Pgc suppresses the zygotically acting RNA decay pathway to protect germ plasm RNAs in the Drosophila embryo.

Specification of germ cells is pivotal to ensure continuation of animal species. In many animal embryos, germ cell specification depends on maternally supplied determinants in the germ plasm. Drosophila polar granule component (pgc) mRNA is a component of the germ plasm. pgc encodes a small protein that is transiently expressed in newly formed pole cells, the germline progenitors, where it globally represses mRNA transcription. pgc is also required for pole cell survival, but the mechanism linking transcriptional repression to pole cell survival remains elusive. We report that pole cells lacking pgc show premature loss of germ plasm mRNAs, including the germ cell survival factor nanos, and undergo apoptosis. We found that pgc- pole cells misexpress multiple miRNA genes. Reduction of miRNA pathway activity in pgc- embryos partially suppressed germ plasm mRNA degradation and pole cell death, suggesting that Pgc represses zygotic miRNA transcription in pole cells to protect germ plasm mRNAs. Interestingly, germ plasm mRNAs are protected from miRNA-mediated degradation in vertebrates, albeit by a different mechanism. Thus, independently evolved mechanisms are used to silence miRNAs during germ cell specification.

dTcf/Pangolin suppresses growth and tumor formation in Drosophila.

Wnt/Wingless (Wg) signaling controls many aspects of animal development and is deregulated in different human cancers. The transcription factor dTcf/Pangolin (Pan) is the final effector of the Wg pathway in Drosophila and has a dual role in regulating the expression of Wg target genes. In the presence of Wg, dTcf/Pan interacts with ß-catenin/Armadillo (Arm) and induces the transcription of Wg targets. In absence of Wg, dTcf/Pan partners with the transcriptional corepressor TLE/Groucho (Gro) and inhibits gene expression. Here, we use the wing imaginal disk of Drosophila as a model to examine the functions that dTcf/Pan plays in a proliferating epithelium. We report a function of dTcf/Pan in growth control and tumorigenesis. Our results show that dTcf/Pan can limit tissue growth in normal development and suppresses tumorigenesis in the context of oncogene up-regulation. We identify the conserved transcription factors Sox box protein 15 (Sox15) and Ftz transcription factor 1 (Ftz-f1) as genes controlled by dTcf/Pan involved in tumor development. In conclusion, this study reports a role for dTcf/Pan as a repressor of normal and oncogenic growth and identifies the genes inducing tumorigenesis downstream of dTcf/Pan.

Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut.

Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. Here we present evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions.

ER stress potentiates insulin resistance through PERK-mediated FOXO phosphorylation.

Endoplasmic reticulum (ER) stress is emerging as a potential contributor to the onset of type 2 diabetes by making cells insulin-resistant. However, our understanding of the mechanisms by which ER stress affects insulin response remains fragmentary. Here we present evidence that the ER stress pathway acts via a conserved signaling mechanism involving the protein kinase PERK to modulate cellular insulin responsiveness. Insulin signaling via AKT reduces activity of FOXO transcription factors. In some cells, PERK can promote insulin responsiveness. However, we found that PERK also acts oppositely via phosphorylation of FOXO to promote FOXO activity. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. We suggest that the protein kinase PERK may be a promising pharmacological target for ameliorating insulin resistance.

Oncogenic cooperation between SOCS family proteins and EGFR identified using a Drosophila epithelial transformation model.

MicroRNAs (miRNAs) are emerging as cooperating factors that promote the activity of oncogenes in tumor formation and disease progression. This poses the challenge of identifying the miRNA targets responsible for these interactions. In this study, we identify the growth regulatory miRNA bantam and its target, Socs36E, as cooperating factors in EGFR-driven tumorigenesis and metastasis in a Drosophila model of epithelial transformation. bantam promotes growth by limiting expression of Socs36E, which functions as a negative growth regulator. Socs36E has only a modest effect on growth on its own, but behaves as a tumor suppressor in combination with EGFR activation. The human ortholog of SOCS36E, SOCS5, behaves as a candidate tumor suppressor in cellular transformation in cooperation with EGFR/RAS pathway activation.

Identification and characterization of novel conserved RNA structures in Drosophila.

