The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription. This study identifies the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. Genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. Evidence is provided that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. An initial characterization is presented of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes. This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development (Beckstead, 2005).
To identify genes that alter their expression in synchrony with the late third instar and prepupal pulses of 20E, RNA was isolated from w1118 animals staged at -18, -4, 0, 2, 4, 6, 8, 10, and 12 hours relative to pupariation, labeled, and hybridized to Affymetrix Drosophila Genome Arrays. The sensitivity and accuracy of the array data were determined by comparing the expression patterns of known 20E-regulated genes with previously published developmental Northern blot data. A subset of this analysis reveals that the temporal expression pattern of key regulatory genes - EcR, usp, E74A, DHR3, FTZ-F1, and DHR39 - are faithfully reproduced in the temporal arrays, as well as the 20E-regulated switch from Sgs glue genes to L71 late genes in the larval salivary glands, and the expression of representative IMP and Edg genes in the imaginal discs and epidermis. This comparison demonstrates that the microarrays accurately reflect the temporal patterns of 20E-regulated gene expression at the onset of metamorphosis and have sufficient sensitivity to detect rare transcripts such as EcR and E74A (Beckstead, 2005).
Roles for brat during metamorphosis were examined because, unlike the other six 20E primary-response genes described above, a brat mutant allele is available (bratk06028) that allows an assessment of its functions during later stages of development. The bratk06028 P-element maps to the fourth exon of the brat gene. Precise excisions of this transposon result in viable, fertile animals, demonstrating that the transposon is responsible for the mutant phenotype. Lethal phase analysis of bratk06028 mutants revealed that 61% of the animals survive to pupariation, with the majority of these animals pupariating 1 to 2 days later than their heterozygous siblings. Of those mutants that pupariated, 11% died as prepupae, 8% died as early pupae, 46% died as pharate adults, and the remainder died within a week of adult eclosion. Phenotypic characterization of bratk06028 mutant prepupae and pupae revealed defects in several ecdysone regulated developmental processes, including defects in anterior spiracle eversion (29%), malformed pupal cases (15%), and incomplete leg and wing elongation (12%). Northern blot hybridization of RNA isolated from staged bratk06028 mutant third instar larvae or prepupae revealed a disruption in the 20E-regulated transcriptional hierarchy. In wild type animals, brat mRNA is induced in late third instar larvae and 10 hour prepupae, similar to the temporal profile determined by microarray analysis, with reduced levels of brat mRNA in bratk06028 mutants, consistent with it being a hypomorphic allele. βFTZ-F1 is unaffected by the brat mutation in mid-prepupae, while E74 mRNA is reduced at 10 hours after pupariation. BR-C, E93, EcR, DHR3, and L71-1 are expressed at higher levels in late third instar larvae and early prepupae, with significant upregulation of BR-C. In addition, the smallest BR-C mRNA, encoding the Z1 isoform, is under-expressed in brat mutant prepupae. It is unlikely that brat exerts direct effects on transcription since it encodes a translational regulator. Nonetheless, these effects on 20E-regulated gene expression are consistent with the late lethality of bratk06028 mutants. In particular, the rbp function provided by the BR-C Z1 isoform is critical for developmental responses to 20E, and overexpression of BR-C isoforms can lead to lethality during metamorphosis. Thus, not only are the brat mutant phenotypes consistent with it playing an essential role during metamorphosis, but it may exert this function through the regulation of key 20E-inducible genes. Efforts are currently underway to address the roles of the remaining six new 20E primary-response regulatory genes in transducing the hormonal signal at the onset of metamorphosis (Beckstead, 2005).
Nanos and Pumilio are required to recruit Brat to Hunchback mRNA. To test the biological significance of the interaction between Brat and the Nos/Pum/NRE ternary complex, it was asked whether substitutions in Pum and Nos that interfere with Brat recruitment also abrogate HB mRNA regulation in vivo. The impetus for these experiments derives from two properties of the Pum680 mutant, which bears the G1330D substitution in the seventh repeat of its RNA-binding domain. (1) PumG1330D binds RNA normally and recruits Nos into a ternary complex, but is defective in regulating HB in embryos; (2) when tested in a yeast four-hybrid experiment, PumG1330D does not recruit Brat. Seven additional Pum mutations were engineered by site-directed mutagenesis into both yeast and Drosophila expression vectors. Residues adjacent to 1330 or at analogous positions in other repeats within the RNA-binding domain were chosen for mutagenesis. The capacity of each Pum mutant to recruit Nos to the NRE or to recruit Brat to the Pum/Nos/NRE complex was assayed in transformed yeast. And the capacity of each Pum mutant to regulate HB mRNA translation in embryos (and thereby direct the development of abdominal segmentation) was assayed in transgenic flies. Brat recruitment in yeast correlates with HB mRNA regulation in embryos, suggesting that contacts between Pum and Brat are essential in vivo (Sonoda, 2001).
To test the role of Nos in Brat recruitment, mutations in the Nos carboxy-terminal domain were screened that were identified originally in a genetic screen for defective nos alleles. Most of the Nos mutants are not recruited by Pum into a ternary complex with the NRE. However, one mutant, NosM379K, is incorporated into a ternary complex normally, but this complex does not interact with Brat. When expressed in appropriately engineered transgenic embryos, the M379K derivative is stable but inactive, consistent with the idea that contacts between Nos and Brat are also essential for HB mRNA regulation in vivo (Sonoda, 2001).
All of the protein-protein interaction experiments described above involve indirect assays performed in yeast. To determine whether the interaction between Brat and the ternary complex is direct and independent of yeast factors, binding experiments in vitro were performed using purified components. Ternary complexes containing the Pum RNA-binding domain, the carboxy-terminal domain of Nos and the wild-type NRE can be captured on glutathione agarose beads (which bind to the GST moeity attached to Pum). Under the same reaction conditions, Brat is recruited into a quaternary complex that, by three criteria, has the same properties as the complex detected in yeast experiments. (1) Retention of either Nos or Brat is substantially reduced (~10-fold and 6-fold, respectively) if the NRE bears a mutation that abrogates Nos binding; (2) Brat is not detectably retained by a binary Pum/NRE complex in the absence of Nos; (3) the PumG1330D mutant captures Nos but not Brat. Taken together, these results demonstrate that Nos and Pum act jointly and directly to recruit Brat to the NRE (Sonoda, 2001).
All of the recessive brat alleles are associated to a greater or lesser extent with a variety of phenotypes, including a dramatic (~10-fold) overgrowth of the larval brain (Arama, 2000); early oogenesis defects; metastasis of transplanted brain and imaginal tissue, and a maternal effect on embryonic viability. This last class of 'female sterile' (fs) alleles appears to interfere preferentially with function in the female germ line, although class members also exhibit the other brat phenotypes. Unlike lethal alleles that encode truncated proteins, bratfs1 and bratfs3 encode proteins with single amino acid substitutions at conserved residues within the NHL domain (Sonoda, 2001 and Arama, 2000).
It was of interest to determine whether the substitutions in the Bratfs1 and Bratfs3 mutant proteins interfere with recruitment to the Nos/Pum/NRE ternary complex. To this end, ternary complexes were assembled in vitro with purified GST-Pum, Nos, and NRE-bearing RNA; these were subsequently incubated with embryonic extracts prepared from embryos derived from either wild-type or bratfs mutant females (henceforth referred to as bratfs mutant embryos). Complexes were captured on glutathione-agarose beads, and bound proteins displayed on a Western blot probed with Brat-specific antibodies (Sonoda, 2001).
About 5% of full-length Brat+ is retained under the conditions of the experiment, but neither Bratfs1 nor Bratfs3 binds appreciably to the ternary complex. The mutant proteins accumulate to normal levels and are stable in vivo. Thus, the single amino acid substitutions in the Bratfs mutant proteins prevent efficient recruitment to the Nos/Pum/NRE complex (Sonoda, 2001).
The consequences of altered Brat function on embryonic development were examined. bratfs mutant embryos have defects in abdominal segmentation that are essentially indistinguishable from those in embryos with reduced nos or pum function. These defects arise as a result of incomplete translational repression of HB mRNA in the posterior of the preblastoderm embryo, as is the case for nos or pum mutants. The level and distribution of Nos, Pum, and HB mRNA appear to be normal in bratfs mutant embryos, suggesting that Brat does not act indirectly to regulate abdominal segmentation. Taken with the interaction data, it is concluded that recruitment of Brat jointly by Nos and Pum to the NRE is required for the normal regulation of HB mRNA in the early embryo (Sonoda, 2001).
The Brain Tumor (Brat) protein is recruited to the 3' untranslated region (UTR) of hunchback mRNA to regulate its translation. Recruitment is mediated by interactions between the Pumilio RNA-binding Puf repeats and the NHL domain of Brat, a conserved structural motif present in a large family of growth regulators. The crystal structure of the Brat NHL domain is described and a model is presented of the Pumilio-Brat complex derived from in silico docking experiments and supported by mutational analysis of the protein-protein interface. A key feature of the model is recognition of the outer, convex surface of the Pumilio Puf domain by the top, electropositive face of the six-bladed Brat ß-propeller. In particular, an extended loop in Puf repeat 8 fits in the entrance to the central channel of the Brat ß-propeller. Together, these interactions are likely to be prototypic of the recruitment strategies of other NHL-containing proteins in development (Edwards, 2003).
One feature of the Brat-Pum model is common to protein complexes formed by other ß-propellers: interaction along the top surface, particularly around the central channel. The WD40 domain of the transcriptional corepressor Tup1 interacts with the DNA-binding factor Matalpha2 to regulate mating-type genes in budding yeast. Although the structure of this complex is unknown, all the mutations in Tup1 that interfere with Tup1-Matalpha2 interaction are located on the top surface of its seven-bladed ß-propeller around the central channel, analogous to the mutations described for Brat. The ubiquitin-conjugating enzyme Cdc4 similarly uses the top surface of its eight-bladed ß-propeller to bind a peptide ligand derived from the Cdk inhibitor, Sic1. In the case of Cdc4, the peptide-binding site is relatively small (buried surface area of ~750 Å2) when compared to the large interface in the docked Brat-Pum complex (2,900 Å2). However, in each case, a flexible peptide (or a loop in the case of Pumilio) docks in and around the central pore, suggesting an emerging recognition theme for ß-propeller molecules (Edwards, 2003).
Brat is normally recruited not to Pum alone, but to a ternary complex of Pum and Nos bound to the NRE. The model of the Pum-Brat subassembly suggests that the 'edge' of the Brat ß-propeller is available to interact with Nos, much as Gß and the scaffolding protein clathrin use the sides of their seven-bladed ß-propellers to bind cofactors. Although its location in the repressor complex is not yet well defined, Nos is probably recruited to the Pum-RNA complex via contacts made by the C terminus of the Pum RNA-binding domain. The proximity of the NHL domain to the presumptive Nos-binding site on Pum extends the likelihood of cooperative Brat-Nos interactions, and may explain why Brat is only recruited subsequent to Pum and Nos binding to the hb 3' UTR (Edwards, 2003).
The Drosophila proteome contains two additional NHL domain proteins [MeiP-26 and Dappled (Dpld)] that, based on genetic evidence, appear to be tumor suppressors and growth regulators like Brat. It is tempting to speculate they interact with cofactors or regulatory targets much as Brat interacts with Pum. However, neither seems likely to use Pum as a cofactor. The NHL domains of Brat and MeiP-26 are very similar: There are no major insertions or deletions in the DA loops of MeiP-26, and the top surface of its ß-propeller, like that of Brat, is electropositive. Thus, based on structural considerations, MeiP-26 might interact with Pum; however, genetic experiments suggest it does not do so in vivo. In contrast, structural considerations suggest the NHL domain of Dpld, which governs the growth of larval organs, is unlikely to bind Pum due to substantial differences in its predicted surface charge distribution and the presence of large insertions in the DA loops on the top surface. Therefore, although Brat, MeiP-26, and Dpld may use their NHL domains in a similar manner, each probably binds to distinct partners (Edwards, 2003).
