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.

Dissociation of mRNA cytoplasmic polyadenylation from translational activation by structural modification of the 5'-UTR.

During early metazoan development, certain maternal mRNAs are translationally activated by elongation of their poly(A) tails. Bicoid ( bcd ) mRNA is a Drosophila maternal mRNA that is translationally activated by cytoplasmic polyadenylation during the first hour after egg deposition. The sequences necessary and sufficient to promote its poly(A) elongation, and hence translation, are contained within its 3'-untranslated region (UTR). The mechanism by which poly(A) elongation at the 3'-end affects translational initiation at the 5'-end remains unknown. To investigate this question, we have analyzed a bicoid mRNA whose 5'-UTR contains a short antisense sequence directed against a portion of the coding region. This mutated RNA is efficiently translated in vitro. After injection into Drosophila embryos, this RNA is stable and polyadenylated, but inefficiently translated. These experiments show that structural modification of the 5'-end of an mRNA can perturb the translational activation normally conferred by polyadenylation in vivo.

Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA.

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, we first examined the mechanism by which maternal hunchback mRNA is translationally activated. In the absence of nanos activity, the poly(A) tail of hunchback mRNA is elongated 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. To determine if nanos activity represses translation by altering the polyadenylation state of hunchback mRNA, we injected various in vitro transcribed RNAs into Drosophila embryos and determined changes in polyadenylation. Nanos activity reduced the polyadenylation status of injected hunchback RNAs by accelerating their deadenylation. Pumilio activity, which is necessary to repress the translation of hunchback, 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 deadenylation of maternal hunchback mRNA, thereby repressing its translation.

Oocyte selection of mutations affecting cytoplasmic polyadenylation of maternal mRNAs.

Translational activation by cytoplasmic polyadenylation is a conserved mechanism in metazoan early development. In Xenopus and mouse, the regulatory sequences that control this process during oocyte meiotic maturation have been identified in the 3' untranslated region (3'-UTR) of a class of maternal messenger RNAs (mRNAs). In this report, we have investigated sequences controlling cytoplasmic polyadenylation of a mouse maternal mRNA. Pools of RNAs, transcribed from DNA randomly mutated by a PCR-based method, were micro-injected into the cytoplasm of mouse primary oocytes to allow in vivo selection of inefficiently polyadenylated transcripts. After oocyte maturation, the nonelongated RNAs were gel-isolated, and single base substitutions that alter poly(A) addition were identified. Analysis of these mutant RNAs identified single nucleotides that influence efficiency of cytoplasmic polyadenylation during mouse oocyte maturation. In addition, this strategy should facilitate identification of yet unknown sequence elements responsible for basic biological mechanisms during and after early development.

Evolutionary conservation of sequence elements controlling cytoplasmic polyadenylylation.

Cytoplasmic polyadenylylation is an evolutionarily conserved mechanism involved in the translational activation of a set of maternal messenger RNAs (mRNAs) during early development. In this report, we show by interspecies injections that Xenopus and mouse use the same regulatory sequences to control cytoplasmic poly(A) addition during meiotic maturation. Similarly, Xenopus and Drosophila embryos exploit functionally conserved signals to regulate polyadenylylation during early post-fertilization development. These experiments demonstrate that the sequence elements that govern cytoplasmic polyadenylylation, and hence one form of translational activation, function across species. We infer that the requisite regulatory sequence elements, and likely the trans-acting components with which they interact, have been conserved since the divergence of vertebrates and arthropods.

Isolation and characterization of two novel, cytoplasmically polyadenylated, oocyte-specific, mouse maternal RNAs.

During early development in mouse and Xenopus, translational activation of stored maternal mRNAs by cytoplasmic polyadenylation requires both the nuclear polyadenylation signal AAUAAA and U-rich cis-acting adenylation control elements (ACEs), also termed cytoplasmic polyadenylation elements, located in the 3' UTR. Using an ACE-based PCR strategy (Sallés et al., 1992) we have isolated two novel cDNAs from mouse oocytes: OM2a and OM2b (for Oocyte Maturation). Each message contains an ACE consensus sequence upstream of AAUAAA, is specifically transcribed in the growing oocyte, and is cytoplasmically polyadenylated upon oocyte maturation. Comparison of the mouse and rat homologs reveals considerable nucleotide sequence homology and conservation of overall gene organization. However, the predicted open reading frames are far less conserved, suggesting that these genes may not be functioning as proteins. The tissue specificity and tight temporal regulation of the RNAs suggest a role for these genes during early development.

Mutagenic alteration of the distal switch II region of RAS blocks CDC25-dependent signaling functions.

We have explored the role of the distal switch II region of the yeast RAS2 protein in determining the response to the nucleotide exchange factor CDC25. We first constructed yeast tester strains in which the deletion of the chromosomal CDC25, RAS1, and RAS2 genes, in combination with the chromosomal suppressor CRI4, resulted in detectable phenotypes in vivo and in vitro. Phenotypes included impaired growth at 37 degrees C, defective glucose-induced cyclic AMP signaling, and low adenylyl cyclase activity of membrane preparations. Tester strains were subsequently used for the reintroduction of various combinations of wild-type and mutated RAS2 and CDC25 genes by genetic techniques, as well as for in vitro reconstitution assays with the corresponding proteins. CDC25 restored both growth and glucose-induced cyclic AMP signaling in the presence, but not in the absence of wild-type RAS2. A gene encoding a RAS2 protein with a mutationally altered switch II region was expressed but was ineffective in reintegrating exchange factor-dependent responses in vivo. Wild-type, but not mutagenically altered, RAS2 proteins were stimulated by exchange factors in vitro. We conclude that the conserved distal switch II region is required for CDC25-dependent activation of RAS.

RAS residues that are distant from the GDP binding site play a critical role in dissociation factor-stimulated release of GDP.

We have previously shown that a conserved glycine at position 82 of the yeast RAS2 protein is involved in the conversion of RAS proteins from the GDP- to the GTP-bound form. We have now investigated the role of glycine 82 and neighbouring amino acids of the distal switch II region in the physiological mechanism of activation of RAS. We have introduced single and double amino acid substitutions at positions 80-83 of the RAS2 gene, and we have investigated the interaction of the corresponding proteins with a yeast GDP dissociation stimulator (SDC25 C-domain). Using purified RAS proteins, we have found that the SDC25-stimulated conversion of RAS from the GDP-bound inactive state to the GTP-bound active state was severely impaired by amino acid substitutions at positions 80-81. However, the rate and the extent of conversion from the GDP- to the GTP-bound form in the absence of dissociation factor was unaffected. The insensitivity of the mutated proteins to the dissociation factor in vitro was paralleled by an inhibitory effect on growth in vivo. The mutations did not significantly affect the interaction of RAS with adenylyl cyclase. These findings point to residues 80-82 as important determinants of the response of RAS to GDP dissociation factors. This suggests a molecular model for the enhancement of nucleotide release from RAS by such factors.

Role of glycine-82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein.

Ras proteins bind either GDP or GTP with high affinity. However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine-82 resides in a region that is highly conserved between the yeast and human proteins. However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H-ras gene product. Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids. This is in agreement with the role of glycine-82 as a pivot point during the transition from the GDP- to the GTP-bound form of the Ras proteins.

Identification of regulatory residues of the yeast adenylyl cyclase.

We have attempted to identify amino acid residues of the yeast adenylyl cyclase that are involved in the regulation of its activity, by isolating adenylyl cyclase-linked spontaneous mutations capable of suppressing the temperature-sensitive phenotype of ras1- ras2-ts1 strains. We previously identified a mutated adenylyl cyclase in which a single point mutation, called CR14, led to the replacement of threonine 1651 with isoleucine. We have now investigated the biological effects of CR14, and of other mutations that cause the replacement of threonine 1651 by distinct amino acids. We have observed that the response of adenylyl cyclase to Ras can be either enhanced or attenuated, without significant effects on the steady-state level of the former enzyme in vivo, depending on the amino acid side chain at position 1651. Therefore, this residue identifies a regulatory region on the adenylyl cyclase molecule. We have also taken advantage of the attenuation of adenylyl cyclase function caused by the replacement of threonine 1651 with aspartic acid to isolate intragenic suppressor mutations. We have identified several point mutations, leading to single amino acid substitutions, individually capable of reactivating the attenuated adenylyl cyclase. The corresponding amino acid changes are located within a relatively small region, including residues 1331, 1345, 1348 and 1374. This region could be physiologically involved in the negative control of the carboxy-terminal catalytic domain.