Over the rainbow to translational control.
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Animals↗
Biomedical subjects
E R Gavis
Publications and source records attributed to E R Gavis.
Recognition and long-range interactions of a minimal nanos RNA localization signal element.
Localization of nanos (nos) mRNA to the germ plasm at the posterior pole of the Drosophila embryo is essential to activate nos translation and thereby generate abdominal segments. nos RNA localization is mediated by a large cis-acting localization signal composed of multiple, partially redundant elements within the nos 3' untranslated region. We identify a protein of approximately 75 kDa (p75) that interacts specifically with the nos +2' localization signal element. We show that the function of this element can be delimited to a 41 nucleotide domain that is conserved between D. melanogaster and D. virilis, and confers near wild-type localization when present in three copies. Two small mutations within this domain eliminate both +2' element localization function and p75 binding, consistent with a role for p75 in nos RNA localization. In the intact localization signal, the +2' element collaborates with adjacent localization elements. We show that different +2' element mutations not only abolish collaboration between the +2' and adjacent +1 element but also produce long-range deleterious effects on localization signal function. Our results suggest that higher order structural interactions within the localization signal, which requires factors such as p75, are necessary for association of nos mRNA with the germ plasm.
3' Untranslated Regions↗
Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism.
Nanos (Nos) protein is required in the posterior of the Drosophila embryo to promote abdominal development, but must be excluded from the anterior to permit head and thorax development [1,2]. Spatial restriction of Nos is accomplished by selective translation of the 4% of nos mRNA localized to the posterior pole and translational repression of the remaining unlocalized mRNA [3-5]. Repression is mediated by a 90-nucleotide translational control element (TCE) in the nos 3' untranslated region (UTR) and the TCE-binding protein Smaug [4,6,7], but the molecular mechanism is unknown. We used sucrose density gradient sedimentation to ascertain whether unlocalized nos mRNA is excluded from polysomes and therefore repressed during translational initiation. Surprisingly, a significant percentage of nos mRNA was found to be associated with polysomes, even in mutants in which all nos mRNA is unlocalized and repressed. Using a regulated Drosophila cell-free translation system, we showed that ribosomes contained within these polysomes are capable of elongation in vitro, under conditions in which synthesis of Nos protein is repressed. Thus, synthesis of ectopic Nos protein is inhibited by a novel regulatory mechanism that does not involve a stable arrest of the translation cycle.
3' Untranslated Regions↗
Overlapping but distinct RNA elements control repression and activation of nanos translation.
Spatially restricted synthesis of Nanos protein in the Drosophila embryo is essential for anterior-posterior patterning. Nanos translation is restricted to the posterior of the embryo by translational repression of nanos mRNA throughout the bulk cytoplasm and selective activation of posteriorly localized nanos mRNA. A 90-nucleotide translational control element (TCE) mediates translational repression. We show that TCE function requires formation of a bipartite secondary structure that is recognized by Smaug repressor and at least one additional factor. We also demonstrate that translational activation requires the interaction of localization factors with sequences that overlap TCE structural motifs. The identification of separate but overlapping recognition motifs for translational repressors and localization factors provides a molecular mechanism for the switch between translational repression and activation.
3' Untranslated Regions↗
Role for mRNA localization in translational activation but not spatial restriction of nanos RNA.
Patterning of the anterior-posterior body axis during Drosophila development depends on the restriction of Nanos protein to the posterior of the early embryo. Synthesis of Nanos occurs only when maternally provided nanos RNA is localized to the posterior pole by a large, cis-acting signal in the nanos 3' untranslated region (3'UTR); translation of unlocalized nanos RNA is repressed by a 90 nucleotide Translational Control Element (TCE), also in the 3'UTR. We now show quantitatively that the majority of nanos RNA in the embryo is not localized to the posterior pole but is distributed throughout the cytoplasm, indicating that translational repression is the primary mechanism for restricting production of Nanos protein to the posterior. Through an analysis of transgenes bearing multiple copies of nanos 3'UTR regulatory sequences, we provide evidence that localization of nanos RNA by components of the posteriorly localized germ plasm activates its translation by preventing interaction of nanos RNA with translational repressors. This mutually exclusive relationship between translational repression and RNA localization is mediated by a 180 nucleotide region of the nanos localization signal, containing the TCE. These studies suggest that the ability of RNA localization to direct wild-type body patterning also requires recognition of multiple, unique elements within the nanos localization signal by novel factors. Finally, we propose that differences in the efficiencies with which different RNAs are localized result from the use of temporally distinct localization pathways during oogenesis.
3' Untranslated Regions↗
Identification of cis-acting sequences that control nanos RNA localization.
The generation of anterior-posterior polarity during development in Drosophila requires the localization of determinant molecules to the anterior and posterior poles of the embryo. Localization of the maternally synthesized nanos RNA to the posterior pole of the embryo is essential to provide a source for a gradient of Nanos protein that directs abdomen formation. nanos RNA localization occurs during oogenesis and requires the function of at least nine genes. cis-acting sequences that direct nanos RNA localization lie within the nanos 3'UTR. In this analysis, we have used nanos 3'UTR deletion mutants to define the localization signal more precisely. Our results indicate that the nanos RNA localization signal is large and complex and that targeting of nanos RNA may be achieved by the combined effects of multiple, partially independent sequences. This idea is supported by evolutionary conservation, both in sequence and in function, of the nanos 3'UTRs of Drosophila melanogaster and Drosophila virilis.
Animals↗
A conserved 90 nucleotide element mediates translational repression of nanos RNA.
Correct formation of the Drosophila body plan requires restriction of nanos activity to the posterior of the embryo. Spatial regulation of nanos is achieved by a combination of RNA localization and localization-dependent translation such that only posteriorly localized nanos RNA is translated. Cis-acting sequences that mediate both RNA localization and translational regulation lie within the nanos 3' untranslated region. We have identified a discrete translational control element within the nanos 3' untranslated region that acts independently of the localization signal to mediate translational repression of unlocalized nanos RNA. Both the translational regulatory function of the nanos 3'UTR and the sequence of the translational control element are conserved between D. melanogaster and D. virilis. Furthermore, we show that the RNA helicase Vasa, which is required for nanos RNA localization, also plays a critical role in promoting nanos translation. Our results specifically exclude models for translational regulation of nanos that rely on changes in polyadenylation.
Animals↗
Pattern formation. Gurken meets torpedo for the first time.
Intercellular communication between oocyte and follicle cells, mediated by the gurken-torpedo/DER signalling pathway, has a crucial role in determining both anterior-posterior and dorsal-ventral polarity in Drosophila.
Animals↗
Localization of oskar RNA regulates oskar translation and requires Oskar protein.
The site of oskar RNA and protein localization within the oocyte determines where in the embryo primordial germ cells form and where the abdomen develops. Initiation of oskar RNA localization requires the activity of several genes. We show that ovaries mutant for any of these genes lack Oskar protein. Using various transgenic constructs we have determined that sequences required for oskar RNA localization and translational repression map to the oskar 3'UTR, while sequences involved in the correct temporal activation of translation reside outside the oskar 3'UTR. Upon localization of oskar RNA and protein at the posterior pole, Oskar protein is required to maintain localization of oskar RNA throughout oogenesis. Stable anchoring of a transgenic reporter RNA at the posterior pole is disrupted by oskar nonsense mutations. We propose that initially localization of oskar RNA permits translation into Oskar protein and that subsequently Oskar protein regulates its own RNA localization through a positive feedback mechanism.
Animals↗
Translational regulation of nanos by RNA localization.
Localization of the maternally synthesized nanos (nos) RNA to the posterior pole of the Drosophila embryo provides the source for a posterior-to-anterior gradient of Nos protein. Correct spatial regulation of nos activity is essential for normal pattern formation. High local concentrations of Nos protein in the posterior of the embryo are necessary to inhibit translation of the transcription factor Hunchback in this region, and thus permit expression of genes required for abdomen formation (see ref. 5 for review). By contrast, misexpression of Nos protein at the anterior of the embryo prevents translation of the anterior morphogen Bicoid, suppressing head and thorax development. Posterior localization of nos RNA is mediated by sequences within the nos 3' untranslated region (3'UTR) and requires the function of eight genes of the 'posterior group'. Although the unlocalized nos RNA is stable in embryos from females mutant for any of the posterior group genes, these embryos appear to lack nos activity because they develop the abdominal defects characteristic of embryos produced by nos mutant females. We report here that unlocalized nos RNA is translationally repressed. Translational repression is mediated by the nos 3'UTR and can be alleviated either by replacement of the 3'UTR with heterologous 3'UTR sequences or by posterior localization. Thus, RNA localization provides a novel mechanism for translational regulation.
Animals↗
Localization of nanos RNA controls embryonic polarity.
Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.
Abdomen↗
Phosphorylation, expression and function of the Ultrabithorax protein family in Drosophila melanogaster.
Alternative splicing of the Ultrabithorax homeotic gene transcript generates a family of five proteins (UBX isoforms) that function as transcription factors. All isoforms contain a homeodomain within a common 99 aa C-terminal region (C-constant region) which is jointed to a common 247 aa N-terminal (N-constant) region by different combinations of three small optional elements. Unlike the UBX proteins expressed in E. coli, UBX isoforms expressed in D. melanogaster cells are phosphorylated on serine and threonine residues, located primarily within a 53 aa region near the middle of the N-constant region, to form at least five phosphorylated states per isoform. Similar, if not identical states can be generated in vitro from purified E. coli UBX protein by a kinase activity in nuclear extracts from D. melanogaster cells. Temporal developmental profiles of UBX isoforms parallel those for the respective mRNAs, and all isoforms are similarly phosphorylated throughout embryogenesis. Analysis by cotransfection assays of the promoter activation and repression functions of mutant UBX proteins with various deletions in the N-constant region shows that repression is generally insensitive to deletion and, hence, presumably to phosphorylation. By contrast, the activation function is differentially sensitive to the different deletions in a manner indicating the absence of a discrete activating domain and instead, the presence of multiple activating sequences spread throughout the region.
Animals↗
An Ultrabithorax protein binds sequences near its own and the Antennapedia P1 promoters.
The homeotic gene Ultrabithorax (Ubx), located in the bithorax complex of Drosophila, encodes a family of closely related proteins that direct the developmental fates of posterior thoracic and anterior abdominal metameres. We have purified a member of the Ubx protein family from an overproducing E. coli strain and have shown that it is sequence-specific DNA binding protein. The protein binds tightly to sequences near its own promoter and near the P1 promoter of Antenna-pedia (Antp), a homeotic gene Ubx is known to repress from genetic studies. The binding sites occur in clusters downstream of the transcription start sites, and far upstream at Antp P1. They range in size from 40 to 90 bp, and contain tandem repeats of the trinucleotide TAA or the related hexanucleotide TAA-TCG. These results suggest that the regulatory activities of Ubx are direct and are mediated by binding of Ubx proteins to promoter region sequences.
Animals↗
Regulation and products of the Ubx domain of the bithorax complex.
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Animals↗
Histone RNA in amphibian oocytes visualized by in situ hybridization to methacrylate-embedded tissue sections.
We present an in situ hybridization method for detecting cellular RNAs in tissue sections using methacrylate as the embedding medium. The technique offers the advantage of superior morphological preservation compared with previously published procedures. Since sections can be cut 1 micron or less in thickness, full advantage is taken of the short path length of 3H electrons. Applying this procedure to developing amphibian oocytes, we investigated the accumulation and localization of RNA complementary to the histone genes and their adjacent spacers. Histone RNA begins to accumulate in the cytoplasm of late pachytene-early diplotene oocytes, rapidly reaching a maximum concentration during Dumont stage 1. After this stage the concentration of histone RNA declines. RNA transcribed from histone coding regions is located almost exclusively in the cytoplasm of oocytes. Transcripts of the spacer regions, which are known to be synthesized on oocyte lampbrush chromosomes, do not accumulate in the oocytes. [3H]RNA complementary to U2 small nuclear RNA, used in these experiments as a control, hybridized predominantly to the nucleus of the oocytes.
Animals↗
Expression of human beta-interferon cDNA under the control of a thymidine kinase promoter from herpes simplex virus.
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Animals↗
Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region.
We have identified a transcriptional control region of the herpes simplex virus tk gene. This finding results from in vivo transcription assays of specific deletion mutants constructed in vitro. A region located between 40 and 100 nucleotides upstream from the putative transcription start site of the tk gene can promote transcription by RNA polymerase form II in the complete absence of the mRNA-coding component of the gene. When the region is deleted enzymatically from a 5' direction, accurate transcriptional expression is reduced by a factor of 50. The small remaining level of accurate transcription is eliminated by deletion of sequences from 32 to 16 nucleotides upstream from the the structural gene. It appears that the sequences between 16 and 32 nucleotides upstream from the 5' terminus of the tk gene are required to specify the exact start site of transcription. Control of both the efficiency of transcription and rough specification of the position of initiation, however, depends on sequences 40--100 nucleotides upstream from the tk structural gene.
Animals↗
Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro.
When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5' flanking and intragenic sequences are deleted. The other set is deleted at the 3' end of the gene. By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5' and 3' termini of the HSV tk gene.
Animals↗