Drosophila Piwi-family proteins have been implicated in transposon control. Here, we examine piwi-interacting RNAs (piRNAs) associated with each Drosophila Piwi protein and find that Piwi and Aubergine bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs. As in mammals, the majority of Drosophila piRNAs are derived from discrete genomic loci. These loci comprise mainly defective transposon sequences, and some have previously been identified as master regulators of transposon activity. Our data suggest that heterochromatic piRNA loci interact with potentially active, euchromatic transposons to form an adaptive system for transposon control. Complementary relationships between sense and antisense piRNA populations suggest an amplification loop wherein each piRNA-directed cleavage event generates the 5' end of a new piRNA. Thus, sense piRNAs, formed following cleavage of transposon mRNAs may enhance production of antisense piRNAs, complementary to active elements, by directing cleavage of transcripts from master control loci.

SUMMARY In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.

Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes [1][2][3][4][5][6][7] . These join Argonaute-1 to regulate gene expression 8,9 . A second endogenous small RNA class, the Piwiinteracting RNAs, bind Piwi proteins and suppress transposons 10,11 . Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of singlestranded RNAs 12,13 . Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious 1,4,5 rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.Drosophila melanogaster expresses five Argonaute proteins, which segregate into two classes. The Piwi proteins (Piwi, Aubergine and AGO3) are expressed in gonadal tissues and act with Piwi-interacting RNAs (piRNAs) to suppress mobile genetic elements 10,11 . The Argonaute class contains AGO1 and AGO2. AGO1 binds microRNAs (miRNAs) and regulates gene expression 8,9 . The endogenous binding partners of AGO2 have remained enigmatic.We generated transgenic flies expressing epitope-tagged AGO2 under the control of its endogenous promoter. Tagged AGO2 localized to the cytoplasm of germline and somatic cells of the ovary (Supplementary Fig. 1). Immunoprecipitated AGO2-associated RNAs differed in their mobility from those bound to AGO1 (Fig. 1a). Deep sequencing of small RNAs from AGO1 and AGO2 complexes yielded 2,094,408 AGO1-associated RNAs and 916,834 AGO2-associated RNAs from Schneider (S2) cells, and 455,227 AGO2-associated RNAs from ovaries that matched perfectly to the Drosophila genome. We also sequenced three libraries derived from 18-29-nucleotide RNAs (936,833 sequences from wild-type ovaries, 1,042,617 sequences from Dicer-2 (Dcr-2) mutant ovaries, and 1,946,339 sequences from loquacious (loqs) mutant ovaries) and an 18-24-nucleotide library from wild-type testes (522,848 sequences). Finally, we added to our analysis 92,363 published sequences derived from 19-26-nucleotide RNAs from S2 cells 15 . We noted that among the ,50% of AGO2-associated RNAs from S2 cells that did not match the genome, ,17% matched the flock house virus (FHV), a pathogenic RNA virus and reported target for RNAi in flies 16,17 . These probably arose because o...

Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells. R NA silencing is a unique RNA-guided gene regulatory mechanism that operates in a wide range of eukaryotic organisms from plants to mammals (1). A feature common to all RNA silencing processes is the production of 21-to 26-nt small RNAs from structured or double-stranded RNA (dsRNA) by the endoribonuclease Dicer (2-6). These small interfering RNAs (siRNAs) control the specificity of RNA silencing in a homology-dependent manner by means of an RNA-induced silencing complex (RISC), of which Argonaute-2 (AGO2) is an essential protein component (1,7,8). RNA silencing in fungi, plants, and worms involves a cellular RNA-dependent RNA polymerase (RdRP); however, the multiple-turnover RISC may mediate RNA silencing in absence of a cellular RdRP in Drosophila and mammalian cells (1, 9-11).We reported recently that infection of cultured Drosophila cells with the plus-strand RNA Nodavirus flock house virus (FHV), triggers specific silencing of FHV RNAs that is associated with accumulation of 22-nt siRNAs (12). Silencing of the replicating viral RNAs is RISC-dependent and sensitive to inhibition by the FHV B2 protein, as shown by the observation that B2 is essential for FHV infection of WT Drosophila cells but dispensable in cells depleted for AGO2 (12). These findings provided an example indicating an antiviral role for RNA silencing in the animal kingdom (12, 13), as has been established in higher plants (14)(15)(16)(17)(18).In this article, we report that specific RNA silencing was induced in mosquito cells in response to viral RNA replication and show that this mosquito antiviral immunity is RISCdependent and sensitive to suppression by the B2 protein encoded by either FHV or nodamura virus (NoV). We demonstrate th...

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, we sought a direct link between the RNA silencing system and heterochromatin components. Here we show that PIWI, an ARGONAUTE/PIWI protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of PIWI. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. We also demonstrate that PIWI, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the PIWI-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome.[Keywords: PIWI; ARGONAUTE; HP1; heterochromatin; epigenetic; RNAi] Supplemental material is available at http://www.genesdev.org. . In TGS, small RNAs are incorporated into specialized effector complexes able to regulate chromatin modification, resulting in reduced access for the transcriptional machinery to chromatin. The TGS system that contributes to initiation and maintenance of heterochromatin formation in Schizosaccharomyces pombe is particularly well characterized (Verdel and Moazed 2005;Grewal and Jia 2007). In S. pombe, the RNA silencing system targets histone 3 Lys 9 (H3K9) methylation and recruits HP1 homolog Swi6 to the MAT locus and to repetitive elements in pericentric heterochromatin, setting up a heterochromatin spreading/maintenance loop that is dependent on the interaction of Swi6 with the Clr4 histone methyltransferase (HMT) (Grewal and Jia 2007). In plants, a specialized small RNA pathway utilizes heterochromatic RNAs to direct DNA methylation and presumably other chromatin modifications to effect silencing of target loci (for review, see Vaucheret 2007). In Caenorhabditis elegans, a single episode of RNA interference (RNAi) exposure can induce silencing inherited over many generations; mutations that abolish inheritance are all involved in chromatin structure, suggesting a chromatin-based mechanism (Vastenhouw et al. 2006). While the work in fission yeast and plants provides a possible paradigm for RNAi-dependent TGS in metazoans, a specialized TGS effector complex has not yet been characterized in an animal.

Summary Animals can detect and consume nutritive sugars without the influence of taste. However, the identity of the taste-independent nutrient sensor and the mechanism by which animals respond to the nutritional value of sugar are unclear. Here, we report that six neurosecretory cells in the Drosophila brain that produce Diuretic hormone 44 (Dh44), a homologue of the mammalian corticotropin-releasing hormone (CRH), were specifically activated by nutritive sugars. Flies in which the activity of these neurons or the expression of Dh44 was disrupted failed to select nutritive sugars. Manipulation of the function of Dh44 receptors had a similar effect. Notably, artificial activation of Dh44 receptor-1 neurons resulted in proboscis extensions, and frequent episodes of excretion. Conversely, reduced Dh44 activity led to decreased excretion. Together, these actions facilitate ingestion and digestion of nutritive foods. We propose that the Dh44 system directs the detection and consumption of nutritive sugars through a positive feedback loop.

Summary Neural systems controlling the vital functions of sleep and feeding in mammals are tightly inter-connected: sleep deprivation promotes feeding, while starvation suppresses sleep. Here we show that starvation in Drosophila potently suppresses sleep suggesting that these two homeostatically regulated behaviors are also integrated in flies. The sleep suppressing effect of starvation is independent of the mushroom bodies, a previously identified sleep locus in the fly brain, and therefore is regulated by distinct neural circuitry. The circadian clock genes Clock (Clk) and cycle (cyc) are critical for proper sleep suppression during starvation. However, the sleep suppression is independent of light cues and of circadian rhythms because starved period mutants sleep like wild type flies. By selectively targeting subpopulations of Clk-expressing neurons we localize the observed sleep phenotype to the dorsally located circadian neurons. These findings show that Clk and cyc act during starvation to modulate the conflict of whether flies sleep or search for food.

Feeding behavior is influenced primarily by two factors: nutritional needs and food palatability. However, the role of food deprivation and metabolic needs in the selection of appropriate food is poorly understood. Here, we show that the fruit fly, Drosophila melanogaster, selects calorie-rich foods following prolonged food deprivation in the absence of taste-receptor signaling. Flies mutant for the sugar receptors Gr5a and Gr64a cannot detect the taste of sugar, but still consumed sugar over plain agar after 15 h of starvation. Similarly, pox-neuro mutants that are insensitive to the taste of sugar preferentially consumed sugar over plain agar upon starvation. Moreover, when given a choice between metabolizable sugar (sucrose or D-glucose) and nonmetabolizable (zero-calorie) sugar (sucralose or L-glucose), starved Gr5a; Gr64a double mutants preferred metabolizable sugars. These findings suggest the existence of a taste-independent metabolic sensor that functions in food selection. The preference for calorie-rich food correlates with a decrease in the two main hemolymph sugars, trehalose and glucose, and in glycogen stores, indicating that this sensor is triggered when the internal energy sources are depleted. Thus, the need to replenish depleted energy stores during periods of starvation may be met through the activity of a taste-independent metabolic sensing pathway.F ood quantity and quality can vary greatly in natural habitats. To survive such variations, animals must be able to search for and detect appropriate food sources under all conditions, especially during times of food scarcity. Peripheral chemosensory neurons, such as sugar taste neurons, allow animals to detect palatable foods (1-5). Additional mechanisms may be necessary for the detection of foods to meet acute nutritional needs. Indeed, animals learn to positively associate a flavor paired with intragastric sugar infusion (6). Recently, studies of Trpm5 −/− mice, which are insensitive to the taste of sugar, also have revealed that these animals develop a preference for a sugar solution on the basis of its caloric content even in the absence of gustatory input (7). Unfortunately, the nature of such mechanisms is currently unknown. It is also not clear whether they function under starvation conditions. To search for mechanisms by which animals can respond to the caloric content of food independently of orosensory cues, we studied the effect of starvation on food choice in Drosophila mutants that are unable to taste sugar. Specifically, we sought to determine whether food-deprived flies carrying mutations in Gr5a and Gr64a (3-5), the sugar receptor genes, and in poxneuro (poxn) (8-10), a gene that specifies chemosensory neurons, develop a preference for the caloric content of sugars in the absence of taste perception. We found that these mutant flies demonstrated a preference for caloric food upon starvation and that this preference correlated with the energy needs of the fly. Furthermore, wild-type (WT) flies showed a shift in preference to metab...