BACKGROUND: Comparative genomics approaches have facilitated the discovery of many novel non-coding and structured RNAs (ncRNAs). The increasing availability of related genomes now makes it possible to systematically search for compensatory base changes - and thus for conserved secondary structures - even in genomic regions that are poorly alignable in the primary sequence. The wealth of available transcriptome data can add valuable insight into expression and possible function for new ncRNA candidates. Earlier work identifying ncRNAs in Drosophila melanogaster made use of sequence-based alignments and employed a sliding window approach, inevitably biasing identification toward RNAs encoded in the more conserved parts of the genome. RESULTS: To search for conserved RNA structures (CRSs) that may not be highly conserved in sequence and to assess the expression of CRSs, we conducted a genome-wide structural alignment screen of 27 insect genomes including D. melanogaster and integrated this with an extensive set of tiling array data. The structural alignment screen revealed ∼30,000 novel candidate CRSs at an estimated false discovery rate of less than 10%. With more than one quarter of all individual CRS motifs showing sequence identities below 60%, the predicted CRSs largely complement the findings of sliding window approaches applied previously. While a sixth of the CRSs were ubiquitously expressed, we found that most were expressed in specific developmental stages or cell lines. Notably, most statistically significant enrichment of CRSs were observed in pupae, mainly in exons of untranslated regions, promotors, enhancers, and long ncRNAs. Interestingly, cell lines were found to express a different set of CRSs than were found in vivo. Only a small fraction of intergenic CRSs were co-expressed with the adjacent protein coding genes, which suggests that most intergenic CRSs are independent genetic units. CONCLUSIONS: This study provides a more comprehensive view of the ncRNA transcriptome in fly as well as evidence for differential expression of CRSs during development and in cell lines.

Everything old is new again: (linc)RNAs make proteins!

Long non-coding RNAs have become the focus of considerable interest over the past few years. Intriguing novel functions have been reported for lincRNAs. Three recent papers identify lincRNAs that work in a more conventional way-encoding protein-in each case a small polypeptide with an interesting biological activity (Magny et al, 2013; Pauli et al, 2014), (Bazzini et al, 2014).

Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover.

Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the ßTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1.

Control of Drosophila Type I and Type II central brain neuroblast proliferation by bantam microRNA.

Post-transcriptional regulation of stem cell self-renewal by microRNAs is emerging as an important mechanism controlling tissue homeostasis. Here, we provide evidence that bantam microRNA controls neuroblast number and proliferation in the Drosophila central brain. Bantam also supports proliferation of transit-amplifying intermediate neural progenitor cells in type II neuroblast lineages. The stem cell factors brat and prospero are identified as bantam targets acting on different aspects of these processes. Thus, bantam appears to act in multiple regulatory steps in the maintenance and proliferation of neuroblasts and their progeny to regulate growth of the central brain.

Metabolic control of PPAR activity by aldehyde dehydrogenase regulates invasive cell behavior and predicts survival in hepatocellular and renal clear cell carcinoma.

BACKGROUND: Changes in cellular metabolism are now recognized as potential drivers of cancer development, rather than as secondary consequences of disease. Here, we explore the mechanism by which metabolic changes dependent on aldehyde dehydrogenase impact cancer development. METHODS: ALDH7A1 was identified as a potential cancer gene using a Drosophila in vivo metastasis model. The role of the human ortholog was examined using RNA interference in cell-based assays of cell migration and invasion. 1H-NMR metabolite profiling was used to identify metabolic changes in ALDH7A1-depleted cells. Publically available cancer gene expression data was interrogated to identify a gene-expression signature associated with depletion of ALDH7A1. Computational pathway and gene set enrichment analysis was used to identify signaling pathways and cellular processes that were correlated with reduced ALDH7A1 expression in cancer. A variety of statistical tests used to evaluate these analyses are described in detail in the methods section. Immunohistochemistry was used to assess ALDH7A1 expression in tissue samples from cancer patients. RESULTS: Depletion of ALDH7A1 increased cellular migration and invasiveness in vitro. Depletion of ALDH7A1 led to reduced levels of metabolites identified as ligands for Peroxisome proliferator-activated receptor (PPARα). Analysis of publically available cancer gene expression data revealed that ALDH7A1 mRNA levels were reduced in many human cancers, and that this correlated with poor survival in kidney and liver cancer patients. Using pathway and gene set enrichment analysis, we establish a correlation between low ALDH7A1 levels, reduced PPAR signaling and reduced patient survival. Metabolic profiling showed that endogenous PPARα ligands were reduced in ALDH7A1-depleted cells. ALDH7A1-depletion led to reduced PPAR transcriptional activity. Treatment with a PPARα agonist restored normal cellular behavior. Low ALDH7A1 protein levels correlated with poor clinical outcome in hepatocellular and renal clear cell carcinoma patients. CONCLUSIONS: We provide evidence that low ALDH7A1 expression is a useful prognostic marker of poor clinical outcome for hepatocellular and renal clear cell carcinomas and hypothesize that patients with low ALDH7A1 might benefit from therapeutic approaches addressing PPARα activity.

A conformation-induced fluorescence method for microRNA detection.

MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3' and 5' ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures.

MicroRNAs and gene regulatory networks: managing the impact of noise in biological systems.

Biological systems are continuously challenged by an environment that is variable. Yet, a key feature of developmental and physiological processes is their remarkable stability. This review considers how microRNAs contribute to gene regulatory networks that confer robustness.

Drosophila miR-14 regulates insulin production and metabolism through its target, sugarbabe.

