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R P Wharton

Publications and source records attributed to R P Wharton.

18 recordsLinked to original sources
PubMed2001

Structure of Pumilio reveals similarity between RNA and peptide binding motifs.

Translation regulation plays an essential role in the differentiation and development of animal cells. One well-studied case is the control of hunchback mRNA during early Drosophila embryogenesis by the trans-acting factors Pumilio, Nanos, and Brain Tumor. We report here 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 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, we identify putative interaction surfaces for hunchback mRNA and the cofactors Nanos and Brain Tumor. This analysis suggests that similar features in helical repeat proteins are used to bind extended peptides and RNA.

Amino Acid Motifs↗
PubMed2001

Drosophila Brain Tumor is a translational repressor.

The Drosophila brain tumor (brat) gene encodes a member of the conserved NHL family of proteins, which appear to regulate differentiation and growth in a variety of organisms. One of the founding family members, Caenorhabditis elegans LIN-41, is thought to control posttranscriptional gene expression. However, the mechanism by which LIN-41, or any other NHL protein, acts has not been clear. Using a yeast "four-hybrid" interaction assay, we show that Brain Tumor is recruited to hunchback (hb) mRNA through interactions with Nanos and Pumilio, which bind to the RNA to repress its translation. Interaction with the Nanos/Pumilio/RNA complex is mediated by the Brat NHL domain; single amino acid substitutions in this domain compromise quaternary complex assembly in vitro and hb regulation in vivo. Thus, recruitment of Brat is necessary for translational repression and the normal development of posterior embryonic pattern. In addition to regulating abdominal segmentation, previous genetic analysis has shown that Brat, Nanos, and Pumilio govern a variety of developmental processes. We examined the role of Brat in two of these processes-regulation of maternal Cyclin B mRNA in the embryo and regulation of imaginal disc development. The results of these experiments suggest that NHL domain proteins are recruited to various mRNAs by combinatorial protein-protein interactions.

Animals↗
PubMed2000

Crystallization and characterization of Pumilo: a novel RNA binding protein.

Axis determination in early Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6(3) with unit cell dimensions of a = b = 94.5 A, c = 228.9 A, alpha = beta = 90 degrees, gamma = 120 degrees and diffract to 2.6 A with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of alpha-helical secondary structure.

Animals↗
PubMed2000

Nanos interacts with cup in the female germline of Drosophila.

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

Animals↗
PubMed1999

Recruitment of Nanos to hunchback mRNA by Pumilio.

Translational regulation of hunchback (hb) 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. Pumilio recognizes the NREs via a conserved binding motif. The mechanism of Nanos action has not been clear. In this report we use protein-protein and protein-RNA interaction assays in yeast and in vitro 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.

Animals↗
PubMed1999

E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila.

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila.

Animals↗
PubMed1999

Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila.

During Drosophila embryogenesis, a gradient of Nanos protein emanating from the posterior pole organizes abdominal segmentation. This gradient arises from translational regulation of nanos mRNA, which is activated in the specialized cytoplasm at the posterior pole of the embryo and repressed elsewhere. Previously, we have defined cis-acting elements in the mRNA that mediate this translational switch. In this report, we identify a factor named Smaug that binds to these elements and represses translation in the bulk cytoplasm. Smaug interacts gentically and biochemically with Oskar, a key component of the pole plasm for activation of nanos mRNA and specification of the germline precursors. These observations suggest that Smaug operates a translational switch that governs the distribution of Nanos protein.

Abdomen↗
PubMed1998

The Pumilio RNA-binding domain is also a translational regulator.

Posterior patterning in the Drosophila embryo requires the action of Nanos (Nos) and Pumilio (Pum), which collaborate to regulate the translation of maternal hunchback (hb) mRNA. Previous work demonstrated that Pum recognizes sites in the 3' UTR of hb mRNA. In this report, we first define the RNA-binding domain of Pum and then show that residues essential for translational repression are embedded within this domain. We also show that 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.

Amino Acid Sequence↗
PubMed1996

The Nanos gradient in Drosophila embryos is generated by translational regulation.

Abdominal segmentation in the Drosophila embryo is governed by a gradient of Nanos (Nos) emanating from the posterior pole. This gradient is derived from translation of the nos mRNA that is localized in the pole plasm; in contrast, unlocalized nos mRNA is translationally repressed. Here we define the essential signals in the 3' untranslated region (UTR) of nos mRNA. Deletion of a 184-nucleotide translational control element (TCE) from the 3' UTR leads to the derepression of nos mRNA in the bulk cytoplasm and the development of lethal anterior defects. Furthermore, a minimal mRNA containing essentially only the TCE in its 3' UTR rescues nos- embryos to adulthood. The TCE is also sufficient to confer on maternal torso mRNA all three aspects of nos mRNA regulation: translational repression in the bulk cytoplasm, localization to the pole plasm, and translational activation at the posterior pole. These three phenomena are coupled intimately, as mutations in a pair of CUGGC pentamers within the TCE simultaneously abrogate all three regulatory events. This coupling suggests a model in which the polarized distribution of nos protein is generated primarily by translational control and that nos mRNA localization is a byproduct of this regulation, at least in part.