Based on the analysis of loss- and gain-of-function experiments, Brat appears to regulate abdominal segmentation (via hb translation), brain size, cell size in the imaginal discs, and the accumulation of rRNA. Strikingly, substitutions that abrogate many of these processes map to the 'same' top surface of the NHL domain, near the central channel. This suggests that Brat may recognize protruding, flexible loops in a number of protein cofactors or regulatory targets, much as it recognizes the loop in Puf repeat 8 that constitutes the core of the Brat-Pum interaction surface (Edwards, 2003).
An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. The Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. It is concluded that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation (Lee, 2006).
The translational repressor Brat directly interacts with the Miranda central domain and is a Miranda cargo protein specifically partitioned into the GMC daughter cell during neuroblast asymmetric cell division. Brat is the first Miranda cargo protein identified since the original finding that Prospero and Staufen were shown to be Miranda cargo proteins over 8 years ago. Prospero is a homeodomain transcriptional repressor, and Staufen is an RNA binding protein that interacts with prospero mRNA. It is unknown whether Miranda has other cargo proteins in addition to Brat, Prospero, and Staufen, and it is unclear whether all three known cargo proteins can associate with a single Miranda protein (Lee, 2006).
It is unknown how Brat promotes Prospero basal localization. A model is favored in which Brat protein stabilizes Prospero/Miranda interactions, so that Prospero protein is cytoplasmic in the absence of Brat. An obviously elevated level of cytoplasmic Prospero is not seen in brat mutant neuroblasts, but delocalization of Prospero protein from the basal crescent might not be visible over background. Alternatively, brat mutant neuroblasts may fail to transcribe or translate prospero in neuroblasts. This would most likely be an indirect effect, since Brat has been shown to only have translational repressor function. It has not been possible to detect prospero mRNA in wild-type larval neuroblasts, despite robust levels in GMCs, so this possibility has not been tested (Lee, 2006).
Some brat mutant neuroblasts show expanded aPKC cortical crescents, in some cases reaching the basal cortex. This phenotype appears specific for aPKC, because other apical cortical proteins (e.g., Baz, Pins) are unaffected. Brat might repress aPKC translation, leading to increased aPKC protein levels in brat mutants. Alternatively, the absence of Prospero or other basal cortical proteins may indirectly affect aPKC localization (Lee, 2006).
brat mutant brains show a dramatic increase in the number of large, proliferating Dpn+ neuroblasts between 48 and 96 hr ALH. Where do these hundreds of extra neuroblasts come from? They are unlikely to come from outside the brain, or from dedifferentiation of neurons or glia, although these models can't formally be ruled out. They are likely to derive from the pool of Dpn+ neuroblasts in the brain, because these are the primary pool of proliferating cells in the larval central brain, and thus the best candidates to generate the thousands of extra cells found in the hypertrophied brat mutant brains (Lee, 2006).
A model is proposed in which a subset of brat mutant “GMCs” enlarge into proliferative neuroblasts. This model is supported by several lines of evidence. (1) brat mutant GMCs maintain neuroblast-specific gene expression (Dpn, Miranda, Worniu); (2) brat mutants show an inverse relationship between increasing neuroblast number and decreasing neuronal number over time, consistent with GMCs forming neuroblasts instead of neurons; (3) brat mutant GMCs can be labeled by a BrdU pulse at their birth, yet most lose BrdU incorporation during the chase interval, showing that they either reenter the cell cycle or undergo cell death, and that cell death is not consistent with the brain overgrowth phenotype; (4) brat mutant telophase profiles show that all GMCs are born as small Miranda+ cells, ruling out physically or molecularly symmetric neuroblast divisions as a mechanism for increasing the neuroblast population; and (5) brat mutants show cell enlargement in other tissues, and a similar cell growth phenotype has been observed in mutants in the C. elegans brat ortholog (Lee, 2006).
What is the cellular origin of the brat mutant phenotype? brat mutant GMCs are compromised in three ways: they lack Brat translational repression activity, lack Prospero, and some may have ectopic aPKC. Loss of Brat translational repression activity could well play a role in the ectopic neuroblast self-renewal phenotype, because all brat mutants disrupting the NHL translational repression domain exhibit a brain tumor phenotype, and Brat has been previously shown to negatively regulate cell growth. Loss of Prospero also plays a role in the brat phenotype: prospero mutant GMCs have a failure to downregulate neuroblast gene expression and a failure in neuronal differentiation, similar to brat mutants. prospero null mutant embryos also show a slight delay in neuronal differentiation, although they appear to undergo normal neuroblast self-renewal. Finally, ectopic aPKC can also mimic aspects of the brat phenotype, including formation of supernumerary large Dpn+ neuroblasts. Interestingly, the mammalian paralogs of Drosophila aPKC (aPKCλ/ζ) are expressed in neural progenitors of the ventricular zone, and the mammalian Prospero ortholog Prox1 is expressed in differentiating neurons of the subventricular zone. Thus, identifying Prospero transcriptional targets and aPKC phosphorylation targets may provide further insight into the molecular mechanism of neural stem cell self-renewal in both Drosophila and mammals (Lee, 2006).
In the early Drosophila embryo, asymmetric distribution of transcription factors, established as a consequence of translational control of their maternally-derived mRNAs, initiates pattern formation. For instance, translation of the uniformly distributed maternal hunchback (hb) mRNA is inhibited at the posterior to form an anterior-to-posterior protein concentration gradient along the longitudinal axis. Inhibition of hb mRNA translation requires an mRNP complex (the NRE-complex) that consists of Nanos (Nos), Pumilio (Pum) and Brain tumor (Brat) proteins, and the Nos responsive element (NRE) present in the 3' UTR of hb mRNA. The identity of the mRNA 5' effector protein that is responsible for this translational inhibition remained elusive. This study shows that d4EHP, a cap-binding protein which represses caudal (cad) mRNA translation (Cho, 2005), also inhibits hb mRNA translation by interacting simultaneously with the mRNA 5' cap structure (m7GpppN, where N is any nucleotide) and Brat. Thus, by regulating Cad and Hb expression, d4EHP plays a key role in establishing anterior-posterior axis polarity in the Drosophila embryo (Cho, 2006).
Transcription is globally repressed in the rapidly-dividing nuclei of early Drosophila embryos, and therefore gene expression is largely regulated by translational control of maternally-provided mRNAs. Translation is often regulated at initiation, which occurs in multiple steps starting with the recruitment of the 40S ribosomal subunit to the 5' end of an mRNA and resulting in the correct positioning of the 80S ribosome at the initiation codon. Recognition of the cap structure by eIF4F (composed of three subunits: eIF4E, eIF4A and eIF4G) is an integral part of this process. Moreover, eIF4G interacts both with eIF4E and the poly(A)-binding protein (PABP), thus circularizing the mRNA, which in turn is believed to promote re-initiation. Consistent with their importance, eIF4E and PABP have emerged as major targets of translational regulatory mechanisms mediated by such modulator proteins as 4E-BPs and Paip2 (Cho, 2006).
Embryonic development in many metazoans requires the activity of various maternal determinants called morphogens, whose spatial and temporal expression is tightly regulated. In Drosophila, local morphogen concentrations are important for the establishment of polarity and subsequent organization of both the antero-posterior and dorso-ventral axes of the embryo. A key morphogen for antero-posterior patterning is the transcription factor Hunchback (Hb); when maternal Hb is allowed to accumulate inappropriately, posterior segmentation is blocked. Two modes of translational control have been proposed for the establishment of the maternal Hb gradient: translational silencing via deadenylation and inhibition at the initiation step in a cap-dependent manner (Cho, 2006 and references therein).
d4EHP, an eIF4E-like cap-binding protein that does not interact with deIF4G and d4E-BP, inhibits the translation of cad mRNA by interacting simultaneously with the cap and Bicoid (Bcd) (Cho, 2005). While many embryos (~41%) produced by females homozygous for the d4EHPCP53 mutation showed anterior patterning defects consistent with mislocalized Cad, some (~7%) also exhibited patterning defects such as missing abdominal segments that cannot be readily explained by ectopic Cad expression. Since inhibition of hb mRNA translation has been linked in one study to the cap structure (Chagnovich, 2001) and since these additional phenotypes could be consistent with inappropriate regulation of Hb, this study investigated the role of d4EHP in Hb expression. Embryos (0-2h) from females homozygous for the d4EHPCP53 mutation (Cho, 2005) were collected and immunostained using anti-Hb antibody. DNA was stained with DAPI to highlight the nuclei). For simplicity, embryos will subsequently be referred to by their maternal genotype. To evaluate the extent of the Hb gradient its signal intensity was measured at 38-50 locations along the anterior-posterior axes of 6-16 embryos of each genotype. The values were corrected for overall signal intensity and then normalized the data for embryo length (EL, anterior pole = 0%, posterior pole = 100%). The normalized values were plotted and average intensity values were calculated to obtain an average trend. It was observed that in OreR embryos, Hb signal intensity drops steeply in the middle of the embryo and reaches 50% maximum intensity at 48% EL. In d4EHPCP53 embryos the Hb expression domain extended substantially further toward the posterior and signal intensity remained at approximately 50% of the maximum throughout the region between 50-75% EL. Normal Hb distribution was restored to d4EHPCP53 mutant embryos by transgene-derived expression of wild-type d4EHP (d4EHPwt, but not by expression of a mutant form of d4EHP (d4EHPW114A), which is unable to bind the cap structure. Expression of another form of d4EHP (d4EHPW85F) which cannot bind Bcd, fully rescued the defective Hb gradient. The expression levels of the wild-type and mutant d4EHP transgenes are essentially equal. Distributions of Nos, Pum, and Brat were unaffected in d4EHPCP53 mutant embryos. Taken together, these data demonstrate that d4EHP plays a key role in establishing the posterior boundary of Hb expression in a manner that requires its cap-binding activity but not an association with Bcd (Cho, 2006).
It was reasoned that Brat might be a candidate partner protein for d4EHP since both are relevant for hb regulation. Thus whether d4EHP and Brat physically interact was investigated in vivo. Extracts prepared from 0-2h Oregon-R (OreR) embryos were treated with RNase and used to examine the interaction between Brat and d4EHP. Western blotting analysis using antibodies against d4EHP and Brat demonstrates that, while anti-d4EHP co-immunoprecipitated endogenous Brat, pre-immune serum did not. To further demonstrate the specificity of this interaction, HA-tagged deIF4EI and the RNA-binding protein La (negative controls) were transfected in HEK293 cells along with FLAG-tagged full-length Brat. While anti-FLAG antibody immunoprecipitated wild-type HA-d4EHP together with FLAG-Brat, deIF4EI and La failed to co-immunoprecipitate. Similarly, other RNA-binding proteins such as hnRNP U and HuR, and a d4EHP mutant (W173A), in which a tryptophan residue that is part of the hydrophobic core and thus affects protein folding is replaced, also failed to interact with Brat, demonstrating that Brat interacts specifically with d4EHP. Since a cell transfection system was used to assay for the d4EHP:Brat interaction, it is possible that other bridging proteins are required for the d4EHP-Brat association (Cho, 2006).
To identify the Brat-interacting domain of d4EHP, a number of individual residues located on its convex dorsal surface were mutated, and co-immunoprecipitation was tested with Brat. From this work no point mutant of d4EHP was identified that abrogated the interaction. As an alternative approach, chimeric proteins were created in which different domains of d4EHP were replaced with their counterparts from deIF4EI, taking advantage of the knowledge that, unlike d4EHP, deIF4EI does not interact with Brat. Mhree mutant forms of d4EHP were produced, with each one of its three dorsal α-helices replaced with that of deIF4EI. It was found that, while helix 1 and 2 mutants failed to disrupt binding to Brat, replacement of d4EHP helix 3 (residues 179 to 194) significantly reduced the interaction with Brat. Consistent with these observations, α-helix 3 is the most divergent between d4EHP and deIF4EI. The overall structure of d4EHP is not affected by the replacement of helix 3 with its deIF4EI counterpart, since the chimeric protein still binds to the cap. Thus, these data demonstrate that Brat interacts with d4EHP on its convex dorsal surface and that this interaction is mediated by the third α-helix of d4EHP (Cho, 2006).
A C-terminal domain of Brat termed the NHL domain is both necessary and sufficient to inhibit hb mRNA translation. The NHL domain contains two large surfaces (defined as top and bottom), that can support protein-protein interactions. While the top surface of the NHL domain binds to Pum and Nos, the bottom surface does not interact with any known protein. Although the Brat NHL domain contains an amino acid sequence that conforms to the YxxxxxxLΦ d4EHP-binding motif (Cho, 2005), the d4EHP:Brat interaction does not require this motif, since a Brat deletion mutant that lacks it can still interact with both d4EHP and the d4EHP W85F mutant. This sequence is most probably masked from interaction with d4EHP because it is located in the hydrophobic core of the NHL domain. To determine whether the d4EHP:Brat interaction requires the NHL domain, a Brat mutant that lacks the domain (Brat ΔNHL) was engineered and used in a co-immunoprecipitation experiment. While wild-type Brat was readily co-immunoprecipitated with d4EHP, the Brat ΔNHL mutant was not. Thus, it is concluded that the NHL domain is the site of d4EHP interaction. To further characterize this interaction, point mutations were designed to replace residues on the two surfaces of the NHL domain, and the mutant proteins were tested for their ability to interact with d4EHP. Mutation of a top surface residue that affects Brat interaction with Pum (G774A) did not affect the d4EHP:Brat interaction. However, when residues on the bottom surface were mutated, the d4EHP:Brat interaction was either significantly reduced (G860D and KE809/810AA), or abrogated (R837D and K882E). Importantly, the Brat NHL R837D mutant can assemble into an NRE-complex, demonstrating that this mutation specifically affects the d4EHP interaction and not the interactions with Pum and Nos (Cho, 2006).
Brat inhibits hb mRNA translation by interacting with the NRE-complex (Sonoda, 2001). Since d4EHP interacts physically with Brat, it was asked whether d4EHP can be co-purified with the NRE complex in vitro. Incubation of recombinant components of the NRE-complex (Brat, Pum, Nos and NRE) together with HA-tagged d4EHP resulted in the retention of d4EHP on glutathione-Sepharose beads through the GST-Pum RNAB fusion protein. The association of Brat with d4EHP was dependent on the ability of d4EHP to bind to Brat, since addition of Pum/Nos/NRE alone or in combination with the Brat R837D mutant failed to capture it. Thus, by interacting with Brat, d4EHP can associate with the NRE complex (Cho, 2006).