Energy homeostasis depends on insulin signaling in metazoans. Insulin levels reflect the nutritional status of the animal to control levels of circulating sugar and regulate storage of resources in the form of glycogen and fat. Over the past several years, evidence has begun to accumulate that insulin production and secretion, as well as cellular responsiveness to insulin, are subject to regulation by microRNAs. Here we present evidence that miR-14 acts in the insulin-producing neurosecretory cells in the adult Drosophila brain to control metabolism. miR-14 acts in these cells through its direct target, sugarbabe. sugarbabe encodes a predicted zinc finger protein that regulates insulin gene expression in the neurosecretory cells. Regulation of sugarbabe levels by nutrients and by miR-14 combines to allow the fly to manage resource mobilization in a nutritionally variable environment.

Regulation of tissue growth through nutrient sensing.

Nutrition is a key regulator of tissue growth. In animals, nutritional status is monitored and signaled at both the cellular and systemic levels. The main mediator of cellular nutrient sensing is the protein kinase TOR (target of rapamycin). TOR receives information from levels of cellular amino acids and energy, and it regulates the activity of processes involved in cell growth, such as protein synthesis and autophagy. Insulin-like signaling is the main mechanism of systemic nutrient sensing and mediates its growth-regulatory functions largely through the phosphatidylinositol 3-kinase (PI3K)/AKT protein kinase pathway. Other nutrition-regulated hormonal mechanisms contribute to growth control by modulating the activity of insulin-like signaling. The pathways mediating signals from systemic and cellular levels converge, allowing cells to combine information from both sources. Here we give an overview of the mechanisms that adjust animal tissue growth in response to nutrition and highlight some general features of the signaling pathways involved.

Drosophila Minus is required for cell proliferation and influences Cyclin E turnover.

Turnover of cyclins plays a major role in oscillatory cyclin-dependent kinase (Cdk) activity and control of cell cycle progression. Here we present a novel cell cycle regulator, called minus, which influences Cyclin E turnover in Drosophila. minus mutants produce defects in cell proliferation, some of which are attributable to persistence of Cyclin E. Minus protein can interact physically with Cyclin E and the SCF Archipelago/Fbw7/Cdc4 ubiquitin-ligase complex. Minus does not affect dMyc, another known SCF(Ago) substrate in Drosophila. We propose that Minus contributes to cell cycle regulation in part by selectively controlling turnover of Cyclin E.

Drosophila miR-124 regulates neuroblast proliferation through its target anachronism.

MicroRNAs (miRNAs) have been implicated as regulators of central nervous system (CNS) development and function. miR-124 is an evolutionarily ancient, CNS-specific miRNA. On the basis of the evolutionary conservation of its expression in the CNS, miR-124 is expected to have an ancient conserved function. Intriguingly, investigation of miR-124 function using antisense-mediated miRNA depletion has produced divergent and in some cases contradictory findings in a variety of model systems. Here we investigated miR-124 function using a targeted knockout mutant and present evidence for a role during central brain neurogenesis in Drosophila melanogaster. miR-124 activity in the larval neuroblast lineage is required to support normal levels of neuronal progenitor proliferation. We identify anachronism (ana), which encodes a secreted inhibitor of neuroblast proliferation, as a functionally important target of miR-124 acting in the neuroblast lineage. ana has previously been thought to be glial specific in its expression and to act from the cortex glia to control the exit of neuroblasts from quiescence into the proliferative phase that generates the neurons of the adult CNS during larval development. We provide evidence that ana is expressed in miR-124-expressing neuroblast lineages and that ana activity must be limited by the action of miR-124 during neuronal progenitor proliferation. We discuss the possibility that the apparent divergence of function of miR-124 in different model systems might reflect functional divergence through target site evolution.

The miRNA machinery targets Mei-P26 and regulates Myc protein levels in the Drosophila wing.

MicroRNAs (miRNAs) have been implicated in cell-cycle regulation and in some cases shown to have a role in tissue growth control. Depletion of miRNAs was found to have an effect on tissue growth rates in the wing primordium of Drosophila, a highly proliferative epithelium. Dicer-1 (Dcr-1) is a double-stranded RNAseIII essential for miRNA biogenesis. Adult cells lacking dcr-1, or with reduced dcr-1 activity, were smaller than normal cells and gave rise to smaller wings. dcr-1 mutant cells showed evidence of being susceptible to competition by faster growing cells in vivo and the miRNA machinery was shown to promote G(1)-S transition. We present evidence that Dcr-1 acts by regulating the TRIM-NHL protein Mei-P26, which in turn regulates dMyc protein levels. Mei-P26 is a direct target of miRNAs, including the growth-promoting bantam miRNA. Thus, regulation of tissue growth by the miRNA pathway involves a double repression mechanism to control dMyc protein levels in a highly proliferative and growing epithelium.

Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing.

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. Here, we present evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and we have identified the Actin regulator Enabled as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary.

Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage.

Drosophila neuroblasts and ovarian stem cells are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim-NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.

MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila.

The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. Here we present the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels.