The extracellular metalloprotease AdamTS-A anchors neural lineages in place within and preserves the architecture of the central nervous system.

The extracellular matrix (ECM) regulates cell migration and sculpts organ shape. AdamTS proteins are extracellular metalloproteases known to modify ECM proteins and promote cell migration, but demonstrated roles for AdamTS proteins in regulating CNS structure and ensuring cell lineages remain fixed in place have not been uncovered. Using forward genetic approaches in Drosophila, we find that reduction of AdamTS-A function induces both the mass exodus of neural lineages out of the CNS and drastic perturbations to CNS structure. Expressed and active in surface glia, AdamTS-A acts in parallel to perlecan and in opposition to viking/collagen IV and ßPS-integrin to keep CNS lineages rooted in place and to preserve the structural integrity of the CNS. viking/collagen IV and ßPS-integrin are known to promote tissue stiffness and oppose the function of perlecan, which reduces tissue stiffness. Our work supports a model in which AdamTS-A anchors cells in place and preserves CNS architecture by reducing tissue stiffness.

Collaborative Control of Cell Cycle Progression by the RNA Exonuclease Dis3 and Ras Is Conserved Across Species.

Dis3 encodes a conserved RNase that degrades or processes all RNA species via an N-terminal PilT N terminus (PIN) domain and C-terminal RNB domain that harbor, respectively, endonuclease activity and 3'-5' exonuclease activity. In Schizosaccharomyces pombe, dis3 mutations cause chromosome missegregation and failure in mitosis, suggesting dis3 promotes cell division. In humans, apparently hypomorphic dis3 mutations are found recurrently in multiple myeloma, suggesting dis3 opposes cell division. Except for the observation that RNAi-mediated depletion of dis3 function drives larval arrest and reduces tissue growth in Drosophila, the role of dis3 has not been rigorously explored in higher eukaryotic systems. Using the Drosophila system and newly generated dis3 null alleles, we find that absence of dis3 activity inhibits cell division. We uncover a conserved CDK1 phosphorylation site that when phosphorylated inhibits Dis3's exonuclease, but not endonuclease, activity. Leveraging this information, we show that Dis3's exonuclease function is required for mitotic cell division: in its absence, cells are delayed in mitosis and exhibit aneuploidy and overcondensed chromosomes. In contrast, we find that modest reduction of dis3 function enhances cell proliferation in the presence of elevated Ras activity, apparently by accelerating cells through G2/M even though each insult by itself delays G2/M. Additionally, we find that dis3 and ras genetically interact in worms and that dis3 can enhance cell proliferation under growth stimulatory conditions in murine B cells. Thus, reduction, but not absence, of dis3 activity can enhance cell proliferation in higher organisms.

BREs mediate both repression and activation of oskar mRNA translation and act in trans.

Asymmetric positioning of proteins within cells is crucial for cell polarization and function. Deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of the oskar mRNA, repression of its translation prior to localization, and finally activation of translation. Translational repression is mediated by BREs, regulatory elements positioned in two clusters near both ends of the oskar mRNA 3' UTR. Here we show that some BREs are bifunctional: both clusters of BREs contribute to translational repression, and the 3' cluster has an additional role in release from BRE-dependent repression. Remarkably, both BRE functions can be provided in trans by an oskar mRNA with wild-type BREs that is itself unable to encode Oskar protein. Regulation in trans is likely enabled by assembly of oskar transcripts in cytoplasmic RNPs. Concentration of transcripts in such RNPs is common, and trans regulation of mRNAs may therefore be widespread.

Dynamic organization and plasticity of sponge bodies.

Sponge bodies, cytoplasmic structures containing post-transcriptional regulatory factors, are distributed throughout the nurse cells and oocytes of the Drosophila ovary and share components with P bodies of yeast and mammalian cells. We show that sponge body composition differs between nurse cells and the oocyte, and that the sponge bodies change composition rapidly after entry into the oocyte. We identify conditions that affect sponge body organization. At one extreme, components are distributed relatively uniformly or in small dispersed bodies. At the other extreme, components are present in large reticulated bodies. Both types of sponge bodies allow normal development, but show substantial differences in distribution of Staufen protein and oskar mRNA, whose localization within the oocyte is essential for axial patterning. Based on these and other results we propose a model for the relationship between P bodies and the various cytoplasmic bodies containing P body proteins in the Drosophila ovary.

miRNA-dependent translational repression in the Drosophila ovary.