To investigate the biological significance of the d4EHP:Brat interaction, the effects of Brat mutants, which are defective for d4EHP binding, were examined in Drosophila embryos. bratfs1 mutant embryos exhibit a significant expansion of the Hb expression domain towards the posterior and display severe abdominal segmentation defects. When a bratWT transgene is expressed in the bratfs1 mutant background, normal Hb distribution and a wild-type segmentation pattern is restored). To investigate whether interaction with d4EHP is essential for the function of Brat in embryonic patterning, transgenes were introduced encoding mutant forms of Brat that affect the d4EHP:Brat interaction (bratR837D and bratK882E) into the bratfs1 mutant background. Despite being expressed at levels similar to the bratWT transgene, these mutant forms fail to fully rescue the normal Hb gradient and, importantly, do not fully rescue the bratfs1 mutant phenotype. Taken together, our data strongly argue that the d4EHP:Brat interaction contributes significantly to hb regulation (Cho, 2006).
Through its interaction with Brat, d4EHP defines and sharpens the posterior boundary of Hb expression. Based on the hypomorphic d4EHPCP53 phenotype, its activity appears most relevant to hb regulation in the region of the embryo from 50-75% EL, although it is possible that a null d4EHP allele would have more drastic effects. The d4EHP:Brat interaction is mediated via residues on the bottom surface of the Brat NHL domain. Thus, as in the established for cad (Cho, 2006), a simultaneous interaction of d4EHP with the cap and Brat results in mRNA circularization and renders hb translationally inactive. Since the interaction between Brat and d4EHP does not involve the previously described 4EHP-binding motif (YxxxxxxLΦ), it is possible that d4EHP interacts with Brat through a bridging protein (Cho, 2006).
The data support a model for the requirement for the 5' cap structure in regulation of endogenous hb mRNA. This is consistent with an earlier study that assessed translation of NRE-containing mRNAs after injection into Drosophila embryos and concluded that the cap structure is functionally significant (Chagnovich, 2001). In contrast, another study reported that Nos and Pum repressed the expression of an engineered transgene containing an internal ribosome entry site (IRES) and a hairpin loop designed to block cap-dependent translation. These results were used to conclude that hb translational repression is cap-independent. However, the phenotypic assay used in that study was indirect and the observed results could also be caused by RNA destabilization. Furthermore, Nos-dependent deadenylation was also shown to be important in establishing the Hb gradient. It is difficult to reconcile all these data without concluding that multiple distinct post-transcriptional mechanisms regulate Hb expression, including two that require Nos. The novel d4EHP-dependent mechanism defined in this study appears important for repressing hb in more central regions of the embryo, while cap-independent regulation involving deadenylation of hb mRNA may predominate in more posterior regions of the embryo. It is noted that mutant forms of Brat that are abrogated for d4EHP interaction retain substantial (but not complete) activity in repressing hb, suggesting some redundancy between these two mechanisms. Analogous overlapping translational control mechanisms have recently been reported for Bruno, which represses Oskar (Osk) expression both through cap-dependent translational regulation and through packaging osk mRNA into translationally silent RNP complexes (Cho, 2006).
Identification of a common inhibitory mechanism which regulates cad and hb mRNA translation simplifies the understanding of how the anterior-posterior axis is organized during early Drosophila embryogenesis. By regulating two classical maternal morphogenetic gradients, d4EHP plays a critical role in early Drosophila embryonic development. It is noteworthy that d4EHP is recruited to these mRNAs through different RNA binding proteins that presumably recognize different sequence elements. In the case of cad, d4EHP becomes associated by binding directly to Bcd, which in turn recognizes a defined 3’UTR element, the BBR. In the case of hb, Bcd binding is not involved in d4EHP recruitment and no element similar to the BBR is present. It remains uncertain whether the interaction between d4EHP and Brat is direct or indirect; since d4EHP and Brat are both uniformly distributed in early embryos, a non-uniformly distributed bridging protein mediating this interaction may be the basis of the spatially-restricted requirement for d4EHP in hb repression. Since d4EHP and some of its interacting partners are evolutionarily conserved in higher eukaryotes and because cap-dependent translation regulation plays such an important role in eukaryotic gene expression, it is predicted that 4EHP-dependent translational inhibitory mechanisms are widespread throughout the animal kingdom (Cho, 2006).
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