Animals↗
PubMed1996

Ectopic E2F expression induces S phase and apoptosis in Drosophila imaginal discs.

Previous experiments suggest that a key event in the commitment of cultured mammalian cells to entering S phase is a rise in activity of the transcription factor E2F. In this report, we study the role of Drosophila E2F in imaginal disc cells in vivo, by examining the distribution of the endogenous protein and studying the consequences of ectopic E2F expression. First, we find that endogenous E217 falls from high to very low levels as cells initiate DNA synthesis during a developmentally regulated G1-S-transition in the eye disc. Second, we find that ectopic E2F expression drives many otherwise quiescent cells to enter S phase. Subsequently, cells throughout the discs express reaper (a regulator of apoptosis) and then die. Third, we find that ectopic E2F expression during S phase in normally cycling cells blocks their re-entry into S phase in the following cell cycle. Although we do not know the fate of these cells, we suspect that ultimately they are killed by ectopic E2F. Taken together, our results show that an elevation in the level of E2F is sufficient to induce imaginal disc cells to enter S phase. Furthermore, they suggest that the downregulation of E2F upon entry into S phase may be essential to prevent the induction of apoptosis.

Animals↗
PubMed1995

Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos.

Posterior patterning in Drosophila embryos is governed by nanos (nos), which acts by repressing the translation of maternal transcripts of the hunchback (hb) gene. Sites in hb mRNA that mediate this repression, named nanos response elements (NREs), have been identified. However, we know of no evidence of a direct interaction between nos, or any other protein, and the NRE. Here, we show that two proteins present in embryonic extracts, neither one nos, bind specifically to the NRE in vitro. Furthermore, we show that binding in vitro correlates with NRE function in vivo. One of the NRE-binding factors is encoded by pumilio (pum), a gene that, like nos, is essential for abdominal segmentation. These and other observations suggest that pum acts by recognizing the NRE and then recruiting nos. Presumably, the resulting complex inhibits some component of the translation machinery.

Animals↗
PubMed1991

RNA regulatory elements mediate control of Drosophila body pattern by the posterior morphogen nanos.

In Drosophila embryos, graded activity of the posterior determinant nanos (nos) generates abdominal segmentation by blocking protein expression from maternal transcripts of the hunchback (hb) gene. When active inappropriately at the anterior pole, nos can also block expression of the anterior determinant bicoid (bcd). We show that 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, we argue that nos acts as a morphogen, controlling hb expression (and hence abdominal pattern) as a function of its concentration-dependent interaction with the NREs.

Animals↗
PubMed1989

Structure of the Drosophila BicaudalD protein and its role in localizing the posterior determinant nanos.

Mutations in the BicaudalD (BicD) gene lead to a global reorganization of the Drosophila body pattern such that the head, thoracic, and anterior abdominal segments are replaced by posterior abdominal segments and terminalia. We first provide evidence that the primary cause of this phenotype is the inhibition of two anterior factors, bicoid and hunchback, by mislocalized activity of the posterior determinant nanos. We then describe the isolation of the BicD gene and show that it encodes a coiled-coil protein similar to the carboxy-terminal portion of the myosin heavy chain. Finally, we find that BicD protein is uniformly distributed throughout wild-type oocytes but is concentrated at the anterior pole of BicD mutant oocytes together with ectopic nanos activity. Taken together, these results suggest that BicD encodes a cytoskeleton-like protein involved in transporting or anchoring the nanos morphogen within the oocyte cytoplasm.

Amino Acid Sequence↗
PubMed1988

Recognition of DNA sequences by the repressor of bacteriophage 434.

The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA.

Bacteriophages↗
PubMed1984

Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor.

It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein.

Base Sequence↗
PubMedDate not supplied

Changing the binding specificity of a repressor by redesigning an alpha-helix.

We replaced amino acids on the 'outside', or solvent-exposed, surface of the DNA recognition alpha-helix of 434 repressor with the corresponding amino acids from the recognition helix of P22 repressor. The binding specificity of the resulting hybrid protein, as measured in vivo and in vitro, was that of P22 repressor.

Amino Acid Sequence↗
PubMedDate not supplied

A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact.

The repressor encoded by bacteriophage 434 binds to its operators by inserting a 'recognition' alpha-helix into the major groove of the DNA. We have identified an amino acid-base pair contact that determines (in part) the DNA-binding specificity of 434 repressor. The identification is based on the properties of a 'new-specificity' mutant, named Repressor [Ala 28], which bears the substitution of Ala for Gln at the first residue of its recognition alpha-helix. Repressor [Ala 28] binds with high affinity to a particular doubly mutant operator bearing the same substitution at position 1 in each half-site, but does not bind to either the wild-type operator or to other mutant operators. We describe molecular models of residue 28-base pair 1 interactions that account for the binding specificities of both the mutant and wild-type proteins.

5-Methylcytosine↗