaling has implications in both niche and GSC aging. If this is the case, Enok activity in the niche should display an age-dependent decline. Furthermore, increasing Enok activity could significantly attenuate the age-dependent decrease in the number of both CpCs and GSCs (Xin, 2013).

In conclusion this paper shows that Enok controls GSC maintenance in the Drosophila ovary at multiple levels. In the case of a cell-autonomous control of GSC self-renewal, Enok acts in a BMP/ Bam-independent manner. Instead, activation of Bruno expression in the GSCs and their differentiating progeny links enok mutations in the germline to the GSC loss. In parallel, Enok plays a non-autonomous role in maintaining the GSC population via regulating the niche size and niche-derived BMP signal output from cap cells. Collectively, these results reveal a novel mechanism underlying a putative epigenetic factor-controlled GSC fate regulation (Xin, 2013).

Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. This study shows that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3' UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells this study shows that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. Increased association of the translational repressor Bru1 with osk mRNA was observed upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. It is concluded that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3' UTR (Dold, 2020).

The data indicates that Mkrn1 is essential for oogenesis, embryonic patterning, and germ cell specification. An essential role for Mkrn1 in oogenesis has also been recently reported (Jeong, 2019). By taking advantage of a new allele that specifically disrupts Mkrn1 binding to RNA, this study demonstrates that Mkrn1 exerts its function in embryogenesis and germ cell specification, primarily via regulating osk translation by antagonizing Bru1 binding (Dold, 2020).

Control of osk translation has been studied in depth, revealing a complex spatio-temporal interplay between repressing and activating factors. Relief of translational repression and activation of osk translation is likely to involve multiple redundant mechanisms. For example, Bru1 can be phosphorylated on several residues, and phosphomimetic mutations in these residues inhibit Cup binding in pulldown assays. However, these do not seem to affect translational repression activity in vivo. Stau, Aub, Orb and pAbp have also been implicated in activating osk translation. However, it is unlikely that Mkrn1 controls osk translation by recruiting Stau, as Stau still colocalizes with osk mRNA in Mkrn1W oocytes. Instead, it is proposed that Mkrn1 exerts its positive activity by competing with Bru1 binding to osk 3' UTR (see Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation). This is evidenced by the overlap of their binding sites, the increased association of Bru1 to osk mRNA upon Mkrn1 knockdown and by the observation that reducing bru1 dosage is sufficient to partially alleviate osk translational repression (Dold, 2020).

Two distinct Bru1 binding regions (AB and C) are present in the osk 3' UTR and are required for translational repression. However, the C region has an additional function in translational activation. Indeed, it was hypothesized that an activator binds the C region to relieve translational repression. This activator was proposed to either be Bru1 itself, or a different protein that can bind the BRE-C, which is what this study observed for Mkrn1. The results suggest that the interaction of pAbp with the nearby AR region, and the consequent stabilization of Mkrn1 binding, contributes to the role of BRE-C in osk translational activation. Other factors may also be involved. For instance, Bicoid Stability Factor (BSF) binds the C region in vitro at the 3' type II Bru1-binding site, at a similar site to where Mkrn1 binds osk. Deletion of this site impacts embryonic patterning, yet depletion of BSF has no effect on Osk protein expression up to stage 10, indicating that initial activation of osk translation is effective even in the absence of BSF. In this case, only late stage oocytes display reduced Osk accumulation. Therefore, it is possible that a concerted action of Mkrn1 and BSF exists at the osk 3' UTR site to activate translation and sustain it at later stages (Dold, 2020).

The binding of Mkrn1 to mRNA seems to be extremely specific. The binding to osk is dependent on a downstream A-rich sequence and on interaction with pAbp. A few other targets identified in this study also display enrichment for downstream AA nucleotides. and human MKRN1 has recently been shown to associate preferentially to such sequences. Relevant to this, Bru1 binds to grk 3' UTR in addition to osk, and several proteins that associate with Mkrn1 also associate with grk mRNA. However, this study found no evidence that Mkrn1 binds specifically to grk, which lacks poly(A) stretches in the proximity of its Bru1 binding sites, and consistently, no regulatory role of Mkrn1 on Grk translation was observed (Dold, 2020).

In addition to pAbp, it is noteworthy that Mkrn1 associates with other proteins previously implicated in osk localization and translational activation. Its interaction with eIF4G would be consistent with a role in alleviating Cup-mediated repression, as it could recruit eIF4G to the cap-binding complex at the expense of Cup. However, interaction between Mkrn1 and eIF4E was not observed. The association between Mkrn1 and Imp is also intriguing as the osk 3' UTR contains 13 copies of a five-nucleotide motif that interacts with Imp. This region is essential for osk translation but Osk accumulation is unaffected in Imp mutants, suggesting the involvement of another factor that binds these motifs. In contrast to pAbp, no alteration was observed of Mkrn1 binding when Imp was depleted, indicating that Imp is not required to stabilize Mkrn1 on osk mRNA (Dold, 2020).

The molecular links uncovered in this study between Mkrn1 and RNA-dependent processes in Drosophila are consistent with recent high-throughput analysis of mammalian MKRN1 interacting proteins. RNA binding proteins, including PABPC1, PABPC4, and eIF4G1, were highly enriched among the interactors. Moreover, human MKRN1 was also recently shown to bind to RNA, dependent on the PAM2 motif and the interaction with PABPC1. In addition, the short isoform of rat MKRN1 was shown to activate translation but the underlying mechanism remained unknown. Since in vertebrates MKRN genes are highly expressed in gonads and early embryos as well, it is possible that similar molecular mechanisms are employed to regulate gene expression at these stages. Consistent with this, MKRN2 was recently found to be essential for male fertility in mice. Thus, this study provides a mechanism that explains the role of Mkrn1 in translation and constitutes a solid framework for future investigations deciphering the roles of vertebrate MKRNs in post-transcriptional control of gene expression during gametogenesis and early development (Dold, 2020).

REGULATION

Protein Interactions

Oskar protein directs the deployment of Nanos, the posterior body-patterning morphogen in Drosophila. To avoid inappropriate activation of nos, osk activity must appear only at the posterior pole of the oocyte, where the OSK mRNA becomes localized during oogenesis. Translation of OSK mRNA is, and must be, repressed prior to its localization; absence of repression allows Osk protein to accumulate throughout the oocyte, specifying posterior body patterning throughout the embryo. Translational repression is mediated by an ovarian protein, Bruno, that binds specifically to Bruno response elements (BREs), present in multiple copies in the OSK mRNA 3'UTR. Addition of BREs to a heterologous mRNA renders it sensitive to translational repression in the ovary (Kim-Ha, 1995).

Bruno physically interacts with Vasa. Repression of translation by Bruno is alleviated once OSK mRNA is localized to the posterior pole of the oocyte. The mechanism of this process is unknown; however, it seems likely that the RNA helicase Vasa is involved, since it is localized to the posterior pole of the oocyte and is required for efficient activation of OSK translation. Immune sera, reactive to Bruno protein, cause a shift in the electrophoretic mobility of Osk protein (Webster, 1997).

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. Regulatory elements for both RNA localization and translational repression are situated in the 3' UTR of OSK mRNA, as they are in NOS. In the case of OSK, premature translation is prevented by Bruno, a 68-kD protein encoded by the arrest (aret) locus. Bruno recognizes a repeated conserved sequence (BRE, for Bruno response element) in the osk 3' UTR, and colocalizes with OSK mRNA to the posterior pole. In contrast to NOS, however, 3' UTR-mediated localization at the posterior pole is not sufficient for translation, as heterologous transcripts localized under the control of the full-length OSK 3' UTR are not translated. This indicates that the OSK 3' UTR, although it may participate, is not sufficient for translational activation, and that sequences elsewhere in the transcript are required for translation of OSK mRNA (Gunkel, 1998).

When OSK mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5' region of the mRNA. This novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the OSK 3' UTR is required for activation of Oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction between trans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. Specific interaction of two proteins with the OSK mRNA 5' region is shown; one of these also recognizes the 3' repressor element. p50 is a BRE binding protein that recognizes 3' repressor motifs similar to those recognized by Bruno. p50 functions as a second translational repressor independent of Bruno. The involvement of a second repressor protein in OSK translational control is not unexpected. Indeed, aubergine (aub), a gene required for efficient OSK mRNA translation, is required even when Bruno-mediated repression is alleviated by mutations in the BRE, leading to the suggestion that the aub gene product enhances translation by counteracting the action of a second repressor. It is interesting to note that the requirement for aub function in OSK translation is conferred not only by the OSK 3' UTR but also involves the 5' end of OSK mRNA. Consistent with this possible involvement of the OSK 5' end in translational repression, it is found that in transgenic flies containing an inefficient BRE, premature translation increases when the 5' end is truncated. Understanding the extent to which the 5' end of the OSKtranscript might contribute to overall translational repression will require mutations that selectively disrupt 5' repressor function without simultaneously affecting derepressor function (Gunkel, 1998).

The second protein interacting with the 5' end, p68, could act as a transcriptional activator. p68 is shown to be independent of Bruno. So far it has not been possible to define a p50-binding specificity distinct from that of p68 and to abolish selectively the binding of one or the other protein. Hence, the data do not allow the affirmation that p50 functions as a repressor, not only by binding to the BRE, but also through its interaction with the OSK 5', or that p68 is the derepressor protein. There are several mechanisms by which OSK could be activated at the posterior pole. The translation repressor proteins Bruno and p50 could be degraded by an activity localized at the posterior pole or else be displaced competitively by a derepressor protein. Alternatively, Oskar protein expression could be activated by concentration of the mRNA, resulting in the accumulation of small amounts of Oskar protein by leaky translation, thus initiating a positive feedback loop in which Oskar protein stimulates its own translation. None of these mechanisms is involved in the initial event of translational derepression. In the absence of the 5' derepressor element, OSK transcripts remain repressed, arguing against a passive, local repressor inactivation model. Therefore, the mode of action of the derepressor element is distinct from that of previously described cases, in which repression is released passively by inactivation of a repressor protein and no additional RNA elements are required. The derepressor element does not coincide with the BRE, suggesting that a competitive displacement of the repressor protein from the BRE is unlikely to be the mechanism leading to derepression. Finally, a combination of leaky translation and positive feedback of Oskar protein on its own translation as a mechanism for derepression is unlikely, as reporter transcripts can be derepressed in the absence of endogenous Oskar. Thus mechanisms by which 3' UTR-binding proteins repress translation are still not understood and it is unclear how the 5' derepressor element overcomes translational repression. The fact that transcripts lacking the derepressor element are localized but not translated demonstrates that the element plays little or no role in RNA localization and that localization does not suffice for translational derepression (Gunkel, 1998).

Translational recruitment of OSK mRNA is always accompanied by posterior localization of the mRNA, indicating that localization may trigger the release from translational repression. It is suggested that RNA localization directs osk transcripts into a cytoplasmic subcompartment containing trans-acting factors that interact specifically with the 5' element to mediate derepression. The spatial restriction of the derepression machinery could be achieved by prelocalization of at least some of the components to the posterior pole, or by the localized activation of uniformly distributed factors. During the early stages of oogenesis, OSK mRNA initially fills the entire cytoplasm of the growing oocyte and yet no Oskar protein is detected, even in the posterior region. This suggests that the derepressor proteins are expressed or activated only at certain stages of oocyte development, possibly through signals from the posterior pole. The existence of localized derepressors is supported by the observation that reporter transcripts bearing the BCD 3' UTR into which the OSK repressor element is inserted are localized to the anterior ofoocytes of embryos and not derepressed, even when they contain the derepressor element. The DEAD-box RNA helicase Vasa (whose SDS-PAGE mobility is similar to that of p68), the 120-kD double-stranded RNA-binding protein Staufen, and Aubergine, whose gene has not yet been cloned, play a role in the translation of OSK mRNA. On the basis of the data presented in this report, Staufen and Aubergine could be required to overcome p50-mediated repression, as both are necessary for osk translation, even in the absence of BRE-mediated repression (Gunkel, 1998).

The coupled regulation of Oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of Oskar mRNA, a mechanism whereby Oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of Oskar mRNA. A cell-free translation system, derived from Drosophila ovaries, has been established that faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. This ovary extract, containing endogenous Bruno, is able to recapitulate Oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, a Drosophila embryo extract lacking Bruno efficiently translates Oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in Oskar mRNA translation. This approach opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level (Castagnetti, 2000).

The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole. Proper expression relies on the coordinated localization and translational control of the Oskar mRNA. Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3' untranslated region of the Oskar mRNA. To begin to understand how Bruno acts in translational repression, a yeast two-hybrid screen was performed to identify Bruno-interacting proteins. One interactor, described here, is the product of the apontic gene. Expression occurs in both the somatic follicle cells and the germline nurse cells and oocyte. APT transcripts are detected as early as stage 2A at low levels in the germarium and at higher levels in the follicle cells. The amount of APT mRNA in the soma decreases during the remainder of oogenesis, while the level in the germline increases. APT mRNA becomes concentrated in the oocyte and also accumulates in the nurse cells at about stage 6. APT transcripts continue to be found in both the oocyte and nurse cells throughout oogenesis. To determine when and where Apt protein is expressed during oogenesis, antisera directed against a recombinant Apt protein were prepared and used for protein detection in whole-mount ovaries by confocal microscopy. Apt protein appears in both the germline and somatic cells of the ovary throughout all stages of oogenesis. In the germline, Apt protein is present in both cytoplasm and nuclei. Within the nurse cells the protein is more concentrated in the cytoplasm, while in the oocyte more protein is found in the nucleus. The protein, however, is not localized to any subdomain within the cytoplasm of either the nurse cells or the oocyte. Although Apt protein is not strictly nuclear or cytoplasmic in cells of the female germline, the protein is highly concentrated in nuclei of the ovarian follicle cells and in post-cellularization-stage embryos. The developmental differences in subcellular location suggest that Apt may have functions, perhaps different, in both nuclei and cytoplasm. Nuclear proteins expressed from maternal mRNAs are sometimes present at high levels in the cytoplasm of early embryos. Examples include the Bicoid, Caudal and Hunchback proteins, which appear in both nuclei and cytoplasm shortly after egg laying. As nuclear divisions progress and the density of nuclei increases, nuclear localization of these proteins remains strong while the fraction of protein in the cytoplasm diminishes. Thus there appears to be no early impediment to nuclear localization, simply a paucity of nuclei. In contrast, the subcellular distribution of Apt protein appears to be actively controlled in early development. Apt protein was monitored in early embryos. Even after migration of nuclei to the surface of the embryo, Apt protein remains evenly distributed between nuclei and cytoplasm, unlike any of the examples described above. This unusual persistence of Apt protein in the cytoplasm suggests the existence of a mechanism to control its distribution, reinforcing the notion of roles for Apt in both cytoplasm and nuclei (Lie, 1999a).

Apt is an RNA binding protein. Remarkably, the regions of the OSK 3' UTR bound by Apt, the AB and C regions, are precisely those bound by Bru. A test of Apt binding was performed to determine if Bru and Apt have the same RNA binding specificity: a series of RNAs was used to map the Bru binding sites, called BREs, within the OSK C region. Three of these RNAs retain the BREs and are bound by Bru, while a fourth RNA, CÆ4, lacks the BREs and fails to bind Bru. Apt binds all four RNAs, including CÆ4, indicating that Apt can bind to sites other than BREs (Lie, 1999a).

Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact in Drosophila. Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes. arrest (aret) mutants are defective for Bru (i.e. aret and bru are the same gene and lead to a developmental arrest early in oogenesis. Mutants in apt are zygotic lethal, and some alleles also cause arrested oogenesis. Several different apt alleles were used for all analyses, since the genetics of apt are complex and different alleles have different effects. Testing for a genetic interaction between the aret and apt mutants, dosages of both genes were reduced to see if this would provide a distinct phenotype. Females heterozygous for aret, heterozygous for any of the five apt alleles, or transheterozygous for both aret and an apt allele, were crossed to wild-type males, and the progeny embryos were then examined for cuticular defects. Females transheterozygous for aret and apt produce a fraction of embryos with head defects. Head defects can result from ectopic or excessive posterior body patterning activity, because this activity interferes with expression of the anterior body patterning morphogen, Bicoid. Consequently, the observed head defects could be explained if both Bru and Apt contribute to repression of OSK mRNA translation. Given this interaction, Apontic is likely to act together with Bruno in translational repression of Oskar mRNA (Lie, 1999a).

Earlier genetic analyses of apt have concentrated on the zygotic phenotype. To define more completely the role of apt in the female germline, females were created with apt minus germline clones using the FLP/DFS method. Ovaries containing germline clones were dissected, stained with DAPI to highlight nuclei, and examined for phenotype. Different apt mutants display dramatically different ovarian phenotypes. One allele is indistinguishable from wild type, because females with germline clones of this allele have phenotypically wild-type ovaries and lay eggs that develop into fertile adults. Females with germline clones of another allele also have phenotypically wild-type ovaries, but a small fraction of the eggs laid developed into embryos with head defects. In contrast, ovaries from females with germline clones from two other mutants have phenotypes that are similar to one another and severe: development is arrested in early oogenesis, and the oocyte fails to differentiate, with all nuclei becoming polyploid. In addition, some of the egg chambers have an abnormal number of nuclei. It is concluded that apt is necessary for oogenesis and that loss of apt activity leads to a developmental arrest during oogenesis. Just as for aret mutants, the arrest occurs too early to allow the ovaries to be examined for defects in OSK mRNA translation (Lie, 1999a).

Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the Oskar mRNA 3' untranslated region. A sequence shared by all of the bound RNAs could not be identified. Thus, despite its ability to efficiently discriminate between different parts of the OSK mRNA, Apt appears to be relatively promiscuous in its binding and may recognize many sites or perhaps a structural feature common to many RNAs (Lie, 1999a).

Translational regulation plays a prominent role in Drosophila body patterning. Progress in elucidating the underlying mechanisms has been limited by the lack of a homologous in vitro system that supports regulation. Global control over all transcripts can be achieved through changes in the activity of the translation machinery. More specific controls often rely on cis-acting regulatory elements present within mRNAs, in many cases in their 3' untranslated regions (3' UTRs). Extracts prepared from Drosophila tissues are competent for translation. Ovarian extracts, but not embryonic extracts, support the Bruno response element-dependent and Bruno-dependent repression of Oskar mRNA translation, which acts in vivo to prevent protein synthesis from transcripts not localized to the posterior pole of the oocyte. Consistent with suggestive evidence from in vivo experiments, regulation in vitro does not involve changes in poly(A) tail length. Moreover, inhibition studies strongly suggest that repression does not interfere with the process of 5' cap recognition. Translational regulation mediated through the Bruno response element is thus likely to occur via a novel mechanism (Lie, 1999b).

Two types of experiments were performed to explore the possibility that OSK translational regulation occurs by changes in the poly(A) tail. First, a direct assay was used to determine the length of the poly(A) tail on OSK ovarian transcripts. The poly(A) tail is short, suggesting that polyadenylation is unlikely to function in regulating translation of OSK mRNA. Second, the distribution of poly(A)-binding protein (PABP) in the Drosophila ovary was determined. Although PABP is present at high levels in the germline cells early in oogenesis, it is noticeably depleted from the oocyte at the stage when OSK mRNA is localized to the posterior pole and translationally activated. Although these experiments are suggestive, neither observation provides a compelling argument against a role for polyadenylation in translational activation. For example, the poly(A) tail could act in a manner not requiring PABP. Furthermore, the fraction of translationally active OSK mRNA may be small, as observed for NOS mRNA, making it difficult to detect the presence of transcripts with longer poly(A) tails. To facilitate biochemical analysis of translational control in Drosophila and definitively address the role of polyadenylation in regulation of OSK mRNA translation, in vitro translation systems were developed from Drosophila tissues. Although similar systems have been prepared from other sources, they are unlikely to contain the factors necessary for specific translational regulation of Drosophila mRNAs. Translational activity of the extracts was monitored using reporter mRNAs encoding luciferase (Lie, 1999b).

The ability of Drosophila extracts to recapitulate regulated translation was tested using the 3' UTR of the OSK mRNA, which contains the BRE control elements that mediate Bru-dependent translational repression. The luciferase (luc) reporter mRNA was modified by addition of the following sequences to its 3' end: either the wild-type OSK 3' UTR (BRE+) or a point-mutated version of the OSK 3' UTR (BRE-) that is unable to bind Bru protein in vitro and fails to support translational repression in vivo. In embryo extracts, as well as reticulocyte lysates, both mRNAs were translated with similar efficiencies, revealing no inherent differences in their abilities to be translated. In contrast, translation of the two mRNAs is markedly different in ovary extracts: the luc BRE+ message is translationally repressed approximately 9-fold, on average, in comparison to the luc BRE- message. These results indicate that the ovary extract supports specific regulation of translation (Lie, 1999b).

To determine whether the dependence of regulated translation on BREs reflects a requirement for Bru, two related experiments were performed. First, ovarian extracts were immunodepleted using anti-Bru antibodies or antibodies purified from normal rat serum and were then tested for translational activity. Notably, the control antibodies have no effect on the relative levels of BRE+ and BRE- translation, but depletion with anti-Bru antibodies largely eliminates BRE-dependent repression. Purified recombinant Bru protein was added to the immunodepleted extracts but was not sufficient to restore translational regulation activity. This result suggests that, in addition to removing Bru, immunodepletion using anti-Bru antibodies may remove other proteins required for BRE-mediated translational regulation. Indeed, a significant fraction of Bru protein is present in a large macromolecular complex. In a second type of experiment, purified Bru was added to embryonic extracts that lack Bru and do not support BRE-mediated regulation of translation. Thus BRE-dependent translational repression in vitro requires Bru. Although addition of Bru to embryo extract promotes BRE-dependent repression, the relative translation of BRE- versus BRE+ RNAs is less (average 3.2-fold) than that measured in ovary extracts (average 9.2-fold). There are at least two likely explanations for this difference: the recombinant Bru protein may not be fully active, although the protein displays RNA-binding activity indistinguishable from that of ovarian Bru; alternatively, additional protein(s) present in ovaries but not in embryos may also contribute to repression and may be required for wild-type levels of activity (Lie, 1999b).

The use of BRE+ and BRE- mRNAs reveals the importance of BREs for translational regulation, but does not address the possible role of other sequences in the OSK mRNA 3' UTR. To determine if BREs alone are sufficient, luc reporter mRNAs bearing multimerized consensus BREs (8 copies), either wild-type or containing point mutations that abrogate Bru binding, were generated and their translation measured in vitro. The BRE+ and BRE- RNAs were translated with only a modest difference in efficiency, suggesting that binding of Bru alone is insufficient for complete repression. The 8x BRE supports translational repression in vivo, but repression is not efficient, consistent with the results of the more quantitative in vitro assay. In contrast, RNAs containing the full OSK AB region (a 124 nt region of the OSK mRNA 3' UTR containing at least four consensus BREs interspersed among other sequences) were translationally regulated with a much higher efficiency. Thus it appears that other cis-acting sequences and presumably other factors contribute to Bru-mediated translational repression, a result consistent with the incomplete repression conferred by the addition of Bru to embryonic extracts (Lie, 1999b).

To begin to explore the mechanism of Bru-mediated repression, the in vitro system was used to rigorously test the role of the poly(A) tail in translational regulation, as well as the role of the 5' cap. A number of maternal mRNAs in Xenopus have been shown to be translationally regulated in a manner involving changes in the length of the poly(A) tail (e.g. cyclin mRNAs and c-mos mRNA), and RNA control elements and proteins that contribute to this process have been identified (e.g. the cytoplasmic polyadenylation element (CPE) and the CPE-binding protein (CPEB). Similar forms of regulation have been reported for maternal mRNAs from other animals such as Bicoid and Hunchback mRNAs in Drosophila and tPA and c-mos mRNAs in mouse, and for mRNAs from somatic tissues such as alpha-CaMKII mRNA. To specifically study the requirement for the poly(A) tail in OSK mRNA translational regulation, BRE+ and BRE- reporter RNAs that lacked poly(A) tails were tested in the in vitro system. The translation of these RNAs was regulated in a manner similar to that of transcripts with a poly(A) tail. Though the overall level of translation from the poly(A)-messages was slightly reduced, BRE-dependent repression remains strong. To address the possibility that reporter mRNAs might be polyadenylated during the course of the reaction, two types of experiments were performed using poly(A)- reporter RNAs bearing only the 5' portion of the OSK 3' UTR, including the AB region. This part of the OSK 3' UTR lacks all of the normal polyadenylation elements, including sequences that resemble the binding site for Xenopus CPEB, a factor involved in cytoplasmic polyadenylation. Initially, the translational regulation of these reporter mRNAs was tested and found to be similar to that of RNAs containing the intact 3' UTR. Then the lengths of reporter mRNAs were monitored over the course of incubation in the in vitro translation extract: no changes in size were observed, confirming that polyadenylation does not occur in vitro. It is concluded from these data that translational regulation of OSK mRNA in vitro is independent of the poly(A) tail and that regulation occurs via a novel mechanism (Lie, 1999b).

Another mRNA feature often implicated in control events is the 5' cap. Cap-dependent translational initiation requires recognition of the cap by the eIF4F complex (or its constituent components), a process that is modulated by several types of regulation. A simple test of the importance of the cap in Bru-mediated regulation is to monitor relative translation efficiencies of BRE+ and BRE- mRNAs lacking cap structures. However, uncapped mRNAs are quite unstable in the extracts. An alternative approach is to inhibit cap-dependent initiation by the addition of excess free cap analog (7-methyl-GpppG). In a preliminary experiment, it was shown that translation in the extracts can be substantially inhibited by free cap analog and is thus largely cap-dependent. To test for cap-dependence of Bru regulation, translation of BRE+ and BRE- mRNAs was tested in the presence of free cap. If Bru interferes with recognition or use of the cap, the levels of BRE- and BRE+ translation would be expected to equalize under conditions where cap-dependent initiation is inhibited. Notably, the ratio of BRE-/BRE+ translation remains similar under all conditions tested, ranging from 0 to 2 mM cap competitor. These results very strongly suggest that Bru interferes with a step in translation that is distinct from cap recognition. Additional evidence supporting this conclusion could come from examination of dicistronic mRNAs, in which translation of one encoded protein is initiated in a cap-independent pathway through the use of an internal ribosome entry site (IRES). However, such an experiment must await the identification of an IRES that is active in Drosophila ovaries (Lie, 1999b).

Prior analysis of OSK mRNA translational regulation has provided strong but indirect evidence that Bru acts as a repressor. Molecular genetic data reveals the essential role for BREs, while genetic evidence demonstrates that Bru acts in controlling the level of OSK activity. However, the complex phenotype of mutants defective in Bru prevented a direct demonstration that the absence of Bru leads to a derepression of OSK translation. The results of the in vitro studies now provide compelling evidence that Bru is in fact required for translational repression mediated through the BREs. How the binding of Bru to the 3' UTR of OSK mRNA leads to translational repression remains uncertain, although the availability of the in vitro system defined here is likely to prove useful in addressing that question. Indeed, a definitive demonstration that the 5' cap and changes in poly(A) tail length are not involved in regulation were only made possible through use of this system (Lie, 1999b).

The Drosophila gene squid (sqd) encodes a heterogeneous nuclear RNA binding protein (hnRNP), also known as hrp40. hnRNPs are a large family of proteins that have been implicated in the processing of nascent mRNA transcripts. Recent studies have demonstrated that a subset of hnRNPs, including human hnRNP A1 and A2, Saccharomyces cerevisiae Nplp3 and Hrp1, and Chironomus tentans Hrp36, rapidly shuttle between the cytoplasm and the nucleus. A specific motif, termed M9, has been shown to mediate this nucleocytoplasmic shuttling, and this motif is present in Sqd. Nuclear import of M9-containing hnRNPs is achieved by an association with the nuclear import protein Transportin. Studies of several of these hnRNPs have indicated that one of their major roles is the nuclear export of mRNAs, suggesting that Sqd may perform a similar function during Drosophila oogenesis (Norvell, 1999 and references).

Sqd protein is detected within the somatic follicle cells and the germ-line-derived nurse cells and oocyte. A germ-line mutation in a squid causes female sterility as a result of mislocalization of Gurken (GRK) mRNA during oogenesis. Alternative splicing produces three isoforms: SqdA, SqdB, and SqdS. These isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in GRK mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. It is argued that SqdS is involved in the transport and localization of GRK mRNA. The ability of SqdA to prevent translation of ventrally localized GRK mRNA and its ability to provide peak levels of Grk protein required on the dorsal side of the egg chamber, strongly suggests that SqdA has a role in the accumulation of Grk protein. Moreover, the role of SqdA is both positive and negative, suggesting that SqdA may influence the association of GRK mRNA with appropriate translational regulators (Norvell, 1999).

Evidence is provided that GRK mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. Because Sqd protein binds GRK mRNA directly and belongs to the class of hnRNPs implicated in nuclear mRNA export, it seems likely that Sqd functions in the nuclear export of GRK mRNA. One complication to this model is that in the sqd mutant, GRK mRNA is still able to leave the nucleus and accumulate in the oocyte cytoplasm but not in the dorso-anterior corner. Therefore, the function of the Sqd protein appears to be in the regulated nuclear export of GRK mRNA, such that Sqd is responsible for delivering the GRK message to a cytoplasmic protein involved in its anchoring, possibly coupled to translation. Thus, one might expect that Sqd protein should interact with some cytoplasmic ovarian proteins (Norvell, 1999).

A number of proteins have been implicated in the translational regulation of GRK mRNA, both positively (e.g., encore and Vasa) and negatively (e.g., Bruno). Therefore, an investigation was carried out to see whether Sqd protein could directly associate with any of these candidates. Using the in vitro association assay, interactions were sought between the Sqd isoforms and Encore, Vasa, or Bruno. Although a direct interaction between Sqd and Encore or Vasa could not be found, Sqd protein associates with Bruno protein in vitro. Although the Sqd-Bruno interaction observed in vitro is not extremely strong, it is very consistently observed over multiple experiments. These data show that Sqd protein and Bruno protein can associate with one another. Moreover, these data provide evidence for a link between GRK mRNA localization and translational regulation (Norvell, 1999).

In addition to its requirement in oogenesis, Sqd is also required somatically. A number of lethal alleles of sqd were generated and the ability of the individual isoforms to restore viability of sqd null alleles was investigated. Again, as with the ability of the specific isoforms to function during oogenesis, the three Sqd isoforms differ in their ability to rescue the viability of a lethal sqd allelic combination. Both SqdS and SqdB were capable of rescuing the essential somatic Sqd function: expression of either of these transgenes allows recovery of 11% and 19% of the expected number of mutant sqd adults, respectively. In contrast, however, SqdA is incapable of restoring the essential somatic function of Sqd, since less than 0.2% of the expected number of sqd adults were recovered. These data further demonstrate that the individual Sqd isoforms are not functionally equivalent (Norvell, 1999).

At least two lines of evidence indicate that Sqd protein must be present within the oocyte nucleus for GRK mRNA to be localized properly during oogenesis. (1) Oof the three Sqd isoforms, only SqdS is detected within the oocyte nucleus; among the Sqd transgenic females, only those expressing the SqdS isoform show properly localized GRK mRNA. The differential nuclear accumulation of the SqdS protein is associated with its ability to interact with Drosophila Transportin. SqdA does not possess a Transportin interaction motif. (2) The relationship between K10 and the distribution of Sqd protein also demonstrates the importance of Sqd accumulation within the oocyte nucleus. Mutations in both fs(1)K10 and sqd, consistently cause a mislocalization of GRK mRNA along the entire anterior cortex of the oocyte and lead to the production of strongly dorsalized eggs. Although the nuclear import of SqdS protein is most likely driven by its association with Transportin, K10 function is required for the stable accumulation of Sqd in the oocyte nucleus. This places K10 function upstream of Sqd in the germ line. Accumulation of Sqd protein in the nurse cells is not affected, and in addition Sqd is detectable within the oocyte cytoplasm of K10 mutants. Since the SqdS protein is the only Sqd isoform that is normally detected within the oocyte nucleus, the major effect of K10 must be on the nuclear retention of SqdS (Norvell, 1999 and references).

At this time it is unclear how K10 performs this function mechanistically. K10 protein will physically interact with the Sqd isoforms. The only known motif within the K10 protein is a potential helix-turn-helix domain in the carboxyl terminus, but site-directed mutagenesis of this domain has revealed that this motif is unnecessary for K10 function. K10 could be responsible for the modification of Sqd protein in such a manner as to promote nuclear retention, or alternatively, K10 could form a complex with Sqd protein that stabilizes its accumulation within the oocyte nucleus. In either case, the finding that Sqd protein requires the presence of K10 to accumulate in the oocyte nucleus further suggests that the phenotype of K10 mutant eggs is attributable to an effect on Sqd (Norvell, 1999 and references).

The ability to investigate the roles of the individual Sqd isoforms in the regulation of Grk during Drosophila oogenesis has revealed that there are two key aspects of Grk regulation: GRK mRNA localization and Grk protein accumulation. Both of these critical aspects of Grk regulation are accomplished by Sqd protein; however, these functions are performed differentially by the SqdS and SqdA isoforms. The severity effects of sqd mutation, therefore, reflect the loss of function of both of these proteins within the germ line, thus causing the mislocalization of GRK mRNA and the inappropriate accumulation of ectopic Grk protein. Restoration of either of these levels of regulation allows partial rescue of the D-V patterning defects of sqd mutants, but full rescue requires the function of both SqdS and SqdA. The data suggest that Sqd protein is a key regulator of both aspects of Grk regulation. The interaction of Sqd with the translational repressor protein Bruno provides a link between GRK mRNA localization and its translational regulation. Bruno has been shown directly to have a role in the translational repression of unlocalized Oskar mRNA. The interaction between Bruno and OSK mRNA is mediated by a specific sequence within the OSK message, which is termed BRE. As of yet, the molecular mechanism of Bruno action is not fully understood. However, correctly localized OSK mRNA must somehow be relieved from the Bruno-mediated repression by specific trans-acting factors localized to the posterior of the embryo (Norvell, 1999).

The interaction between Sqd and Bruno suggests that Bruno may play a role in the translational regulation of GRK mRNA. In support of this, GRK mRNA is known to contain a BRE within its 3' UTR, and Bruno has been shown to bind the GRK message. In addition, it has been demonstrated that during late stages of oogenesis, Bruno protein is concentrated at the anterior end of the oocyte, in a position that is coincident with localized GRK mRNA. On the basis of protein interaction data, it is suggested that Bruno may serve as a translational repressor of unlocalized GRK mRNA. As is the case for localized OSK mRNA, the appropriately localized GRK message would be relieved of its Bruno-mediated repression by other localized trans-acting factors. Moreover, the physical association between Sqd protein and Bruno protein suggests an appealing model to explain the similarity of the sqd and K10 mutant phenotypes. These two female sterile mutations represent the only cases in which mislocalized GRK mRNA is translated consistently and efficiently in all egg chambers. It is proposed that the role of Sqd protein is to take GRK mRNA from the oocyte nucleus, recruit Bruno in the cytoplasm, and deliver GRK mRNA to an anchor. In the absence of nuclear Sqd, in either sqd or K10 egg chambers, GRK RNA exits the nucleus by a generalized export mechanism, but does not associate efficiently with the repressor Bruno nor with the anchor. Because the interaction between GRK mRNA and Bruno does not occur, even the unlocalized GRK mRNA is translated efficiently. Using this model, Sqd protein provides the physical link between GRK mRNA transport, localization, and its appropriately regulated translation (Norvell, 1999).

Bruno regulates gurken during Drosophila oogenesis

Translational regulation of localized transcripts is a powerful mechanism to control the precise timing and localization of protein expression within a cell. In the Drosophila germline, oskar transcript must be translationally repressed until its localization at the posterior pole of the oocyte, since ectopic production of Oskar causes severe patterning defects. Translational repression of oskar mRNA is mediated by the RNA-binding protein Bruno, which binds to specific motifs in the oskar 3'UTR. Bruno over-expression is shown to cause defects in antero-posterior and dorso-ventral patterning, consistent with a role of Bruno in both oskar and gurken mRNA regulation. Bruno and gurken interact genetically. Finally, Bruno is shown to bind specifically to the gurken 3'UTR; the dorso-ventral defects caused by Bruno over-expression are due to a reduction of Gurken levels in the oocyte. It is concluded that Bruno plays similar roles in translational regulation of gurken and oskar (Filardo, 2003).

Bru has mainly been studied with regard to its role in translational repression of the posterior determinant Osk. However, the most obvious effect of aret mutations in females is premature arrest of oogenesis, a phenotype unrelated to translational misregulation of osk mRNA. During early oogenesis, the cystoblast fails to develop into a 16-cell cyst in the presence of strong aret mutant alleles. In contrast, weak aret alleles produce apparently normal egg chambers, which then undergo degeneration at stage 9. Hence, Bru affects a number of cellular processes that take place in the germline, including osk translational regulation. By analogy to osk regulation, the aret phenotype might therefore be caused by misregulation of target RNAs which, in the wild-type, are tightly regulated by Bru. Another not mutually exclusive possibility is that lack of functional Bru impairs other processes in which the protein is involved and that are unrelated to its RNA-binding activity. The fact that Bru over-expression causes phenotypes similar to Bru loss-of-function, and the fact that these defects can be modulated by simultaneous over-expression of BRE-containing RNA, supports the hypothesis that at least some of the aret early oogenesis phenotypes are indeed the result of RNA mis-regulation (Filardo, 2003).

Bru over-production, like Bru loss-of-function, impairs ovarian development. Most remarkably, Bru-over-expressing egg chambers that develop beyond the earliest stages undergo an extra round of division with incomplete cytokinesis, suggesting a role of Bru in regulation of the cystocyte divisions. Another gene, encore (enc), has also been shown to be involved both in regulation of germline mitoses and in establishment of oocyte polarity, the latter due to its role in grk mRNA localization and translation. enc encodes a 210 kDa protein with one conserved R3H domain, a single-stranded nucleic acid-binding domain. Thus, Bruno is not the only RNA-binding protein to be involved in regulation of the cystoblast divisions and in establishment of polarity. Given the nature of the proteins, it is likely that both Enc and Bru mediate their oogenesis effects through RNA binding. In contrast to Enc, which is required for Grk accumulation, Bru appears to negatively regulate Grk levels, most likely at the level of translation (Filardo, 2003).

Bru also provides a new example of genes whose activity affects establishment of both the A/P and the D/V axis. Bru has previously been shown to repress osk mRNA translation and new results show that Bru negatively regulates grk as well. The grk 3'UTR contains a single BRE. The interaction between Bruno and grk is most likely responsible for the observed reduction in Grk signal in egg chambers in which Bru is over-expressed. Another protein involved in regulation of both osk and grk is the DEAD-box RNA helicase Vasa, although in this case mutant alleles show a reduction in Osk and Grk levels, suggesting a positive role for Vas in osk and grk mRNA translation. oo18 RNA binding (orb), encoding the Drosophila cytoplasmic polyadenylation element binding protein (CPEB) is also required for both osk and grk mRNA localization and translation. Thus, regulation of the mRNAs encoding the embryonic polarity determinants Osk and Grk appears to be intimately related, involving many of the same RNA regulatory proteins (Filardo, 2003).

Cup is an eIF4E binding protein that associates with Bruno and recruits Barentsz in the regulation of oskar mRNA translation in oogenesis

Translational control is a critical process in the spatio-temporal restriction of protein production. In Drosophila oogenesis, translational repression of oskar1 (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation. This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive. An ovarian protein, Cup, is required to repress precocious osk translation. Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins. A mutant Cup protein lacking this sequence fails to repress osk translation in vivo. Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner. These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex (Nakamura, 2004).

In a search for new components of the oskar RNP complex, this study identified the 147-kD protein of this complex as the product of the female sterile gene cup. Surprisingly, cup is required both for translational repression and localization of oskar mRNA. Cup was found to bind to eukaryotic initiation factor 4E (eIF4E) and is necessary to recruit the localization factor Barentsz to the complex. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. Because of its interactions with both the localization and translational control complexes, it is proposed that Cup is a likely regulatory target for the coupling machinery (Nakamura, 2004).

Cup has been identified as a component of an eight-protein complex that contains oskar mRNA (Wilhelm, 2000). cup is also required for oskar mRNA localization and is necessary to recruit the plus end-directed microtubule transport factor Barentsz to the complex. eIF4E is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. It is proposed that Cup coordinates localization with translation (Wilhelm, 2003).

During localization, osk RNA forms cytoplasmic granules in both nurse cells and the oocyte. The granules contain several proteins, including the DEAD-box protein Maternal expression at 31B (Me31B), the Y-box protein Ypsilon schachtel (Yps), and Exuperantia (Exu). Genetic evidence has shown that Exu is involved in the proper localization of bcd and osk RNAs in oogenesis, although the molecular function of Exu remains unknown. Both Yps and Me31B are involved, directly or indirectly, in the translational silencing of osk RNA in oogenesis. Yps antagonizes Orb, a positive regulator of osk RNA localization and translation. In egg chambers lacking me31B, osk RNA is prematurely translated in early oogenesis (Nakamura, 2001). These data indicate that the granules are maternal ribonucleoprotein (RNP) complexes containing proteins required for both RNA localization and translational control. The complex is highly enriched in eIF4E and a germline protein, Cup. Cup is required to repress osk translation. Evidence is provided that Cup-mediated translational repression is achieved by preventing the assembly of the eIF4F complex at the 5' end of osk RNA, and that Cup acts together with Bru to repress osk translation (Nakamura, 2004).

To identify new proteins in the Me31B complex, ovarian extracts from wild-type females were immunoprecipitated on a preparative scale using an affinity-purified anti-Me31B antibody (α-Me31B). α-Me31B specifically coprecipitatesmany proteins from the extracts. Mass spectrometric analyses of these proteins revealed that both Exu and Yps, the known components in the Me31B complex (Nakamura, 2001), are present in the immunoprecipitates. The analyses also revealed that the 35 kDa protein was eIF4E and the 150 kDa protein is Cup, a germline-specific protein required for oogenesis. Cup is expressed from early oogenesis and present until the blastoderm stage of embryogenesis. Numerous cup alleles have been isolated as female sterile mutants, which show a wide range of phenotypes. However, the biochemical function of Cup has remained elusive (Nakamura, 2004).

To examine the association among Me31B, eIF4E and Cup in vivo, ovaries expressing a GFP-Me31B fusion protein were stained for eIF4E and Cup. The GFP-Me31B form cytoplasmic particles in the germline, and the distribution patterns of the fusion protein are indistinguishable from those of endogenous Me31B (Nakamura, 2001). α-eIF4E stains cytoplasmic particles that are positive for GFP-Me31B. This colocalization is observed throughout oogenesis. Cup colocalized with GFP-Me31B is also found throughout oogenesis. Thus, eIF4E, Cup, and Me31B all form a complex during oogenesis (Nakamura, 2004).

To better understand the interactions between Me31B, eIF4E, and Cup, ovarian extracts were immunoprecipitated by α-Me31B and α-eIF4E, and the precipitates were analyzed by Western blotting. α-Me31B coprecipitates eIF4E and Cup, and α-eIF4E coprecipitates Me31B and Cup, indicating that they all form a complex. However, in the presence of RNase during immunoprecipitation, α-Me31B fails to coprecipitate eIF4E or Cup. Thus, the Me31B-eIF4E and the Me31B-Cup interactions are indirect and probably mediated through RNA in the complex. In contrast, α-eIF4E coprecipitates Cup even in the presence of RNases, suggesting a direct interaction between eIF4E and Cup in vivo (Nakamura, 2004).

The interaction of Cup and eIF4E in vitro was studied using a GST pull-down assay. GST-eIF4E pulls down Cup synthesized in vitro. The association is unaffected by RNase. These results indicate that Cup associates with eIF4E in vitro and that the interaction is RNA independent (Nakamura, 2004).

The results show that Cup is an eIF4E binding protein that is involved in translational repression of osk RNA during oogenesis. The conserved YxxxxLφ motif in Cup is important for eIF4E binding and Cup and eIF4G are likely to bind the same surface of eIF4E. These results suggest that Cup competes with eIF4G for eIF4E binding, and hence inhibits translation initiation. Cup^Δ212 protein, which lacks the conserved eIF4E binding sequence, is unable to bind eIF4E in vivo, and fails to repress osk translation. These results strongly suggest that the Cup-eIF4E interaction is essential for the Cup-mediated repression of osk translation, although it is possible that other of Cup's functions are also affected in the cup^Δ212 mutant. Furthermore, Cup was found to interact with Bru in a yeast two-hybrid assay and that the Cup-eIF4E complex associates with Bru in an RNA-independent manner. Based on these results, it is speculated that the Bru-mediated repression of osk translation is operated, at least in part, through the interaction with Cup, which binds eIF4E and prevents the eIF4E-eIF4G interaction at the 5' end of osk RNA (Nakamura, 2004).

Because btz mutants display a late stage oskar mRNA localization defect similar to that of cup mutants (van Eeden, 2001), the effect of cup mutants on the distribution of Btz was examined. Normally, Btz protein is present on the nuclear envelope in nurse cells and colocalizes with oskar mRNA in the oocyte. However, in cup¹/cup⁴⁵⁰⁶ egg chambers, the accumulation of Btz protein within in the oocyte is greatly reduced from stage 1 onward, whereas the Btz present on the nuclear envelope in the nurse cells is unaffected. The failure in the transport of Btz to the oocyte is not due to a general defect in assembly of the oskar RNP since cup¹/cup⁴⁵⁰⁶ egg chambers localize Yps and oskar mRNA normally during early oogenesis. Thus, Cup is specifically required to localize Btz to the oocyte. This result, together with the findings that Cup and Btz colocalize as well as share similar oskar mRNA localization defects, argues that cup mutants fail to localize oskar mRNA because Cup is required to recruit Btz to the complex (Wilhelm, 2003).

Since all mutations isolated to date that disrupt oskar mRNA localization also block oskar translation, the role of cup in oskar translation was examined. Surprisingly, Oskar protein accumulated prematurely in the oocyte during stages 6 and 7 in cup¹/cup⁴⁵⁰⁶ egg chambers, indicating that cup is required to translationally repress oskar mRNA during these stages. It is also worth noting that in cup mutants accumulation of Oskar protein was observed at only those sites where oskar mRNA is most enriched. This may be due to the fact that the cup alleles used in this study are hypomorphic alleles. The effects of cup are specific for oskar mRNA since the localized translation of gurken mRNA at the dorsal anterior region of the oocyte during stage 9 is unaffected in a cup¹/cup⁴⁵⁰⁶ mutant background. Thus, cup is not a general translational regulator of localized messages (Wilhelm, 2003).

To better understand the role of Cup in maintaining the translational repression of oskar mRNA, attempts were made identify components of the translation machinery that were present in the complex by testing likely candidates. Immunoprecipitation of GFP-Exu and Yps show that eIF4E, the 5' cap binding component of the translation initiation complex, is specifically associated with these components of the oskar RNP complex. eIF4E and other components of the translation initiation machinery are generally thought of as being homogenously distributed due to their critical role in translation throughout the cell. Surprisingly, eIF4E is localized in a dynamic pattern within the oocyte. eIF4E is localized to the posterior of the oocyte early in oogenesis during stages 1-6. At stages 7 and 8, eIF4E redistributed to the anterior of the oocyte, and during stages 9 and 10, eIF4E accumulated at the posterior of the oocyte. This pattern of localization was also observed with a GFP-eIF4E protein trap line. Thus, eIF4E localizes in a temporal-spatial pattern identical to that of Cup, suggesting that it is a component of the complex in vivo (Wilhelm, 2003).

Since Cup is required for the correct localization of Btz to the oocyte, whether Cup is required for eIF4E localization was investigated. Immunostaining of cup¹/cup⁴⁵⁰⁶ mutant egg chambers reveals that Cup is required for localization of eIF4E to the posterior of the oocyte from stage 1 onward. Disruption of cup function does not significantly affect the level of unlocalized eIF4E, indicating that the defect is primarily in the recruitment of eIF4E to the complex (Wilhelm, 2003).

Because Cup shares limited homology with 4E-T, a known eIF4E binding protein and a translational repressor in mammals, whether Cup binds to eIF4E was tested using a two-hybrid interaction assay. This assay showed a direct interaction between Cup and eIF4E. Cup interacted equally with both isoforms of eIF4E. Deletion analysis of Cup using the two-hybrid assay identified an eIF4E interaction domain that contains a canonical eIF4E binding motif. This motif is found in eIF4G as well as translational repressors (e.g., 4E-T) that block translation by preventing the eIF4E-eIF4G interaction. Thus, Cup is an eIF4E binding protein that acts directly to repress oskar translation (Wilhelm, 2003).

Thus, the assignment of Cup as a novel component of the oskar RNP complex is based on a number of findings: (1) Cup copurifies with both Exu and Yps, which have both been shown to be in a biochemical complex with oskar mRNA; (2) Cup protein exhibits the same dynamic localization pattern as that seen for oskar mRNA as well as other components of the complex; (3) Cup colocalizes with Yps and Btz particles, indicating that this these proteins form a complex in vivo; (4) the relevance of the biochemical association is supported by genetic studies of cup function, demonstrating a role for cup in translational repression of oskar mRNA as well as recruitment of Btz and eIF4E to the RNP complex (Wilhelm, 2003).

Because Cup is a translational repressor that is also required to assemble the oskar mRNA localization machinery, it is proposed that the coupling between localization and translation occurs by regulating these two functions of Cup. In this model, Cup is required early in the assembly of the transport complex in order to recruit components, such as Btz, that will later be used to dock to kinesin. This is consistent with the results that cup is required to localize Btz to the posterior pole and that cup mutants exhibit oskar mRNA localization defects comparable to those observed in btz mutants. The fact that mammalian Btz and 4E-T are nucleocytoplasmic shuttling proteins suggests that the defect in particle assembly in cup mutants may occur in the nucleus rather than in the cytoplasm. However, further studies will be necessary to determine the site of assembly (Wilhelm, 2003).

Because Btz is normally part of the transport complex throughout oogenesis even though it is only required for the kinesin-mediated transport step during stages 9 and 10, it is further proposed that the complex undergoes rearrangement in order to activate Btz and switch from minus end-directed transport to kinesin-mediated transport. Since the direct binding of Cup to Btz or Btz to kinesin has not yet been established it is unclear how many components of the complex may be involved in this reorganization (Wilhelm, 2003).

Once the complex reaches the posterior pole, it is argued that the localization machinery is disassembled and the interaction between Cup and eIF4E is broken to allow translational activation. Because Cup is stably maintained at the posterior pole after stage 9, whereas Btz is not, it is proposed that the trigger that disrupts the binding of Cup to eIF4E also leads to partial disassembly of the localization machinery via Cup. The molecular trigger for such rearrangements is unknown, however, the ability of 4E-T to bind eIF4E is regulated by phosophorylation (Pyronnet, 2001). Studies directed at identifying regulators of the Cup-eIF4E interaction might lead to greater mechanistic insights into the coupling mechanism (Wilhelm, 2003).

One of the attractive features of this model is that it suggests how coupling might be accomplished in other systems. Recent work in neurons on the translational regulator CPEB suggests that it can promote the transport of mRNA into dendrites. Since CPEB represses translation by recruiting the eIF4E binding protein, maskin, to transcripts, it is possible that the observed transport effect is due to a requirement for maskin to assemble the localization machinery. Thus, Cup may be representative of a general class of eIF4E binding proteins whose role is to couple mRNA localization to translational activation (Wilhelm, 2003).

Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles

Prior to reaching the posterior pole of the Drosophila oocyte, oskar mRNA is translationally silenced by Bruno binding to BREs in the 3' untranslated region. The eIF4E binding protein Cup interacts with Bruno and inhibits oskar translation. Validating current models, the mechanism proposed for Cup-mediated repression has been directly demonstrated: inhibition of small ribosomal subunit recruitment to oskar mRNA. However, 43S complex recruitment remains inhibited in the absence of functional Cup, uncovering a second Bruno-dependent silencing mechanism. This mechanism involves mRNA oligomerization and formation of large (50S-80S) silencing particles that cannot be accessed by ribosomes. Bruno-dependent mRNA oligomerization into silencing particles emerges as a mode of translational control that may be particularly suited to coupling with mRNA transport (Chekulaeva, 2006).

Tight restriction of Oskar protein to the posterior pole of the Drosophila oocyte is crucial for development of the future embryo and is largely achieved by posterior localization of oskar mRNA and its translational inhibition prior to localization. This molecular analysis of oskar mRNA translational repression and of the relative roles of Bruno and Cup in this process has demonstrated the existence of two distinct modes of repression by Bruno and their mechanistic basis. This study has demonstrated directly the mechanism hypothesized for Bruno/Cup function, whereby cap-dependent 43S complex recruitment is inhibited. It has also been discovered that Bruno exerts its function through a second mechanism that does not require functional Cup and its interaction with eIF4E. This mode of repression involves Bruno-dependent oskar mRNA oligomerization and assembly into silencing particles, unusually large RNPs in which oskar remains inaccessible to the translation machinery (Chekulaeva, 2006).

This analysis of ribosomal complexes assembled on oskar reporter mRNA in vitro revealed that 48S initiation complex formation is inhibited both in the presence and in the absence of Cup-eIF4E interaction. This result is compatible with either of two possible mechanisms: (1) inhibition of small ribosomal subunit recruitment and (2) blocking of the following step: scanning of the 5'UTR by the small ribosomal subunit. Indeed, such scanning complexes in which the 43S subunit moves along the mRNA searching for the initiation codon are not stable and can easily dissociate during centrifugation in the sucrose density gradient. Therefore, as with a failure in recruitment of the small ribosomal subunit, interfering with scanning would also result in a reduction of the 48S peak (Chekulaeva, 2006).

The first of the two oskar repression mechanisms requires the interaction of Cup and eIF4E. This Cup-dependent repression process also requires a m⁷GpppN cap on the mRNA. Since binding of the small ribosomal subunit represents the cap-dependent step in translation initiation, these results provide a direct demonstration of the hypothesized mechanism for Cup regulation of oskar mRNA: a block of cap-dependent 43S recruitment mediated by a functional interaction between Cup-eIF4E and Bruno. Interestingly, it was observed that Cup recruits eIF4E to the mRNA in a cap-independent manner, suggesting an unexpected role for Cup, over and beyond its role in translational repression. Recruitment of eIF4E to oskar mRNA complexes by Cup might ensure colocalization and local enrichment of this otherwise limiting translation factor at the posterior pole, where oskar mRNA is translationally activated (Chekulaeva, 2006).

The second mechanism of oskar regulation revealed by this analysis also involves Bruno but requires neither Cup-eIF4E interaction nor a m⁷GpppN cap. It is therefore unlikely that this mechanism directly interferes with cap-dependent recruitment of the 43S complex (Chekulaeva, 2006).

This analysis shows that repressed oskar reporter mRNA forms unusually heavy complexes sedimenting between the 48S and 80S peaks. Importantly, these complexes form in the absence of the Cup-eIF4E interaction and of ribosomal subunit binding, as revealed by their persistence upon addition of cap analog. It is therefore proposed that oskar mRNA is sequestered in such large RNP complexes and hence inaccessible to the 43S preinitiation complex. Consistent with such a sequestration hypothesis, the repressed mRNA is selectively protected from the degradation machinery. Interestingly, a model of 'masked' (translationally inactive, stable) mRNAs was put forward 40 years ago. Masking factors were proposed to bind to mRNA and promote aggregation into higher-order condensed particles, protected from any processive events, including translation, degradation and polyadenylation/deadenylation (Chekulaeva, 2006).

The current experiments reveal that assembly of oskar mRNA into RNP complexes as large as monoribosomes can occur without any involvement of the RNA with the ribosomal subunits. These findings shed an unexpected light on the published literature, where complexes of 80S and larger can be intuitively taken as an indication of ribosomal association and translation elongation. Based on the co-sedimentation of oskar mRNA with polysomes and experiments involving the polysome-disrupting agent puromycin, it has been concluded that in the ovary, repressed oskar mRNA is associated with translating ribosomes (Braat, 2004). The current data challenge this conclusion, because it is shown directly that heavy RNPs (up to 80S in vitro) can form on oskar reporter mRNA without ribosomal subunit binding. The Braat study employed experimental conditions in which more than one variable was simultaneously changed. Specifically, the Mg²⁺ concentration, which can affect both polysome and RNP stability, differed by an order of magnitude between the puromycin-treated samples (2.5 mM Mg²⁺) and the cycloheximide control (25 mM Mg²⁺). This experiment was repeated, altering only one variable (puromycin). When the Mg²⁺ concentration is kept constant, puromycin does not affect the heavy RNPs that were previously interpreted as being 'polysomal'. It is suggested that oskar mRNA is engaged in puromycin-insensitive, heavy silencing particles that are sequestered from ribosomal engagement and that cosediment with polysomes (Chekulaeva, 2006).

Remarkably, oskar silencing particles comprise not single mRNA molecules but mRNA oligomers, whose formation is dependent on the specific association of Bruno with the BREs. The fact that the same components, Bruno and BREs, are responsible for both translational repression and mRNA oligomerization into silencing particles suggests a causal relationship between oligomerization and translational silencing (Chekulaeva, 2006).

The interesting finding that Cup is present in the heavy but not in the light RNP peak highlights the role of silencing particles in oskar repression. The sucrose gradient analysis of repressed complexes in cup^Δ212 extract demonstrates that Cup-4E interaction is not required for silencing particle formation. However, the fact that Cup is exclusively associated with the silencing particles but not with the light RNP peak of repressed mRNA suggests that particle formation may contribute not only to Cup-independent repression but also to Cup-dependent repression (Chekulaeva, 2006).

Consistent with the in vitro demonstration of oskar mRNA multimerization in silencing particles, it has been demonstrated that oskar mRNA molecules can self-associate through the 3′UTR for localization to the posterior pole of the oocyte. Since oskar mRNA is translationally repressed prior to posterior localization, it is tempting to speculate that the large silencing complexes that have been identified as containing oskar mRNA multimers are related to oskar mRNA localization complexes. It should be noted, however, that at present, there is no evidence for a role of the translational repressor Bruno in oskar mRNA localization. It is also possible that direct intermolecular RNA-RNA interactions might contribute to oskar oligomerization, as in the case of bicoid mRNA (Chekulaeva, 2006).

The current work suggests that silencing particles in Drosophila ovary extracts form by Bruno-mediated mRNA oligomerization from lower complexity precursors. Recent reports have described the presence of large particles, P bodies, in both yeast and mammalian cells. From these P bodies, silenced mRNAs may either return to the translating pool or be targeted for degradation. Furthermore, this work has suggested that RNP particles may aggregate from precursors into higher-order structures. In this regard, it is notable that both Cup and Me31B [Me31B has been implicated in translational regulation of Oskar mRNA during early oogenesis (Nakamura, 2001) and is a homolog of the S. cerevisiae P body component and translational repressor Dhh1p (Coller, 2005)] are present in silencing particles -- it has recently been shown that the mammalian eIF4E binding protein, 4E-T, and Dhh1p, the S. cerevisiae homolog of Me31B, are P body components. While the factors that promote P body aggregation in mammals and yeast are currently unknown, Bruno has been identified as a critical factor for silencing particle formation. Interestingly, this analysis shows that while Bruno is associated with the repressed mRNA both in silencing particles and lighter RNPs, Cup associates only with the mRNA in silencing particles. The fact that Bruno does not recruit Cup in the light RNP peak suggests that effectors may exist that regulate this interaction and cause RNP transition to silencing particles by addition/modification of factors and/or conformational change. It will be interesting to further explore the relationship between silencing particles and P bodies (Chekulaeva, 2006).

The exciting finding that oskar silencing particles comprise not single mRNA molecules, but mRNA multimers, suggests a mode of mRNA translational control that seems particularly suited to coupling of translational repression with mRNA transport within the cell. Such a repression mechanism would also allow coordinate repression of multiple oskar mRNAs, as well as coordinate derepression of the mRNAs within the silencing mRNP, upon its localization at the oocyte posterior pole. The particles could in principle contain other RNAs regulated and assembled into RNPs by common components. It will be interesting to determine if gurken mRNA, which is translationally repressed by Bruno (but not Cup) and colocalizes with oskar mRNA during the early stages of oogenesis, is coassembled with oskar mRNA in silencing particles (Chekulaeva, 2006).

DEVELOPMENTAL BIOLOGY

Bruno possesses sex specific isoforms. There are three female-specific transcripts of 2.7, 3.3 and 3.7 kb, as well as a single male-specific transcript of 4.0 kb. These transcripts are present in ovaries and testes, respectively, but are not detectable in the remaining somatic tissue. The three ovarian mRNAs are abundant in ovaries and in 0-2 hour embryos, but are extremely reduced or absent during the rest of embryogenesis and the larval stages of the cell cycle. Two pupal transcripts are evident. One migrates slightly slower than the male-specific message and one slightly faster than the 3.3-kb female specific message. The structural bases of these differences in mRNA sizes is not yet known (Webster, 1997).

During oogenesis, BRU mRNA is first expressed in all of the germ cells in region 2A of the germarium and continues to be found throughout the cytoplasm of both the nurse cells and oocyte as oogenesis progresses. Bru protein is also expressed throughout the nurse cells. In contrast, the distribution of Bru protein in the oocyte is highly restricted, showing striking colocalization with OSK mRNA: at stages when OSK transcripts accumulate in discrete regions of the oocyte, Bru protein is highly concentrated in the same regions. Bru protein first appears in all germ cells in region 2A of the germarium and rapidly becomes concentrated in the presumptive oocyte. Bru quickly resolves as a crescent at the oocyte posterior, following a dynamic pattern similar to that of OSK mRNA, including a transient accumulation at the anterior of the oocyte infrequently detected during stages 7 and 8 of oogenesis. In early embryos, however, although OSK mRNA continues to be localized to the posterior pole, Bru protein is no longer detectable in whole-mount tissue. Bru protein is also localized to a distinct anterodorsal zone in stage 10 oocytes, a region where OSK mRNA does not appear. This localization is intriguing, as it coincides with the position of Gurken mRNA. Although Bru protein binds in vitro to GRK mRNA, the significance of this interaction is unknown (Webster, 1997).

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. GFP-tagged versions of Vasa and Aubergine were used to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. Perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes (Snee, 2004).

Perinuclear nuage contains, in addition to Vas and Aub, the Maelstrom (Mael), and Gustavus (Gus) proteins. Another component, Bruno (Bru), is a protein that acts in translational repression of osk and gurken (grk) mRNAs. By immunolocalization and expression of a GFP-tagged version of this protein, it was found that Bru is concentrated in perinuclear clusters, similar to the distribution of known nuage components. Double labelling experiments with GFPAub confirmed that Bru colocalizes with nuage. However, Bru is also present at high levels in the cytoplasm, raising the question of whether the colocalization reveals an association with nuage or simply reflects random overlap of an abundant protein with the more narrowly distributed nuage. Evidence that Bru is specifically associated with nuage comes from analysis of Bru distribution in vas mutants: as for other nuage components, the perinuclear clusters of Bru are strongly reduced. Given this identification of Bru as a nuage-associated protein, arrest (aret) mutants (the aret gene encodes Bru) were included in a genetic analysis of nuage. The other genes tested were vas, tud, aub and spindle E (spnE), each of which encodes a nuage component or has been shown to be required for nuage formation, or both (Snee, 2004).

Live imaging was used to better characterize the perinuclear nuage defects seen in static images and to extend the analysis to include cytoplasmic nuage particles. GFPAub was used as the nuage marker to test the role of vas, aret and tud, and VasGFP was used to test the roles of aub and spnE. The live imaging confirmed, for the most part, the basic observations from analysis of fixed samples. In vas mutants perinuclear nuage is almost completely absent, with only a few nuage clusters visible. Loss of spnE activity has a less extreme effect: the perinuclear nuage clusters are largely missing, but a perinuclear zone of VasGFP remains. Consistent with the results by using fixed samples, the persistent perinuclear zone of VasGFP is qualitatively different from wild type, appearing almost completely uniform and lacking any visible discontinuities. Similar results were obtained with the aub mutant, except that the VasGFP perinuclear clusters remain present up to stage 8 of oogenesis, after which they disappear. In aret and tud mutants no significant alteration of perinuclear nuage was detected (Snee, 2004).

In mutants whose perinuclear VasGFP is uniform (spnE^- and later stage aub^-), the protein undergoes rapid exchange with cytoplasmic pools, just as for VasGFP in perinuclear clusters of wild-type egg chambers. In photobleaching experiments the fluorescence-recovery half-time is 50 seconds in aub^- and 48.5 seconds in spnE^-, similar to the t_1/2=59 seconds for wild type (Snee, 2004).

Cytoplasmic nuage particles are affected differently in the vas, aub and spnE mutants. The vas and spnE mutants have few or no cytoplasmic nuage particles. By contrast, aub mutants have no dramatic reduction in the abundance of cytoplasmic nuage particles, even at times well after the disappearance of perinuclear nuage clusters at stage 8, and the particles have a fairly typical size distribution. These particles do not simply represent the default appearance of VasGFP; they are absent in the spnE mutant. Thus, it seems unlikely that perinuclear nuage clusters are required for the formation of cytoplasmic nuage particles, a conclusion consistent with the observation that cytoplasmic particles are produced only infrequently by detachment of perinuclear nuage clusters (Snee, 2004).

The consequences of loss of vas activity were examined in the male germ line. Just as in nurse cells, Vas appears to be concentrated in nuage in spermatocytes. Given the crucial role for Vas in the nuage of other cell types, either male nuage must differ in this requirement or nuage is not essential in the male germ line for fertility. To distinguish between these possibilities vas^AS spermatocytes were tested for the presence of nuage, using GFPAub as a marker. Although GFPAub was present in the cytoplasm, there were no visible perinuclear nuage clusters, indicating that nuage does not form in the vas mutant and is therefore not required for spermatocyte function. An alternate and less probable interpretation is that a rudimentary form of nuage, lacking Aub, is present and is sufficient to provide a minimal requirement for nuage in males (Snee, 2004).

In Drosophila, two types of function, not mutually exclusive, have been proposed for nuage. In one model nuage has been suggested to serve as a precursor to polar granules, a view initially based on ultrastructural similarities of the two organelles and supported by the identification of shared components. Another possible role for nuage is based on its position at the periphery of the nucleus, at or near nuclear pores. Specifically, nuage might act in some aspect of remodelling RNPs when RNAs are exported from the nucleus. Analysis of the movements and genesis of nuage particles provides two arguments against the first model: (1) the rate of release of perinuclear nuage clusters in the nurse cells is very low, much lower than expected if the clusters form polar granules; (2) no nuage particles arriving at the posterior pole of the oocyte and becoming incorporated into polar granules were detected. An additional observation that argues against a model where nuage is a precursor for polar granules, is the presence of cytoplasmic nuage particles in aub mutants, despite the fact that these mutants do not assemble polar granules. However, this evidence does not exclude the first model, because the nuage particles in the mutant might not be fully functional. A third argument is provided by the evidence that Osk cannot interact with nuage, leaving de novo assembly of polar granules as the only reasonable option. Overall, the results strongly suggest that nuage is not the precursor to polar granules, and it is believed that the shared features are simply indicative of similar biochemical activities, rather than a precursor-product relationship (Snee, 2004).

The data do not directly test the model that nuage might function as a transition zone in the movements of mRNAs from the nucleus to the cytoplasm, where RNP components might be exchanged or otherwise modified. However, new properties of nuage, and these relate to possible functions, have been identified. It was found that Bruno, an RNA binding protein that acts as a translational repressor of osk and grk mRNAs, is associated with nuage. This extends the correlation of nuage components with factors that act in some aspect on mRNA localization or translational control. Of the previously identified nuage components, Vas and Gus are involved in the regulation of grk mRNA localization and translation, Aub is required for efficient translation of osk mRNA and has also been implicated in RNAi, and mael mutants display defects in the early stages of mRNA localization. Moreover, spnE, which is necessary for normal nuage formation, is required for the localization of multiple mRNAs and acts in RNAi. Thus, every known nuage component has a role in one or more types of post-transcriptional control of gene expression (Snee, 2004).

EFFECTS OF MUTATION

Genetic analysis of the role of bruno in oogenesis is made difficult by the lack of ovaries in bruno mutants. Flies sensitized to changes in the level of Osk protein were examined for the affects of reducing wild-type Bru protein levels. If Bru acts to repress OSK translation, a reduction in Bru protein might lead to a partial derepression of OSK translation and, subsequently, to elevated Osk activity. A transgene was used which encodes a form of OSK mRNA that retains Bruno response sequences but is mislocalized to the anterior of the oocyte. Flies bearing this genotype produce embryos with modest head defects caused by the misexpressed OSK mRNA. Reduction in Bru level enhances this phenotype, resulting in progeny with extensive anterior deletions, often accompanied by duplication of posterior pattern elements (Webster, 1997).

In females hemizygous for either aretPA62 or aretPD41(alleles encoding missense mutations that alter the first of the three RNA-binding domains) oogenesis appears to proceed normally until approximately stage 9, at which time the egg chambers degenerate. arrestPA62/arrestPD41 transheterozygotes do complete oogenesis and lay eggs, some of which hatch into viable larvae. However the majority of the embryos from these mothers display variable and complex cuticle defects involving partial or complete fusion of adjacent segments (Webster, 1997).

Genetic interactions of Drosophila melanogaster arrest reveal roles for translational repressor Bruno in accumulation of Gurken and activity of Delta

arrest mutants have pleiotropic phenotypes, ranging from an early arrest of oogenesis to irregular embryonic segmentation defects. One function of arrest is in translational repression of oskar mRNA; this biochemical activity is presumed to be involved in other functions of arrest. To identify genes that could provide insight into how arrest contributes to translational repression or that may be targets for arrest-dependent translational control, deficiency mutants were screened for dominant modification of the arrest phenotype. Only four of the many deficiencies tested, which cover ~30% of the genome, modified the starting phenotype. One enhancer, identified fortuitously, is the Star gene. Star interaction with arrest results in excess Gurken protein, supporting the model that gurken is a target of repression. Two modifiers were mapped to individual genes. One is Lk6, which encodes a protein kinase predicted to regulate the rate-limiting initiation factor eIF4E. The second is Delta. The interaction between arrest and Delta mimics the phenotype of homozygous Delta mutants, suggesting that arrest could positively control Delta activity. Indeed, arrest mutants have significantly reduced levels of Delta protein at the interface of germline and follicle cells (Yan, 2004).

A screen of third chromosome deficiencies was screened for dominant modifiers of aret mutants. About three-quarters of the third chromosome was screened, corresponding to ~30% of the genome. Only four deficiencies dominantly modified the aret mutant phenotype, suggesting that the total number of genes in the genome with this property is small. For two of the four deficiencies the gene responsible for the interaction was identified, and a third interacting gene was fortuitously discovered while preparing for the screen. It was anticipated that two different types of modifiers might be detected by the screen: those in genes that act in the same process as Bru and those in genes that are themselves regulated by Bru or act in a process in which a limiting component is regulated by Bru. Characterization of the interacting genes suggests that examples of each type of modifier were discovered (Yan, 2004).

Bru has been proposed to translationally regulate grk mRNA. The supporting evidence includes (1) binding of Bru to grk mRNA in vitro and indirect evidence of binding in vivo; (2) rare dorsoventral patterning defects as a consequence of overexpression of Bru, and enhancement of this phenotype by reduction of grk gene dosage, and (3) evidence that localized Grk is present at reduced levels when Bru is overexpressed, although unlocalized Grk appears more abundant. However, there has been no evidence of excess Grk protein in aret mutants. Star is required for grk activity, and it acts post-translationally in either trafficking or secretion of Grk protein. When flies were both homozygous for aret and heterozygous for S¹ they accumulated Grk protein in nurse cells, while ectopic accumulation could not be detected in either aret mutants or S¹ heterozygotes alone. This synthetic effect on Grk protein accumulation is simple to rationalize. In aret mutants Grk protein is excessively translated, but an S-dependent delivery step could efficiently clear the protein from the nurse cells. When S activity is reduced, a detectable level of Grk remains in the nurse cells. The distribution of the ectopic Grk, both in cytoplasm and at the nurse cell boundaries, could correspond to the sites where the protein might stall during delivery. The actual site of S action is not known, and two different sites of S concentration, in endoplasmic reticulum or on the plasma membrane, have been reported. Although this explanation has some appeal, it is important to note that none of the evidence firmly establishes a role for Bru in translational repression of grk mRNA, and it remains possible that Bru could, for example, influence the site of translation rather than its efficiency (Yan, 2004).

Although the combination of S¹ and aret mutations does affect Grk expression or distribution, there are no precedents that clearly demonstrate how excess or ectopic Grk would enhance the oogenesis arrest phenotype of aret mutants. Thus the explanation for the enhancement remains unknown and could involve the effects on grk or on other genes that are subject to regulation by Bru (Yan, 2004).

The eIF4E protein binds to the cap at the 5' end of mRNAs. It is a rate-limiting component of translational initiation, and its activity is under tight control. One form of regulation is phosphorylation, which is thought to control the mRNA cap-binding activity of eIF4E. Several lines of correlative evidence suggest that this phosphorylation is important for cell proliferation, and mutation of the Drosophila eIF4E to prevent phosphorylation results in reduced viability and poor growth (Yan, 2004 and references therein).

A transgene expressing a mutant and constitutively activated version of eIF4E, in which the regulatory phosphorylation is mimicked by an amino acid change, can suppress the aret phenotype. This result raises the possibility that Bru has a positive role in initiation of translation. Specifically, in the aret mutant one or more target mRNAs that require Bru for activation of translation may be underexpressed, and increasing translation suppresses this defect (Yan, 2004).

However, the aret mutant phenotype is also suppressed by a mutation of Lk6 and enhanced by overexpression of Lk6. Lk6 is the Drosophila protein most closely related to mammalian mitogen-activated protein kinase-interacting protein kinase 1 (MNK1), which phosphorylates translation initiation factor eIF4E after activation by either the p44/42 or p38 MAPKs. Thus mutation of Lk6 might be expected to reduce eIF4E phosphorylation and thereby decrease translational capacity. By this view the suppression of the aret phenotype would be consistent with an interaction between eIF4E and Bru that involves the known function of Bru in translational repression. In favor of this notion Bru has been shown to physically interact with Cup, an eIF4E-binding protein that is required for repression of osk mRNA translation. To explore this possibility further it was asked if suppression of the aret phenotype by EP(3)0886 was accompanied by a change in the levels of Osk or Grk proteins, or if homozygous EP(3)0886 females have abnormal amounts of either protein. No change was seen in either case. Thus it is not known if the Lk6 mutation impacts the function of aret in repression of osk or grk mRNAs (Yan, 2004).

Given the similar consequences on the aret phenotype of the constitutively active eIF4E and the mutant predicted to reduce eIF4E activity, the simplest explanation is that Lk6 may affect aret function by a means other than phosphorylation of eIF4E. Suppression of the aret phenotype by the mutant eIF4E clearly suggests a link between Bru and the initiation of translation, although this need not be direct (Yan, 2004).

The combination of aret^PD/aret^QB with Dl^9P/+ produces a variety of ovarian defects, complicating interpretation of the phenotype. Nevertheless, one striking feature is the similarity of many of the defects to those seen when Dl activity is largely or completely eliminated, suggesting that the aret mutations are enhancing the Dl phenotype. Dl is a component of the Notch/Dl signaling pathway, which acts in many signaling events in a wide range of cell types. In the ovary Dl is required in the germline cells for control of differentiation and proliferation of the somatic follicle cells and for setting up anteroposterior polarity. The earliest and, at least initially, most dramatic consequence of loss of Dl activity is the fusion of cysts—the phenotype most apparent in the aret^PD/aret^QB; Dl^9P/+ ovaries (Yan, 2004).