BACKGROUND: The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary. METHODOLOGY/PRINCIPAL FINDINGS: Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed. CONCLUSIONS/SIGNIFICANCE: Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.

Bicaudal C and trailer hitch have similar roles in gurken mRNA localization and cytoskeletal organization.

Bicaudal C and trailer hitch are both required for dorsoventral patterning of the Drosophila oocyte. Each mutant produces ventralized eggs, a phenotype typically associated with failure of the oocyte to provide a dorsalization signal--the Gurken protein--to the follicle cells. Bicaudal C and trailer hitch are both implicated in post-transcriptional gene regulation. Bicaudal C acts in recruiting a deadenylase to specific mRNAs, leading to translational repression. The role of trailer hitch is less well defined, but mutants have defects in protein secretion, and show aberrant distribution of an endoplasmic reticulum exit site marker whose mRNA is associated with Trailer hitch protein. We show that Bicaudal C and trailer hitch interact genetically. Mutants of these two genes have shared defects in localization of gurken and other anteriorly-localized mRNAs, as well as altered microtubule organization which may underlie the mRNA localization defects. Bicaudal C and trailer hitch mutants also share a syndrome of actin-related abnormalities, including the formation of ectopic actin cages near the anterior of the oocyte. The cages sequester Gurken protein, blocking its secretion and thus interfering with signaling of the follicle cells to specify dorsal fate.

A late phase of Oskar accumulation is crucial for posterior patterning of the Drosophila embryo, and is blocked by ectopic expression of Bruno.

In Drosophila, posterior embryonic body patterning and germ cell formation rely on Oskar, a protein that is concentrated at the posterior pole of the oocyte. A program of mRNA localization and translational regulation ensures that Oskar is only expressed at the proper location. One key regulatory factor is Bruno, which represses translation of oskar mRNA before its localization. Ectopic expression of a bruno cDNA prolongs repression, even after oskar mRNA is localized, and posterior body patterning is efficiently and selectively blocked. Surprisingly, the initial accumulation of Oskar, while frequently reduced, is not eliminated, arguing that levels of Oskar previously thought to be sufficient for patterning do not suffice, or that Bruno acts at a downstream step in patterning. Expression of the bruno cDNA does not inhibit posterior patterning when Oskar is expressed independent of Bruno-mediated regulation, ruling out a downstream requirement for Bruno. Notably, an Oskar::GFP reporter protein reveals continual accumulation during the late phases of oogenesis. Taken together, these results strongly argue that a late phase in accumulation of Osk protein, typically not monitored because of imperviousness of late stage oocytes to antibodies, is crucial for body patterning.

Recognition of the bcd mRNA localization signal in Drosophila embryos and ovaries.

The process of mRNA localization, often used for regulation of gene expression in polarized cells, requires recognition of cis-acting signals by components of the localization machinery. Many known RNA signals are active in the contexts of both the Drosophila ovary and the blastoderm embryo, suggesting a conserved recognition mechanism. We used variants of the bicoid mRNA localization signal to explore recognition requirements in the embryo. We found that bicoid stem-loop IV/V, which is sufficient for ovarian localization, was necessary but not sufficient for full embryonic localization. RNAs containing bicoid stem-loops III/IV/V did localize within the embryo, demonstrating a requirement for dimerization and other activities supplied by stem-loop III. Protein complexes that bound specifically to III/IV/V and fushi tarazu localization signals copurified through multiple fractionation steps, suggesting that they are related. Binding to these two signals was competitive but not equivalent. Thus, the binding complexes are not identical but appear to have some components in common. We have proposed a model for a conserved mechanism of localization signal recognition in multiple contexts.

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere.

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

Live imaging of nuage and polar granules: evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components.

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. We used GFP-tagged versions of Vasa and Aubergine to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. We found that perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. We also find that Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes.

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein.

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.