In Drosophila embryos, graded activity of the posterior determinant Nanos (nos) generates abdominal segmentation by blocking protein expression from maternal transcripts of hb. When active inappropriately at the anterior pole, NOS can also block expression of the anterior determinant Bicoid. Both regulatory interactions are mediated by similar sequences in the 3' untranslated region of each transcript. These NOS response elements (NREs) are both necessary and sufficient to confer nos-dependent regulation, the degree of regulation determined by the number and quality of the elements and the level of NOS in vivo. Based on these and other results, it can be argued that NOS acts as a morphogen, controlling HB expression (and hence abdominal pattern) as a function of its concentration-dependent interaction with the NREs. Thus, it would seem that the requirements for NOS mRNA localization, involving proteins of the posterior group are imposed by the presence of the NRE in BCD and HB mRNAs (Wharton, 1991).
The Drosophila Nanos protein is a localized repressor of Hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. One equivalent of zinc is bound with high affinity by each of the CCHC motifs. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. Full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the Hunchback mRNA target sites for Nanos-dependent translational repression are found to disrupt translational repression in vivo, but have little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target Hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex (Curtis, 1997).
Posterior patterning in Drosophila embryos is governed by Nanos repression of the translation of maternal transcripts of the hunchback gene. Sites in HB mRNA that mediate this repression, named Nanos response elements (NREs), have been identified. Two proteins present in embryonic extracts, neither one NOS, bind specifically to the NRE in vitro. Binding in vitro correlates with NRE function in vivo. One of the NRE-binding factors is encoded by pumilio, like nos, a gene essential for abdominal segmentation. This suggests that PUM acts by recognizing the NRE and then recruiting NOS. Presumably, the resulting complex inhibits some component of the translation machinery (Murata, 1995).
Nanos protein promotes abdominal structures in Drosophila embryos by repressing the translation of maternal hunchback mRNA in the posterior. To study the mechanism of nanos-mediated translational repression, the mechanism by which maternal Hunchback mRNA is translationally activated was examined. In the oocyte from wild-type females, where no HB translation is detected, the mRNA has a poly(A) tail length of approximately 30 nucleotides. However, concomitant with translation of the mRNA at between 0.5 and 1.5 hours after egg deposition, the poly(A) tail is elongated to approximately 70 nt. In the absence of nanos activity, the poly(A) tail of Hunchback mRNA is elongated to approximately 100 nt concomitant with its translation, suggesting that cytoplasmic polyadenylation directs activation. However, in the presence of nanos the length of the Hunchback mRNA poly(A) tail is reduced via the nanos response element present in the HB 3'UTR. To determine if nanos activity represses translation by altering the polyadenylation state of Hunchback mRNA, various in vitro transcribed RNAs were injected into Drosophila embryos and changes in polyadenylation were determined. nanos activity reduces the polyadenylation status of injected Hunchback RNAs by accelerating their deadenylation. Pumilio activity, which is necessary to repress the translation of Hunchback mRNA, is also needed to alter polyadenylation. An examination of translation indicates a strong correlation between poly(A) shortening and suppression of translation. These data indicate that nanos and pumilio determine posterior morphology by promoting the de-adenylation of maternal Hunchback mRNA, thereby repressing its translation (Wreden, 1997).
Posterior patterning in the Drosophila embryo requires the action of Nanos and Pumilio, which collaborate to regulate the translation of maternal Hunchback mRNA. Pum recognizes sites in the 3' UTR of HB mRNA. The RNA-binding domain of Pum has been defined and residues essential for translational repression are shown to be embedded within this domain. Nos and Pum can repress cap-independent translation from an internal ribosome entry site (IRES) in vivo, suggesting that they act downstream of the initial steps of normal, cap-dependent translation (Wharton, 1998).
Translation of Hunchback(mat) (HB[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the HB[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of Pum to the NRE is thought to target HB(mat) mRNA for repression. The RNA-binding domain of Pum is an evolutionarily conserved, 334 amino acid region at the carboxy terminus of the approximately 158 kDa Pum protein. This contiguous region of Pum retains the RNA binding specificity of full length Pum protein. Proteins with sequences homologous to the Pum RNA binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the Pum RNA binding domain in other far flung species suggests that the domain is an ancient protein motif, and conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila Pum (Zamore, 1997).
Translational repression of Hunchback mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The Pumilio protein is thought to bind the NREs and thereby repress HB translation. The RNA-binding domain of Pum defines an evolutionarily conserved family of RNA-binding proteins: the Pum-Homology Domain (Pum-HD) proteins, which have been identified in yeast, plants, and animals. The Pum RNA-binding domain [the Drosophila PUM-HD (DmPUM-HD)] has been shown to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of Pum might recognize one NRE. The RNA-binding affinity and stoichiometry of the DmPUM-HD has been analyzed and it is found that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). No cooperative interactions is detected between DmPUM-HD monomers bound at adjacent sites. These results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on an estimate of the intraembryonic concentration of PUM (>40 nM), it is proposed that in vivo nearly all NREs are occupied by a PUM monomer (Zamore, 1999).
Translational regulation of Hunchback mRNA is essential for posterior patterning of the Drosophila embryo. This regulation is mediated by sequences in the 3'-untranslated region of HB mRNA (the Nanos response elements or NREs), as well as two trans-acting factors -- Nanos and Pumilio. Pum binds to a pair of 32-nucleotide sequences (named Nanos response elements -- NREs) in the 3'-UTR of maternal HB mRNA in order to repress HB translation in the posterior of the embryo. This translational repression is essential for normal abdominal segmentation. The RNA-binding domain of Pum is structurally similar to that of another translational regulator, FBF (fem-3 mRNA-binding factor) found in C. elegans (Zhang, 1997). The minimal RNA-binding domain of each protein consists of eight imperfect repeats plus flanking residues. These structural similarities define a conserved 'Puf' motif (Pum and FBF) that is found in proteins from diverse organisms from yeast to humans. However, the RNA partner of no other Puf domain protein has been identified, nor is it clear whether other Puf proteins regulate translation or some other aspect of RNA metabolism. Thus, Pumilio recognizes the NREs via a conserved binding motif. The mechanism of Nanos action has not been clear. In this report protein-protein and protein-RNA interaction assays in yeast and in vitro were used to show that Nanos forms a ternary complex with the RNA-binding domain of Pumilio and the NRE. Mutant forms of the NRE, Nos, and Pum that do not regulate HB mRNA normally in embryos do not assemble normally into a ternary complex. In particular, recruitment of Nos is dependent on bases in the center of the NRE, on the carboxy-terminal Cys/His domain of Nos, and on residues in the eighth repeat of the Pum RNA-binding domain. These residues differ in a closely related human protein that also binds to the NRE but cannot recruit Drosophila Nos. Taken together, these findings suggest models for how Nos and Pum collaboratively target HB mRNA. More generally, they suggest that Pum-like proteins from other species may also act by recruiting cofactors to regulate translation (Sonoda, 1999).
In one model, Nos simultaneously makes specific contacts with Pum and nucleotides 17-20 of the NRE. On their own, neither the Nos-Pum nor the Nos-NRE contacts are strong enough to recruit Nos to HB mRNA (at least in the presence of competitor proteins and RNAs), because binary complexes with Nos are not detectable. In another model, unbound Pum cannot interact with Nos, but binding to the NRE induces a conformational change in Pum, which subsequently recruits Nos via protein-protein contacts. In this model, nucleotides 17-20 of the NRE interact with Pum to induce the conformational change without affecting its affinity for the RNA, and nonspecific interactions between Nos and other portions of the RNA help stabilize the complex. Either model is consistent with the nonspecific RNA-binding activity reported for the carboxy-terminal portion of Nos in vitro and the RNA-Nos cross-link found in this study. Further structural and biochemical experiments will be required to distinguish between these (or alternative) models (Sonoda, 1999 and references therein).
The mechanism by which the ternary complex blocks translation is not yet clear. mRNAs subject to Nos- and Pum-dependent repression are deadenylated in vivo. In addition, Nos and Pum have been shown to regulate internal ribosome entry site (IRES)-dependent translation in imaginal disc cells, suggesting that their regulatory target lies downstream of cap recognition and scanning. It is assumed that some surface of the ternary complex, formed jointly by Nos and Pum, targets a component of the polyadenylation or translation machinery. This surface appears to be altered in the Pum680 mutant protein, which binds the NRE normally but is defective in regulating HB translation in the embryo. The Pum680 mutant recruits Nos into a ternary complex normally and thus apparently is defective in a subsequent step of the repression reaction. The RNA-binding domain of Pum therefore appears to have at least three different functions in regulating HB: recognizing the NRE, recruiting Nos, and acting as a corepressor (with Nos) to block translation (Sonoda, 1999 and references therein).
In the experiments reported in this study, focus was placed on discrete regions of both Nos (the carboxy-terminal 97 amino acids) and Pum (the minimal RNA-binding domain), which play an essential role in formation of the ternary complex. However, other regions of Nos are known to be required for its function in repressing translation in the embryo. In addition, residues elsewhere in Pum play an unknown role in augmenting the intrinsic translational repression activity of the RNA-binding domain. Thus, the ternary complex formed by the 157-kD, full-length Pum protein may be stabilized by auxillary protein-protein or protein-RNA interactions in addition to those that mediate recruitment of the carboxy-terminal domain of Nos by the RNA-binding (or Puf domain) of Pum. The results suggest that Puf domain proteins generally may act by recruiting cofactors to specific RNA binding sites. Cofactor specificity may be mediated, at least in part, by the eighth repeat of the Puf domain. Although Puf domain proteins have been described in organisms from yeast to humans, for only one protein other than Drosophila Pum, C. elegans FBF, is the relevant RNA regulatory target known. FBF regulates the sperm/oocyte switch in the hermaphrodite germ line by governing the translation of fem-3 mRNA (Zhang, 1997). The FBF RNA-binding domain interacts with one of the C. elegans Nos homologs (Kraemer, 1999). Further experiments will be required to determine whether the Pum/fly Nos complex and the FBF/worm Nos complex function in a similar manner (Sonoda, 1999 and references therein).
Maternally derived HB mRNA is uniformly distributed throughout the embryo; the mRNA is translationally repressed in the posterior, giving rise to an anterior-to-posterior gradient of Hb protein. Failure of this repression results in the abnormal accumulation of Hb in the posterior, which inhibits abdominal segmentation. Two conserved RNA-binding proteins, Pumilio (Pum) and Nanos (Nos), are specifically required to repress HB translation. Pum, which is distributed uniformly throughout the embryo, is the founding member of a large family of RNA-binding proteins. Pum binds to 32 nucleotide sites in the 3' UTR of HB (Nos Response Elements, NREs) to regulate HB translation. Nos, which initially is distributed as a gradient emanating from the posterior pole of the embryo, contains a conserved zinc finger that mediates nonspecific RNA binding. Nos is selectively recruited into a ternary complex on HB mRNA by NRE-bound Pum. The mechanism by which the resulting Nos/Pum/NRE complex regulates translation is not yet understood, although deadenylation is thought to play a role (Sonoda, 2001 and references therein).
To identify targets or cofactors of the Nos/Pum/NRE ternary complex, a yeast 'four-hybrid' experiment was performed; a Gal4 activation domain fusion library was screened for proteins that interact with the ternary complex. The bait contained the RNA-binding domain of Pum, full-length Nos, and NRE-bearing RNA. As anticipated, factors that interact with individual components in isolation were identified. However, one factor, which proved to be a fragment of Brain tumor (Brat), interacts only with the ternary complex and not with either Nos alone, Pum alone, or a Pum/NRE binary complex. Deletion analysis revealed that recruitment of Brat is dependent on the conserved carboxy-terminal domain of Nos that mediates its interaction with Pum on HB mRNA, and not the amino-terminal domain of Nos that mediates interaction with Cup during early oogenesis. Mutational analysis further showed that a fragment of Brat consisting of little more than the NHL domain is recruited to the ternary complex. Protein-protein interaction experiments show that Nanos and Pumilio are required to recruit Brat to HB mRNA and genetic experiments show that Brat is required for repression of HB mRNA (Sonoda, 2001).