Large germline clones of strong Dl mutant alleles cause a complete fusion of egg chambers into a single egg chamber with multiple cysts, reminiscent of the complete fusions described here. Smaller clones retain a more regular ovariole organization. Individual egg chambers with Dl germline clones often fuse with the adjacent anterior wild-type egg chamber. Fusion can be incomplete, resulting in a double layer of follicle cells that separate the egg chambers, much as observed for the A/P partial fusions reported in this study. However, the similarities are not perfect. For example, Dl mutant clones upregulate FasIII in the follicular epithelium, but aret^PD/aret^QB; Dl^9P/+ egg chambers do not. Other features of the Dl mutant phenotype, such as the defects in anteroposterior polarity, are difficult to detect in the aret^PD/aret^QB; Dl^9P/+ ovaries, because of their arrest of oogenesis. The lack of perfect correspondence between the Dl germline clones and the aret^PD/aret^QB; Dl^9P/+ ovaries is not surprising for several reasons: (1) there is substantial phenotypic variation even among the Dl germline clones, if both large and small clones are considered; (2) the clones are homozygous for Dl^–, while in the aret mutant background one wild-type copy of Dl remains; (3) the Dl-like defects in aret^PD/aret^QB; Dl^9P/+ ovaries are superimposed on the aret mutant phenotype (Yan, 2004).

The simplest interpretation of these results is that the aret mutations are reducing the activity of the N/Dl signaling pathway, which in combination with mutation of one copy of Dl leads to phenotypes similar to those resulting from loss of Dl. This model is fully supported by the finding that in aret mutants the amount of Dl protein concentrated at the border between germline cells and follicle cells is reduced. What remains unclear is how this reduction occurs. Assuming that Bru is acting as a translational repressor, in the aret mutant the target protein should be present at elevated levels. By this model the target should be a gene that normally has a negative effect on Dl expression or delivery to the membrane. Alternatively, Bru could also have a role in translational activation, in which case Dl could be a direct target. This seems quite unlikely, as the Dl 3'-UTR lacks any recognizable BREs, the sequences to which Bru is known to bind. Nevertheless, a role for Bru in translational activation is possible, and the target could normally have a positive effect on provision of Dl activity (Yan, 2004).

Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis; Identification of dominant suppressors and enhancers of a muscleblind overexpression phenotype

Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene. This study used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematodes to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors leads to an externally rough eye morphology. This phenotype has been used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog arrest, exon junction complex components tsunagi and always early, and pro-apoptotic genes Traf1 and reaper. This study further investigated Muscleblind implication in apoptosis and splicing regulation. Missplicing of troponin T was found in muscleblind mutant pupae, and Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing was confirmed in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression. Taken together these genetic approaches identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell (Vicente-Crespo, 2008).

Using Drosophila as a model organism, this study reports the first screen specifically addressed to identify gene functions related to the biomedically important protein Muscleblind. In support of the relevance of the results, the strong functional conservation between fly and vertebrate Muscleblind proteins is shown. Furthermore, data is presented supporting that Muscleblind can induce apoptosis in vivo in imaginal disc tissue, and a conserved motif in the MblC protein isoform was identified that conferred pro-apoptotic activity in Drosophila cell culture when mutated. Noteworthy, this is the first conserved motif (besides CCCH zinc fingers) that is associated with a particular function in Muscleblind proteins (Vicente-Crespo, 2008).

Whereas most vertebrates include three muscleblind paralogues in their genomes, a single muscleblind gene carries out all muscleblind-related functions in Drosophila. These functions are probably accomplished through alternative splicing, which generates four Muscleblind protein isoforms with different carboxy-terminal regions. An evolutionary analysis was performed with isoform-specific protein sequences in order to assess conservation of alternative splicing within protostomes. MblC-like isoforms have been detected even in the nematodes C. elegans and Ascaris suum but not MblA, B or D, that were only consistently found within Drosophilidae. Interestingly, also vertebrate Mbnl1 genes included MblC-like sequences. This finding, together with previous studies that shown that mblC is the isoform with the strongest activity in a muscleblind mutant rescue experiment and α-actinin minigene splicing assay point to mblC as the isoform performing most of muscleblind functions in the fly. Despite this, Muscleblind isoforms are partially redundant. Both mblA and B partially rescue the embryonic lethality of muscleblind mutant embryos and were able to similarly promote foetal exon exclusion in murine TnnT3 minigene splicing assays. MblD showed no activity in splicing assays or in vivo overexpression experiments. However, we show a marginal increase in cell viability in cell death assays. Using isoform-specific RNAi constructs we plan to re-evaluate the function of Muscleblind isoforms both in vivo and in cell culture (Vicente-Crespo, 2008).

Although the regulation of alternative splicing by Muscleblind proteins is an established fact, the cellular processes in which the protein participates are largely unknown. Genetic screens provide a way to approach those processes as they interrogate a biological system as a whole. Overexpression of MblC in the Drosophila eye originated an externally rough eye phenotype that is temperature sensitive, thus indicating sensitization to the muscleblind dose. A deficiency screen was performed, and several candidate mutations were tested for dominant modification of the phenotype. Nineteen were identifed genes of which more that half can be broadly classified as involved in apoptosis regulation (rpr, th and Traf1), RNA metabolism (Aly, tsu, aret and nonA) or transcription regulation (jumu, amos, Dp, CG15435 and CG15433), whereas the rest do not easily fall into defined classes. muscleblind has been shown to regulate α-actinin and troponinT alternative splicing both in vivo and in cell culture. The genetic interaction with the Drosophila homolog of human splicing factor CUG-BP1 (aret) and nonA supports a functional relationship in flies. The antagonism between MBNL1 and CUG-BP1 has actually been shown in humans, whereas RNA-binding protein NonA might be relevant to Muscleblind sequestration by CUG repeat RNA in flies (Vicente-Crespo, 2008 and references therein).

Reduction of dose of exon junction complex (EJC) components tsunagi and Aly also modify MblC overexpression phenotype. EJC provides a binding platform for factors involved in mRNA splicing, export and non-sense mediated decay (NMD). This suggests a previously unforeseen relationship between Muscleblind and EJC, perhaps helping to couple splicing to mRNA export. Consistently, Aly mutations enhanced a CUG repeat RNA phenotype in the Drosophila eye. A similar coupling between transcription and splicing might explain the identification of a number of transcription factors in the screen. Of these, the effect of jumu alleles in the eye and wing MblC overexpression phenotypes were studied in some detail. Loss of function jumu mutations suppress both wing defects and rough eye, whereas they have no effect on unrelated overexpression phenotypes thus suggesting that the interaction is specific (Vicente-Crespo, 2008).

Mutations in the Drosophila homolog of vertebrate Inhibitor of Apoptosis (Diap1 or thread) dominantly enhanced the rough eye phenotype. Consistently with the specificity of the interaction, a second Drosophila paralog, Diap2, did not interact. Also, a deficiency that removes the Drosophila proapoptotic genes hid, reaper and grim (which inhibit thread) was a dominant suppressor while reaper overexpression in eye disc enhanced the phenotype. Interestingly the human homolog of Drosophila Hsp70Ab, Hsp70, has been related to apoptosis as it directly interacts with Apaf-1 and Apoptosis Inducing Factor (AIF) resulting in the inhibition of caspase-dependent and caspase-independent apoptosis. All these genetic data are consistent with MblC overexpressing eye discs being sensitized to enter apoptosis, although no increase in caspase-3 activation was detected in third instar eye imaginal disc overexpressing MblC (Vicente-Crespo, 2008).

Human MBNL1 and CUB-BP1 cooperate to regulate the splicing of cardiac TroponinT (cTNT). The current study detected splicing defects in Drosophila troponinT mRNA in muscleblind mutant pupae. Interestingly, an abnormal exclusion of exon 3 was detected in muscleblind mutant pupae, encoding a glutamic acid-rich domain homologous to the foetal exon of cTNT regulated by human MBNL1. Drosophila exon 3 is only absent in the troponinT isoform expressed in TDT and IFM muscles and probably confers specific functional properties much like the foetal exon does in humans. This identifies troponinT as a new target of Muscleblind activity in flies (Vicente-Crespo, 2008).

CUG-BP1 protein antagonizes MBNL1 exon choice activity in IR and cTNT pre-mRNAs. Moreover, a genetic interaction has been detected between MblC overexpression and aret loss of function mutations. In order to further characterize the functional interaction between Muscleblind and Bruno proteins, their ability to regulate murine TnnT3 was examined in human cell culture. MblA, B and C showed strong activity on TnnT3 mRNA but no significant activity was detected for any Bruno protein. This shows a strong functional conservation between fly and vertebrate Muscleblind proteins as Drosophila isoforms can act over a murine target in a human environment. In contrast, Bruno proteins might not conserve the regulatory activity over troponinT mRNA described for their vertebrate homologues or at least they were not functional in the cellular environment used in this assay. Because GFP-tagged Bruno proteins were only weakly expressed in HEK cells under the experimental conditions used, the level of expression might be insufficient to overcome endogenous Muscleblind activity in cell culture. Furthermore, Bruno proteins might antagonize Muscleblind on a different subset of RNA targets. Although bruno1 has been shown to regulate splicing of some transcripts in S2 cell culture and Bruno3 binds the same EDEN sequence than human CUG-BP, no in vivo experiments have addressed the functional conservation between fly and vertebrate Brunos. Bruno1 is expressed in the germ line where it acts as translational repressor of oskar and gurken mRNAs (Vicente-Crespo, 2008).

Wing imaginal discs stained with anti-caspase-3 and with TUNEL showed that activation of apoptosis was not general in cells expressing MblC but restricted to defined regions within the disc, in particular the wing blade. The spatial constraints that were observed within the imaginal disc might explain the small effect detected when expressing Muscleblind proteins in S2 cells. MblC might require the presence of other factors to be able to unleash programmed cell death. Alternatively, the level of overexpression may be critical and transfected Muscleblind proteins may not reach a critical threshold in Drosophila S2 cells. MblC activation of apoptosis could reveal a direct regulation of apoptotic genes at RNA level or be an indirect effect. Several apoptotic genes produce pro-apoptotic or anti-apoptotic isoforms depending on the regulation of their alternative splicing. MblC could be similarly regulating protein isoforms originating from one or a number of key apoptotic genes at the level of pre-mRNA splicing. Alternatively, MblC could be regulating isoform ratio of a molecule indirectly related to programmed cell death, for example a cell adhesion molecule causing apoptosis by inefficient cell attachment to the substrate. Furthermore, human MBNL proteins are implicated not only in splicing but also in RNA localization, a process that if conserved in flies can potentially impinge in apoptosis regulation (Vicente-Crespo, 2008).

The analysis of MblC-specific sequence revealed a region conserved in Muscleblind proteins from nematodes to humans. Post-translational prediction programs found a motif (FKRP) weakly resembling a sumoylation target site. However, results in S2 cells suggest that sumoylation, if actually taking place, modifies only a small fraction of MblC proteins. FKRP may alternatively participate in an interaction with a Muscleblind partner potentially regulating activity or location in cell compartments, assist in protein dimerization, or others functions. The FKRP site was mutated and a number of functional assays were performed using the mutant MblC. Whereas MblC^K202I excluded foetal exon in TnnT3 minigene splicing assays and bound CUG repeat RNA like its wild type counterpart, the mutant protein showed a different preferential distribution in human cells and significantly increased cell death activation upon overexpression. The mechanism by which the FKRP site influences subcellular distribution and cell death-inducing activities is currently unknown, but nevertheless constitutes the first motif, other than zinc fingers, that is associated with a function within Muscleblind proteins (Vicente-Crespo, 2008).

EVOLUTIONARY HOMOLOGS

C. elegans Bruno homolog

While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using C. elegans as a model for studying neuron/target communication, this study reports that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. Human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, a new neuronal, putative pre-mRNA splicing factor, UNC-75, has been identified and it has been shown that UNC-75, as well as the C. elegans homolog of ELAV, are each required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function (Loria, 2003).

The vertebrate orthologs of UNC-75, CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5, have been shown to be involved in splicing in an in vitro assay. To investigate whether the function of these proteins is conserved (a notion that was expected from the level of primary sequence similarity), UNC-75 and its human orthologs were compared in more detail. mRNA samples derived from a variety of different human tissues were hybridized with probes specific to three of the four human orthologs. CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5 each show highly similar expression patterns that are largely restricted to the nervous system. Within the nervous system, every region tested shows expression of CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5. Thus, the pan-neuronal expression of the human orthologs of UNC-75 mirrors the pan-neuronal expression of C. elegans UNC-75 (Loria, 2003).

In addition to their similar tissue distribution, the function and subcellular localization of the worm and human proteins are also conserved. A human CELF4/BrunoL4 cDNA, expressed under control of the unc-75 promoter, is able to rescue the uncoordinated phenotype of unc-75 mutants as well as the resistance to the acetylcholine esterase inhibitor aldicarb ('ric' phenotype), which likely results from a disruption in signaling between cholinergic ventral cord motor neurons and their body wall muscle targets. Furthermore, translational fusions of CELF4/BrunoL4 with GFP (punc75::gfp::L4) show localization to subnuclear speckles reminiscent of the UNC-75::GFP speckles. Since loss of UNC-75 can be rescued by a human ortholog that acts as a splicing factor in vitro, it is reasonable to suggest that UNC-75 also acts as a pre-mRNA splicing factor, a notion further corroborated by its subnuclear localization (Loria, 2003).

Functional comparison of UNC-75 and EXC-7 proteins was extended by analyzing exc-7 null mutant animals. In contrast to fly Elav, which severely affects neuronal development and viability, exc-7 mutants are viable and show no locomotory or defecation defects. Also in contrast to fly Elav, exc-7 is only expressed in a subset of neurons in the nervous system, several of which are cholinergic neurons. The development and morphology of several cholinergic neuron classes were assessed by using cell-specific gfp markers; no obvious defects were found. Moreover, synaptic vesicles in the cholinergic SAB neurons cluster normally in exc-7 null mutants, leading to the conclusion that EXC-7 has no significant impact on neuronal development. However, when cholinergic motorneuron function was tested in more detail, it was found that exc-7(rh252) animals show a synaptic transmission defect similar to unc-75 (Loria, 2003).

The relation of the ric phenotype of unc-75 and exc-7 was assessed. If these two genes act in a similar process, their null phenotypes should not enhance one another. It was found, however, that the synaptic transmission defect of the double mutant is significantly enhanced compared to the single mutants. Moreover, although exc-7 mutant animals show no locomotory defects on their own, unc-75; exc-7 double mutant animals are smaller and appear significantly more uncoordinated than unc-75 single mutants. Lastly, it was found that the ric phenotype of exc-7 null mutants is not rescued by an elevation in ambient temperature. This lack of temperature sensitivity is similar to that of unc-17 mutants, which are affected in synaptic vesicle loading. It is concluded that unc-75 and exc-7 have nonredundant and distinct roles in cholinergic synaptic transmission and likely regulate the pre-mRNA splicing of a distinct set of target genes (Loria, 2003).

A bruno-like gene is required for stem cell maintenance in planarians

The regenerative abilities of freshwater planarians are based on neoblasts, stem cells maintained throughout the animal's life. A member of the Bruno-like family of RNA binding proteins is critical for regulating neoblasts in the planarian Schmidtea mediterranea. Smed-bruno-like (bruli) mRNA and protein are expressed in neoblasts and the central nervous system. Following bruli RNAi, which eliminates detectable bruli protein, planarians initiate the proliferative response to amputation and form small blastemas but then undergo tissue regression and lysis. The neoblast population was characterized by using antibodies recognizing SMEDWI-1 and Histone H4 (monomethyl-K20) and cell-cycle markers to label subsets of neoblasts and their progeny. bruli knockdown results in a dramatic reduction/elimination of neoblasts. These analyses indicate that neoblasts lacking bruli can respond to wound stimuli and generate progeny that can form blastemas and differentiate; yet, they are unable to self-renew. These results suggest that bruli is required for stem cell maintenance (Guo, 2006).

Bruno-like is involved in asymmetric distribution of maternal mRNAS in zebrafish

Asymmetric distribution of maternal mRNAs has not been well documented in zebrafish. dazl mRNA is localized at the vegetal pole. A novel zebrafish gene, bruno-like (brul) is described that provides another example of vegetal mRNA localization. brul encodes an Elav-type RNA-binding protein that belongs to the Bruno-like family that includes mammalian CUG-BP, Xenopus EDEN-BP, and Drosophila Bruno. At 24 hpf, brul mRNA is abundant in lens fiber cells. At the onset of embryogenesis, maternal brul mRNA is detected at the vegetal pole, and it then migrates rapidly toward the blastoderm through yolk cytoplasmic streams. During oogenesis, brul mRNA becomes localized at the vegetal cortex at stage II, later than dazl mRNA. Anchoring of brul mRNA was dependent on microfilaments (Suzuki, 2000).

etr-1, a possible Bruno homolog in Xenopus

In Xenopus development, dorsal mesoderm is thought to play a key role in both the induction and patterning of the nervous system. Noggin, which is expressed in dorsal mesoderm, can mimic that tissue's neural-inducing activity without inducing mesoderm. Neural tissue induced in ectodermal explants by noggin has been further characterized using four neural-specific genes: two putative RNA-binding proteins, nrp-1 and etr-1; the synaptobrevin sybII; and the lipocalin cpl-1. The expression domain of each gene during embryogenesis was determined and then expression of these genes was examined in noggin-treated explants. All markers, including the differentiated marker sybII, were expressed in noggin-induced neural tissue. cpl-1, a marker of dorsal brain, and etr-1, a marker absent in much of the dorsal forebrain, were both expressed in non-overlapping territories within these explants. It is concluded that the despite the absence of mesoderm, noggin-induced neural tissue shows considerable differentiation and organization, which may represent dorsal-ventral patterning of the forebrain (Knecht, 1995).

Mammalian Bruno homologs

Expansion of trinucleotides repeats is associated with a number of neurodegenerative diseases. Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease associated with a (CTG)n nucleotide repeat expansion in the 3'-untranslated region of the myotonin protein kinase (Mt-PK) gene. These CTG repeats are translated into CUG repeats in messenger RNA. While an unstable CTG triplet repeat expansion is responsible for myotonic dystrophy, the mechanism by which this genetic defect induces the disease remains unknown. To detect proteins binding to CTG triplet repeats, bandshift analysis was performed using as probes double-stranded DNA fragments having CTG repeats [ds(CTG)6-10] and single-stranded oligonucleotides having CTG repeats ss(CTG)8 or RNA CUG triplet repeats (CUG)8. The source of protein was nuclear and cytoplasmic extracts of HeLa cells, fibroblasts and myotubes. Proteins binding to the double-stranded DNA repeat [ds(CTG)6-10] are inhibited by nonlabeled ds(CTG)6-10, but not by a non-specific DNA fragment (USF/AD-ML). Another protein binding to ssCTG probe and RNA CUG probe is inhibited by nonlabeled (CTG)8 and (CUG)8. Nonlabeled oligos with different triplet repeat sequences, either ss(CAG)8 or ss(CGG)8, do not inhibit binding to the ss(CTG)8 probe. However, when labeled as probes, the (CAG)8 and (CGG)8 bind to proteins distinct from the CTG proteins and binding is inhibited by nonlabeled (CAG)8 or (CGG)8 respectively. The protein binding only to the RNA repeat (CUG)8 is inhibited by nonlabeled (CUG)8 but not by nonlabeled single- or double-stranded CTG repeats. The CUG-BP exhibits no binding to an RNA oligonucleotide of triplet repeats of the same length but having a different sequence, CGG. The CUG binding protein is localized to the cytoplasm, whereas dsDNA binding proteins are localized to the nuclear extract. Thus, several trinucleotide binding proteins exist and their specificity is determined by the triplet sequence. The novel protein, CUG-BP, is particularly interesting since it binds to triplet repeats known to be present in myotonin protein kinase mRNA, which is responsible for myotonic dystrophy (Timchenko, 1996a).

This study reports the isolation and characterization of a (CUG)n triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based on their ability to bind specifically to (CUG)8 oligonucleotides in vitro. While CUG-BP1 is the major (CUG)8-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is associated with polyadenylated RNAs in vivo. In vitro RNA-binding/photocrosslinking studies demonstrate that CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3'-UTR. It is proposed that the (CUG)n repeat region in Mt-PK mRNA is a binding site for CUG-BP/hNab50 in vivo, and triplet repeat expansion leads to sequestration of this hnRNP on mutant Mt-PK transcripts (Timchenko, 1996b).

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. A protein has been isolated with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. The phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patients. CUG-BP/hNab50 is a substrate for DMPK (Drosophila homolog: Genghis Khan) both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK ( homozygous DM patients and DMPK knockout mice) shows that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in both the DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patients. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in the dramatic reduction of CUG-BP2, the hypophosphorylated isoform, accumulation of which is observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter the phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. These results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs (Roberts, 1997).

The post-transcriptional regulation of gene expression by RNA-binding proteins is an important element in controlling both normal cell functions and animal development. The diverse roles are demonstrated by the Elav family of RNA-binding proteins, where various members have been shown to regulate several processes involving mRNA. Another family of RNA-binding proteins distantly related to the Elav family but closely related to Bruno, a translational regulator in Drosophila melanogaster, has been discovered. In humans, six Bruno-like genes have been identified, whereas other species such as Drosophila, Xenopus laevis, and Caenorhabditis elegans have at least two members of this family, and related genes have also been detected in plants and ascidians (phylum Urochordata). The human BRUNOL2 and BRUNOL3 are 92% identical in the RNA-binding domains, although the BRUNOL2 gene is expressed ubiquitously whereas BRUNOL3 is expressed predominantly in the heart, muscle, and nervous system. Both of these proteins bind the same target RNA, the Bruno response element. The RNA-binding domain that recognizes the Bruno response element is composed of two consecutive RNA recognition motifs at the amino terminus of vertebrate Bruno protein. The possible involvement of the Bruno family of proteins in the CUG repeat expansion disease, myotonic dystrophy, is discussed (Good, 2000).

REFERENCES

Search PubMed for articles about Drosophila bruno

Braat, A. K., et al. (2004). Localization-dependent Oskar protein accumulation control after the initiation of translation. Dev. Cell 7: 125-131. 15239960

Castagnetti, S., et al. (2000). Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development 127: 1063-1068. PubMed Citation: 10662645

Castrillon, D. H., et al. (1993). Toward a molecular genetic analysis of of spermatogenesis in Drosophila melanogaster: Characterization of male-sterile mutatnts generated by single P element mutagenesis. Genetics 135: 489-505. PubMed Citation: 8244010

Chekulaeva, M., Hentze, M. W. and Ephrussi, A. (2006). Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles. Cell 124(3): 521-33. 16469699

Coller, J. and Parker, R. (2005). General translational repression by activators of mRNA decapping. Cell 122: 875-886. 16179257

Dold, A., Han, H., Liu, N., Hildebrandt, A., Bruggemann, M., Ruckle, C., Hanel, H., Busch, A., Beli, P., Zarnack, K., Konig, J., Roignant, J. Y. and Lasko, P. (2020). Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation. PLoS Genet 16(1): e1008581. PubMed ID: 31978041

Filardo, P. and Ephrussi, A. (2003). Bruno regulates gurken during Drosophila oogenesis. Mech. Dev. 120: 289-297. 12591598

Good, P. J., et al. (2000). A family of human RNA-binding proteins related to the Drosophila Bruno translational regulator. J. Biol. Chem. 275(37): 28583-92. PubMed Citation: 10893231

Gunkel, N., Yano, T., Markussen, F.-H., Olsen, L. C. and Ephrussi, A. (1998). Localization-dependent translation requires a functional interaction between the 5' and 3' ends of oskar mRNA. Genes Dev. 12: 1652-1664. PubMed Citation: 9620852

Guo, T., Peters, A. H. and Newmark, P. A. (2006). A bruno-like gene is required for stem cell maintenance in planarians. Dev. Cell 11(2): 159-69. Medline abstract: 16890156

Jeong, E. B., Jeong, S. S., Cho, E. and Kim, E. Y. (2019). Makorin 1 is required for Drosophila oogenesis by regulating insulin/Tor signaling. PLoS One 14(4): e0215688. PubMed ID: 31009498

Kim-Ha, J., Kerr, K. and Macdonald, P. M. (1995). Translational regulation of oskar mRNA by Bruno, an ovarian RNA-binding protein, is essential. Cell 81(3): 403-412. PubMed ID: 7736592

Knecht, A. K., et al. (1995). Dorsal-ventral patterning and differentiation of noggin-induced neural tissue in the absence of mesoderm. Development 121(6): 1927-1935

Scott, E. K., Lee, T. and Luo, L. (2001). enok encodes a Drosophila putative histone acetyltransferase required for mushroom body neuroblast proliferation. Curr Biol 11: 99-104. PubMed ID: 11231125

Lie, Y. S. and Macdonald, P. M. (1999a). Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation. Development 126: 1129-1138. PubMed ID: 10021333

Lie, Y. S. and Macdonald, P. M. (1999b). Translational regulation of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts. Development 126: 4989-4996. PubMed ID: 10529417

Loria, P. M., et al. (2003). Two neuronal, nuclear-localized RNA binding proteins involved in synaptic transmission. Curr. Biol. 13: 1317-1323. 12906792

Munro, T. P., Kwon, S., Schnapp, B. J. and St Johnston, D. (2006). A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP. J Cell Biol 172(4): 577-588. PubMed ID: 16476777

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