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.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. Here, we systematically evaluate the minimal requirements for functional miRNA–target duplexes in vivo and distinguish classes of target sites with different functional properties. Target sites can be grouped into two broad categories. 5′ dominant sites have sufficient complementarity to the miRNA 5′ end to function with little or no support from pairing to the miRNA 3′ end. Indeed, sites with 3′ pairing below the random noise level are functional given a strong 5′ end. In contrast, 3′ compensatory sites have insufficient 5′ pairing and require strong 3′ pairing for function. We present examples and genome-wide statistical support to show that both classes of sites are used in biologically relevant genes. We provide evidence that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3′ ends are key determinants of target specificity within miRNA families.

Cell proliferation, cell death, and pattern formation are coordinated in animal development. Although many proteins that control cell proliferation and apoptosis have been identified, the means by which these effectors are linked to the patterning machinery remain poorly understood. Here, we report that the bantam gene of Drosophila encodes a 21 nucleotide microRNA that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. We identify the pro-apoptotic gene hid as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity.

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.

MicroRNAs are small noncoding RNAs that serve as posttranscriptional regulators of gene expression in higher eukaryotes. Their widespread and important role in animals is highlighted by recent estimates that 20%-30% of all genes are microRNA targets. Here, we report that a large set of genes involved in basic cellular processes avoid microRNA regulation due to short 3'UTRs that are specifically depleted of microRNA binding sites. For individual microRNAs, we find that coexpressed genes avoid microRNA sites, whereas target genes and microRNAs are preferentially expressed in neighboring tissues. This mutually exclusive expression argues that microRNAs confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions.

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...

Increasingly complex networks of small RNAs act through RNA-interference (RNAi) pathways to regulate gene expression, to mediate antiviral responses, to organize chromosomal domains, and to restrain the spread of selfish genetic elements. Historically, RNAi has been defined as a response to double-stranded RNA. However, some small RNA species may not arise from double-stranded RNA precursors. Yet, like microRNAs and small interfering RNAs, such species guide Argonaute proteins to silencing targets through complementary base-pairing. Silencing can be achieved by corecruitment of accessory factors or through the activity of Argonaute itself, which often has endonucleolytic activity. As a specific and adaptive regulatory system, RNAi is used throughout eukarya, which indicates a long evolutionary history. A likely function of RNAi throughout that history is to protect the genome from both pathogenic and parasitic invaders.

SummaryEukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression.