Coordinated control of lumen size and collective migration in the salivary gland

Drosophila Smaug is a sequence-specific RNA-binding protein that can repress the translation and induce the degradation of target mRNAs in the early Drosophila embryo. Our recent work has uncovered a new mechanism of Smaug-mediated translational repression whereby it interacts with and recruits the Argonaute 1 (Ago1) protein to an mRNA. Argonaute proteins are typically recruited to mRNAs through an associated small RNA, such as a microRNA (miRNA). Surprisingly, we found that Smaug is able to recruit Ago1 to an mRNA in a miRNA-independent manner. This work suggests that other RNA-binding proteins are likely to employ a similar mechanism of miRNA-independent Ago recruitment to control mRNA expression. Our work also adds yet another mechanism to the list that Smaug can use to regulate its targets and here we discuss some of the issues that are raised by Smaug’s multi-functional nature.     

Smaug destroys a huge treasure

Smaug, a protein repressing translation and inducing mRNA decay, directly controls an unexpectedly large number of maternal mRNAs driving early Drosophila development.See related research, http://genomebiology.com/2014/15/1/R4Regulation of translation and mRNA stability is a key aspect of early metazoan development. One of the best studied factors involved in these processes is the Drosophila protein Smaug. In this issue of Genome Biology, Chen et al. [1] report that a large number of maternal mRNAs in the fly embryo are probably regulated directly by Smaug.     

microRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA‐binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA‐independent manner, thereby repressing translation.     

Fine-scale genetic breaks driven by historical range dynamics and ongoing density-barrier effects in the estuarine seaweed Fucus ceranoides L.

Background

In the laboratory, the Drosophila melanogaster heat shock protein Hsp90 can buffer the phenotypic effects of genetic variation. Laboratory experiments either manipulate Hsp90 activity pharmacologically, or they induce mutations with strong effects in the gene Hsp83, the single-copy fly gene encoding Hsp90. It is unknown whether observations from such laboratory experiments are relevant in the wild.

Results

We here study naturally occurring mutations in Hsp83, and their effects on fitness and phenotypic buffering in flies derived from wild populations. We examined more than 4500 flies from 42 Drosophila populations distributed world-wide for insertions or deletions of mobile DNA in or near the Hsp83 gene. The insertions we observed occur at low population frequencies, and reduce Hsp83 gene expression. In competition experiments, mutant flies performed much more poorly than wild-type flies. Mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. Specifically, in Hsp83 mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. At even slightly elevated temperatures, inbred mutant Hsp83 populations went extinct, whereas inbred wild-type populations persisted.

Conclusions

Our work shows that Hsp90, a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress.     

Mei-P26 Cooperates with Bam,Bgcn and Sxl to Promote Early Germline Development in the Drosophila Ovary

In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. Here we present data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam.     

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.     

Multiple mechanisms collaborate to repress nanos translation in the Drosophila ovary and embryo

Translational control of gene expression is essential for development in organisms that rely on maternal mRNAs. In Drosophila, translation of maternal nanos (nos) mRNA must be restricted to the posterior of the early embryo for proper patterning of the anterior-posterior axis. Spatial control of nos translation is coordinated through the localization of a small subset of nos mRNA to the posterior pole late in oogenesis, activation of this localized mRNA, and repression of the remaining unlocalized nos mRNA throughout the bulk cytoplasm. Translational repression is mediated by the interaction of a cis-acting element in the nos 3' untranslated region with two proteins, Glorund (Glo) and Smaug (Smg), that function in the oocyte and embryo, respectively. The mechanism of Glo-dependent repression is unknown. Previous work suggests that Smg represses translation initiation but this model is not easily reconciled with evidence for polysome association of repressed nos mRNA. Using an in vitro translation system, we have decoupled translational repression of nos imposed during oogenesis from repression during embryogenesis. Our results suggest that both Glo and Smg regulate translation initiation, but by different mechanisms. Furthermore, we show that, during late oogenesis, nos translation is also repressed post-initiation and provide evidence that Glo mediates this event. This post-initiation block is maintained into embryogenesis during the transition to Smg-dependent regulation. We propose that the use of multiple modes of repression ensures inactivation of nos RNA that is translated at earlier stages of oogenesis and maintenance of this inactivate state throughout late oogenesis into embryogenesis.     

Rapid ATP-dependent deadenylation of nanos mRNA in a cell-free system from Drosophila embryos

Shortening of the poly(A) tail (deadenylation) is the first and often rate-limiting step in the degradation pathway of most eukaryotic mRNAs and is also used as a means of translational repression, in particular in early embryonic development. The nanos mRNA is translationally repressed by the protein Smaug in Drosophila embryos. The RNA has a short poly(A) tail at steady state and decays gradually during the first 2-3 h of development. Smaug has recently also been implicated in mRNA deadenylation. To study the mechanism of sequence-dependent deadenylation, we have developed a cell-free system from Drosophila embryos that displays rapid deadenylation of nanos mRNA. The Smaug response elements contained in the nanos 3'-untranslated region are necessary and sufficient to induce deadenylation; thus, Smaug is likely to be involved. Unexpectedly, deadenylation requires the presence of an ATP regenerating system. The activity can be pelleted by ultracentrifugation, and both the Smaug protein and the CCR4.NOT complex, a known deadenylase, are enriched in the active fraction. The same extracts show pronounced translational repression mediated by the Smaug response elements. RNAs lacking a poly(A) tail are poorly translated in the extract; therefore, SRE-dependent deadenylation contributes to translational repression. However, repression is strong even with RNAs either bearing a poly(A) tract that cannot be removed or lacking poly(A) altogether; thus, an additional aspect of translational repression functions independently of deadenylation.     

Glorund interactions in the regulation of gurken and oskar mRNAs

Precise temporal and spatial regulation of gene expression during Drosophila oogenesis is essential for patterning the anterior-posterior and dorsal-ventral body axes. Establishment of the anterior-posterior axis requires posterior localization and translational control of both oskar and nanos mRNAs. Establishment of the dorsal-ventral axis depends on the precise restriction of gurken mRNA and protein to the dorsal-anterior corner of the oocyte. We have previously shown that Glorund, the Drosophila hnRNP F/H homolog, contributes to anterior-posterior axis patterning by regulating translation of nanos mRNA, through a direct interaction with its 3′ untranslated region. To investigate the pleiotropy of the glorund mutant phenotype, which includes dorsal-ventral and nuclear morphology defects, we searched for proteins that interact with Glorund. Here we show that Glorund is part of a complex containing the hnRNP protein Hrp48 and the splicing factor Half-pint and plays a role both in mRNA localization and nurse cell chromosome organization, probably by regulating alternative splicing of ovarian tumor. We propose that Glorund is a component of multiple protein complexes and functions both as a translational repressor and splicing regulator for anterior-posterior and dorsal-ventral patterning.     

A role for Phospholipase D in Drosophila embryonic cellularization

Background

Cellularization of the Drosophila embryo is an unusually synchronous form of cytokinesis in which polarized membrane extension proceeds in part through incorporation of new membrane via fusion of apically-translocated Golgi-derived vesicles.

Results

We describe here involvement of the signaling enzyme Phospholipase D (Pld) in regulation of this developmental step. Functional analysis using gene targeting revealed that cellularization is hindered by the loss of Pld, resulting frequently in early embryonic developmental arrest. Mechanistically, chronic Pld deficiency causes abnormal Golgi structure and secretory vesicle trafficking.

Conclusion

Our results suggest that Pld functions to promote trafficking of Golgi-derived fusion-competent vesicles during cellularization.     

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.     

The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells

Background

When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response.

Results

In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization.

Significance

We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.