A model is presented of how Nos, Pum, and Brat act to regulate gene expression. The model involves combinatorial interactions among cis-acting sequences in regulated mRNAs, proteins that recognize these sequences, and the NHL domain of Brat. Recruitment of Brat occurs through protein-protein interactions with RNA-bound Pum and Nos; formation of the resulting quaternary complex is essential for translational control of HB. Recruitment of Brat to the NRE jointly by Nos and Pum is essential for regulation of HB mRNA. Three lines of evidence show that the NHL domain plays a key role in this process: (1) the NHL domain is sufficient to mediate interaction with the Nos/Pum/NRE complex, thereby targeting Brat to HB mRNA; (2) single amino acid substitutions within the NHL domain attenuate interaction with the ternary complex and regulation of HB in vivo; (3) maternal expression of the wild-type NHL domain alone is sufficient to restore HB regulation in bratfs mutant embryos. This result suggests that the NHL domain contains intrinsic translation regulatory activity. However, activity of the isolated NHL domain is (necessarily) assayed in the presence of Bratfs mutant protein, and thus, the possibility that the amino-terminal BCC domain participates somehow in HB mRNA regulation cannot be ruled out (Sonoda, 2001).
Translation regulation plays an essential role in the differentiation and development of animal cells. One well-studied case is the control of Hunchback mRNA during early Drosophila embryogenesis by the trans-acting factors Pumilio, Nanos, and Brain Tumor. This study reports a crystal structure of the critical region of Pumilio, the Puf domain, that organizes a multivalent repression complex on the 3' untranslated region of Hunchback mRNA. The similarity between Pum RBD and that of another translation regulator FBF, which binds to the 3'UTR of fem-3 mRNA in C. elegans, defines a Puf (Pum and FBF) domain, which is conserved in organisms as diverse as plants, yeast, and humans. The Puf domain is characterized by eight imperfect repeats of ~36 amino acids (Puf repeats), followed by a C-terminal extension. All eight repeats appear to be required for proper folding of the Puf domain, since limited proteolysis fails to yield stable smaller fragments. The Puf domain is thus amongst the largest sequence-specific RNA binding motifs to be discovered; the RRM, the KH domain (70 residues), and the dsRBD (65 residues), are much smaller. The PUF domain structure reveals an extended, rainbow shaped molecule, with tandem helical repeats that bear unexpected resemblance to the armadillo repeats in beta-catenin and the HEAT repeats in protein phosphatase 2A. Based on the structure and genetic experiments, putative interaction surfaces for Hunchback mRNA and the cofactors Nanos and Brain Tumor are identified. This analysis suggests that similar features in helical repeat proteins are used to bind extended peptides and RNA (Edwards, 2001).
Two lines of evidence from mutagenesis studies support the idea that the Pum concave surface binds RNA. (1) A gene encoding the 322 residue minimal Pum RBD was randomly mutagenized and variants were isolated that bind normally to the wild-type NRE in yeast. Collectively, these variants bear substitutions at 61 residues, 55 of which map to the structure; the remaining six are in the putative 9th repeat, not in this crystal structure. Of these, only 3 (presumably silent) substitutions fall on the solvent exposed concave surface, with the remaining 52 lying elsewhere. The relative paucity of substitutions within the inner surface is consistent with this being the area that contacts the RNA. (2) Based on the structure, single substitutions were introduced in solvent-exposed residues along the inner surface in five of the eight Puf domains and RNA binding activity was tested in yeast. Each of these mutants is inactive. Thus, the concentration of positive charge and the distribution of both silent and inactivating substitutions together suggest that the RNA interacts with the inner concave surface (Edwards, 2001).
It is proposed that HB mRNA binds to this inner surface in an extended single-stranded conformation. Algorithms that predict RNA structure suggest the NRE does not adopt a stable secondary or tertiary structure. The minimal NRE for high affinity Pum binding consists of nucleotides 3-27, which bracket specific contacts with nucleotides 9, 11-13, and 21-24. The length of this minimal NRE, in an extended single-stranded conformation (112 Å), agrees approximately with the contour length (90 Å) of the concave surface of the Puf domain. It is noteworthy that beta-catenin also has the highest concentration of positive charge within its concave surface (or groove), which is the proposed binding site for segments of cadherins, APC, and members of the LEF-1/TCF family of transcription factors. A recent crystal structure of a beta-catenin/TCF complex shows the TCF segment tethered along the positively charged groove. In the case of karyopherin-alpha, the concave surface is the binding site for the NLS peptide. Taken together, the binding of ligands to concave surfaces is a recurring theme in helical repeat proteins. The Pum Puf domain shows that this type of extended surface can be used to bind RNA, as well as peptides (Edwards, 2001).
Repression of HB mRNA depends not only on Pum, but also on the recruitment of Nanos and Brat to form a quaternary complex. Previous work suggested that Nos is recruited via residues in Puf repeat 8. These residues map to the extra long loop between helices H1 and H2 in repeat 8, that is the main protrusion from an otherwise relatively smooth outer Pum surface. Two different insertions into this loop have no effect on Pum-RNA binding but eliminate recruitment of Nos. To further define the Nos interaction surface, the collection of Pum mutants that bind normally to RNA was tested for Nos recruitment in yeast. Of the 61 substitutions distributed throughout the domain, only two abrogate interaction with Nos. One is a substitution in the putative ninth Puf repeat that is not represented in this structure, while the other changes the solvent exposed phenylalanine on the H1/H2 loop to a serine (F1367S). Thus, the Pum surface that interacts with Nos appears to be limited to a small region that includes the eighth repeat and the C-terminal tail. If this tail indeed does fold into a ninth Puf repeat, then the Pum-Nos interface would span a length of ~15-20 Å on the outer convex surface. It is tempting to think that the C-terminal tail may only fold when Pum binds to the RNA, thereby explaining why Nos is only recruited to the Pum/NRE binary complex and not to Pum alone. The insertions into the long flexible loop in repeat 8 may modify its conformation such that F1367 is no longer exposed for interaction with Nos. The proposed Phe-Nos interaction is reminiscent of the way in which a solvent exposed phenylalanine on the receptor CD4 interacts with the HIV gp120 glycoprotein (Edwards, 2001).
Development of the Drosophila abdomen requires repression of maternal Hunchback (HB) mRNA translation in the posterior of the embryo. This regulation involves at least four components: nanos response elements within the HB 3' untranslated region and the activities of Pumilio (Pum), Nanos (Nos), and Brain tumor. To study this regulation, an RNA injection assay was developed that faithfully recapitulates the regulation of the endogenous HB message. Previous studies have suggested that Nos and Pum can regulate translation by directing poly(A) removal. RNAs that lack a poly(A) tail and cannot be polyadenylated and RNAs that contain translational activating sequences in place of the poly(A) tail are still repressed in the posterior. These data demonstrate that the poly(A) tail is not required for regulation and suggest that Nos and Pum can regulate HB translation by two mechanisms: removal of the poly(A) tail and a poly(A)-independent pathway that directly affects translation (Chagnovich, 2001).
Nos, Pum, and Brat proteins regulate maternal HB translation in the posterior of the Drosophila embryo via the Nos response elements (NREs) in the HB 3' UTR. However, it has been difficult to study the mechanism of regulation in detail because HB translational regulation has not been recapitulated faithfully in in vitro or tissue culture assays. Recently, a number of in vitro translation systems from Drosophila embryos have been established to study the regulation of nos and oskar mRNAs. Unfortunately, it has not been possible to recapitulate HB regulation with these and similar systems. RNA injection assays are a method of choice for analyzing translational efficiency in vivo in a variety of WT and mutant backgrounds by using modified RNAs. To apply this system to Drosophila embryos and increase the sensitivity of the injection assay, F-Luc and R-Luc genes were used as reporters. In this injection assay, two in vitro-transcribed RNAs are coinjected into either the anterior or posterior pole of the preblastoderm embryo. The control mRNA contains the Renilla reniformis luciferase (R-Luc) coding region with short 5' and 3' UTRs that lack regulatory elements. This unregulated mRNA is used to normalize for the amount of RNA injected. The test mRNA (HFH) contains the maternal HB 5' UTR, the F-Luc coding region, and the maternal HB 3' UTR. After incubation, F-Luc and R-Luc activities are determined. The relative translational efficiency of each mRNA in the anterior versus the posterior of the embryo (A/P) then is calculated. An A/P of 1 means the test mRNA is translated equally well in the anterior and the posterior, whereas a ratio of greater than 1 means the test mRNA is translated more efficiently in the anterior than the posterior. Translation of a test mRNA with an intact HB 3' UTR and a WT NRE (HFHWT A25) is repressed in the posterior versus the anterior of WT embryos (A/P 7.8 ± 2.0), mirroring the regulation of endogenous maternal HB mRNA (Chagnovich, 2001).
To determine whether injected RNAs require the same factors for regulation as the endogenous maternal HB transcript, the effect of nos, pum, and NRE mutations on the translation of the HFH mRNAs was studied. HFH transcripts injected into nos or pum mutant embryos are translated equally well in the anterior versus the posterior. This finding is consistent with the requirement for Nos and Pum in regulating HB translation in vivo. Further, HFH transcripts containing mutant NREs in which the six guanosines have been changed to uracil (GU) show no significant difference in translation between the anterior and posterior of the embryo, regardless of the genetic background. This NRE mutation disrupts Pum binding to the NRE in vitro and eliminates translational repression of maternal HB mRNA in vivo. Thus factors that are required for the regulation of the endogenous maternal HB transcript also are required for the regulation of the injected transcripts (Chagnovich, 2001).
Removal of the poly(A) tail is the prevailing model for translational regulation of many maternal mRNAs. Indeed, maternal HB mRNA is polyadenylated in the cytoplasm concomitant with its translational activation. In the posterior, maternal HB is rapidly deadenylated in a Nos, Pum, and NRE-dependent process. To determine whether deadenylation could account for the regulation of maternal HB translation, the effect of the poly(A) tail on HFH translation was examined in the injection assay. HFH mRNAs that lack a poly(A) tail are polyadenylated upon injection and more efficiently translated in the anterior versus the posterior (A/P 7.4 ± 0.8). HFH reporter mRNAs without a poly(A) tail were prepared that carry a point mutation in the polyadenylation signal (AAUAAA to AAUACA) and thus cannot be polyadenylated. These HFH mRNAs are still regulated (A/P 3.5 ± 0.7), although polyadenylation is no longer detectable on transcripts bearing this mutation. To confirm this finding, HFH mRNAs were injected that lack a poly(A) tail and were end-labeled with cordycepin, an ATP analog that lacks a 3' hydroxyl group and, as a result, blocks further elongation of poly(A) tails. These mRNAs are also differentially regulated in the absence of a poly(A) tail (A/P 3.0 ± 1.3). This finding suggests that a poly(A) tail is not absolutely necessary for the regulation of HB translation in the embryo and that the process of deadenylation is not required for HB repression. HFH mRNAs that are not polyadenlyated are less well regulated than reporters with a poly(A) tail (A/P ~3 as compared with A/P ~7). Because RNAs lacking a poly(A) tail are less well translated it is possible that this difference in regulation is caused by lower translational activation of unadenylated RNA in the anterior rather than to less efficient repression of this RNA in the posterior (Chagnovich, 2001).
Injected mRNAs lacking a poly(A) tail are poorly translated and thus may not accurately reflect regulation in vivo. Reporter mRNAs were therefore developed that are efficiently translated independent of a poly(A) tail. To accomplish this HFH reporter mRNAs were synthesized that contained the Drosophila histone H1 3' terminal stem loop (HSL) in place of the poly(A) tail (HFH HSL). Most histone mRNAs do not contain a poly(A) tail, but rather end with this conserved stem-loop structure. It has been shown that the HSL with the stem loop-binding protein regulates the stability and translational activity of histone mRNAs as does the poly(A) tail for other cellular transcripts. To prevent addition of a poly(A) tail to the HSL, the AAUAAA polyadenylation signal was mutated as described. HFHWT HSL mRNAs are translated more efficiently in the anterior than the posterior, demonstrating spatial regulation similar to the endogenous maternal HB (A/P 5.4 ± 2.0). Further, this regulation depends on an intact NRE and the presence of Nos activity. There are several possible explanations for this finding: the NRE complex can direct removal of the HSL; HSL-containing messages are destabilized in an NRE-dependent manner; or the NRE complex can directly repress translation (Chagnovich, 2001).
To determine whether the NRE represses translation of HSL-containing mRNAs by directing removal of the HSL, just as it directs removal of the poly(A) tail, radiolabeled, m7GpppG-capped HB 3' UTRs containing either a poly(A) tail or the HSL were injected into WT embryos. Consistent with previous findings, RNAs containing a poly(A) tail maintain the poly(A) tail in the anterior, but the poly(A) tail is rapidly removed in the posterior. However, when RNAs containing the HSL are injected, the HSL is maintained in the anterior and posterior of the embryo, demonstrating that the NRE complex is not directing removal of HSL. Next tested was whether the differences in translation of the HSL-containing reporters were caused by differential stability of the mRNAs. In these experiments, the levels of HSL or poly(A)+ reporter RNAs compared with a poly(A)+ control RNA containing a mutant NRE were examined. The relative levels of the reporter mRNAs are not significantly altered in the anterior versus the posterior for either the polyadenylated or the HSL-containing mRNAs. Together these data demonstrate that translational regulation of HB does not require removal of the poly(A) tail. It is concluded that NRE-directed repression can be independent of the poly(A) tail (Chagnovich, 2001).
Regulation of poly(A) and HSL containing reporters is quantitatively different, suggesting that there may be differences in some aspects of their regulation. To further compare the translational regulation of polyadenylated and HSL-containing mRNAs, the regulation was followed of HFH reporter mRNAs containing a poly(A) tail versus a HSL at 30, 60, and 90 min after injection. Whereas absolute production of the reporters rises with time, the A/P ratio of test mRNAs containing a poly(A) tail continues to increase up to the 90-min time point. In contrast, the regulation of the HSL-containing transcripts reaches a plateau by 60 min. One explanation for this disparity is that the HSL-containing transcripts are translationally active in both the anterior and the posterior and that active repression has to be maintained in the posterior throughout the test period. In contrast, poly(A)-containing mRNAs are translated at a constant rate at the anterior, but are translationally inactivated in the posterior. To evaluate the stability of the injected mRNAs at the later time point, RNA was isolated at various times after injection and the levels of the reporter mRNAs was determined. There is no significant difference in the stability of the mRNAs containing a WT NRE (HFH) versus those containing a mutant NRE (HCH). This suggests that poly(A) removal inactivates the HB mRNA, targeting it for translational silencing but not for immediate decay tail (Chagnovich, 2001).
It is proposed that the NRE has two effects on HB regulation: poly(A) removal and poly(A) independent translational repression. One potential mechanism to achieve both of these effects would be through the action of the cap-dependent poly(A) nuclease PARN. Human and Xenopus PARN has been shown to compete with eIF4E for binding to the cap, thereby potentially disrupting translational initiation. Once bound to the cap, PARN rapidly removes the poly(A) tail. The NRE complex could recruit PARN to the RNA or destabilize the cap complex, allowing PARN access. To test this possibility, RNAs corresponding to the HB 3' UTR were synthesized that were capped with either m7GpppG or the cap analog ApppG. mRNAs with an ApppG cap are not efficiently deadenylated by PARN. Similarly to what is seen with m7GpppG-capped messages, ApppG-capped messages are deadenylated in the posterior but not the anterior. Additionally, there is no clear PARN homolog in the Drosophila genome. It is concluded that PARN or a PARN-related mechanism is not mediating the effects of the NRE on HB mRNA (Chagnovich, 2001).
To determine whether the cap complex is required for regulation, ApppG-capped HFH reporter mRNAs were injected. These mRNAs were very poorly translated (>20 times less well than the m7GpppG-capped reporters). To obtain luciferase levels that were above background, higher levels of reporter mRNAs (500 pg/µl) were injected. At these RNA levels, m7GpppG-capped mRNAs were differentially regulated (A/P 4.9 ± 1.1). Regulation of ApppG-capped reporters bearing a WT NRE (2.2 ± 0.9) was not significantly different from that for ApppG-capped reporters bearing a mutant NRE. These data may indicate a requirement for the cap in NRE-mediated regulations. It is likely, that because of the low translation levels of ApppG-capped mRNAs, the failure to differentially regulate these mRNAs results from a failure to efficiently activate translation in the anterior as opposed to repress translation in the posterior (Chagnovich, 2001).
Thus, the RNA injection assay has shown that the NRE and its associated factors can repress HB translation independent of the poly(A) tail and in the presence of a heterologous translation activation signal (the HSL). At least two models are compatible with this scenario. In the first, the NRE complex may disrupt a single molecule or step in translation that results in both poly(A) removal and translational repression. Because of its central role in linking the 5' cap complex with the 3' poly(A) tail, PABP would be a likely target in this model. In this scenario, the NRE complex recruits factors to the HB RNA that could modulate PABP activity. Drosophila Paip-2, for example, could be such a factor. Recently, human Paip-2 has been shown to interact with PABP and disrupt its binding to the poly(A) tail, resulting in an increase in deadenylation and a decrease in translation. However, because a poly(A) tail is not absolutely required for HB translational repression, it is more likely that the NRE complex would disrupt the cap complex, and that deadenylation is a consequence rather than the cause of cap complex disruption. It has been demonstrated in plant extracts that interaction between eIF4F and eIF4B increases poly(A) binding by stabilizing PABP on the tail. Thus it is possible that disruption of the cap complex or its interaction with PABP could result in decreased translation and increased deadenylation (Chagnovich, 2001).
In an alternative model, the NRE could act by two discrete pathways: one directly interfering with translation and a second partially redundant pathway directing poly(A) removal. The data as well as the absence of an obvious homolog in the Drosophila genome suggest that the cap-dependent nuclease PARN is not the deadenylase. However, the Drosophila genome does contain CCR4 and CAF1, which have been shown to be components of the major cytoplasmic deadenylase in yeast, and PAN2 and PAN3, which have a role in both nuclear and cytoplasmic deadenylation. Either or both of these complexes could be recruited to the HB mRNA by the NRE complex, thus fulfilling the deadenylation pathway of this model. This study shows that the NRE complex can directly repress translation of mRNAs containing the HSL. The stem loop is a conserved structure that fulfills many of the functions the poly(A) tail serves on other transcripts and is essential for translation of histone mRNA. Recently, stem loop-binding protein has been identified as a sequence-specific factor that interacts with the stem loop and participates in histone mRNA processing and mobilization onto polyribosomes. The mechanism of translational activation by the stem loop and its associated factor(s) remains unclear, although it is believed to mediate interaction between the termini, possibly through an interaction with eIFs. It is speculated that the NRE complex is directly interfering with translation at the same step for mRNAs bearing a histone stem loop or poly(A) tail. Such a common step would likely involve initiation or elongation of the polypeptide rather than PABP. In addition, silencing of polyadenylated RNAs would increase the efficiency of translational repression (Chagnovich, 2001).
Although the best-studied examples of translational regulation require removal of the poly(A) tail, examples of poly(A)-independent regulation have been described. For example, human ribonucleoproteins K and E1 repress translation of erythroid 15-lipoxygenase mRNA by inhibiting assembly of the 80S ribosome. In Xenopus, it has been shown that Maskin interacts with eIF4E, thereby disrupting cap complex assembly. Although Nos has two potential eIF4E binding sites, deletion of these sites does not affect Nos regulation of HB. Nos, ectopically expressed in the fly eye, can repress expression of NRE-containing mRNAs that initiate translation via an internal ribosome entry site, suggesting that Nos, Pum, and Brat may act downstream of the cap. Although the possibility cannot be excluded that these transcripts are degraded in the eye primordia as a result of NRE-mediated removal of the poly(A) tail, it is intriguing to speculate that the NRE complex may act to directly interfere with the function of the general translation machinery on transcripts containing NREs. Interestingly, other maternal RNAs (e.g., Oskar and Nanos) whose translational repression, like HB, is mediated by sequences in the 3' UTR, do not require the poly(A) tail for their regulation. This finding suggests that direct inhibition of the translation machinery may be a common strategy in the Drosophila embryo (Chagnovich, 2001).
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, these 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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