Post-transcriptional regulation of gene expression is a mechanism that governs developmental and cellular events in metazoans. In early embryogenesis, transcriptionally quiescent cells depend upon maternally supplied factors such as RNA binding proteins and RNA that control key decisions. Morphogen gradients form and in turn pattern the early embryo generating different cell types and spatial order. In the nematode Caenorhabditis elegans, the early embryo relies upon several RNA binding proteins that control mRNA stability, translation efficiency, and/or mRNA localization of cell fate determinants essential for proper development. MEX-5 and MEX-3 are two conserved RNA-binding proteins required to pattern the anterior/posterior axis and early embryo. Mutation of either gene results in a maternal effect lethal phenotype with proliferating posterior muscle into the anterior blastomeres (Muscle EXcess). Several cell-fate determinants are aberrantly expressed in mex-5 and mex-3 embryos. Both proteins are thought to interact with cis-regulatory elements present in 3’-UTRs of target RNAs controlling their metabolism. However, previous studies failed to demonstrate that these proteins regulate maternal transcripts directly. This dissertation presents a thorough assessment of the RNA binding properties of MEX-5 and MEX-3. Quantitative biochemical approaches were used to determine the RNA binding specificity of both proteins. MEX-5 has a relaxed specificity, binding with high affinity to linear RNA containing a tract of six or more uridines within an eight-nucleotide window. This is very different from its mammalian homologs Tristetraprolin (TTP) and ERF-2. I was able to identify two amino acids present within the MEX-5 RNA binding domain that are required for the differential RNA recognition observed between MEX-5 and TTP. MEX-3 on the other hand is a specific RNA binding protein, recognizing a bipartite element with flexible spacing between two four-nucleotide half-sites. I demonstrate that this element is required for MEX-3 dependent regulation in vivo. Previous studies only identify a small number of candidate regulatory targets of MEX-5 and MEX-3. The defined sequence specificity of both proteins is used to predict new putative targets that may be regulated by either protein. Collectively, this study examines the RNA binding properties of MEX-5 and MEX-3 to clarify their role as post-transcriptional regulators in nematode development.

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes.

Lung cancer is the leading cause of cancer-related mortality worldwide. The main risk factor associated with lung cancer is cigarette smoking. Research through the years suggests that nicotine in cigarettes promotes lung cancer by activating signaling pathways that lead to cell proliferation, cell survival, angiogenesis, and metastasis. Nicotine’s cellular actions are mediated by its cognate receptors, nicotinic acetylcholine receptors (nAChRs). Here, I describe the expression levels of all known human nAChR subunit genes in both normal and lung cancer cells. Of note, the genes encoding the α5, α3, and β4 subunits (CHRNA5/A3/B4) are over-expressed in small cell lung carcinoma (SCLC), the most aggressive form of lung cancer. This over-expression is regulated by ASCL1, a transcription factor important in normal lung development and lung carcinogenesis. The CHRNA5/A3/B4 locus has recently been the focus of a series of genetic studies showing that polymorphisms in this region confer risk for both nicotine dependence and lung cancer. I show that CHRNA5/A3/B4 depletion results in decreased SCLC cell viability. Furthermore, while nicotine promotes SCLC cell viability and tumor growth, blockade of α3β4 nAChRs inhibits SCLC cell viability. These results suggest that increased expression and function of nAChRs, specifically the α3β4α5 subtype, potentiate the effects of nicotine in SCLC. This dual hit from the carcinogens in tobacco and the cancer-promoting effects of nicotine, may provide a possible mechanism for the increased aggressiveness of SCLC. In addition, nAChRs can be activated by the endogenous ligand, acetylcholine, which acts as an autocrine/paracrine growth factor in SCLC. Increased function of α3β4α5 nAChRs in SCLC could also potentiate acetylcholine’s mitogenic effects. This mechanism, combined with other known autocrine/paracrine growth loops in SCLC, may help explain the ineffectiveness of available therapies against SCLC. In an effort to add to the current arsenal against SCLC, I screened a 1280-compund library using a bioluminescence-based viability assay I developed for high-throughput applications. Primary screening, followed by secondary and tertiary verification, indicate that pharmacologically active compounds targeting neuroendocrine markers inhibit SCLC cell viability.

The nervous system is made up of two major cell types, neurons and glia. The major distinguishing feature between neuronal cells and glial cells is that neurons are capable of transmitting action potentials while glial cells are electrically incompetent. For over a century glial cells were neglected and it was thought they existed merely to provide trophic and structural support to neurons. However, in the past few decades it has become increasingly clear that glial cell functions underlie almost all aspects of nervous system development, maintenance, and health. During development, glia act as permissive substrates for axons, provide guidance cues, regulate axon bundling, facilitate synapse formation, refine synaptic connections, and promote neuronal survival. In the mature nervous system glial cells regulate adult neurogenesis through phagocytosis, act as the primary immune cell, and contribute to complex processes such as learning and memory. In recent years, glial cells have also become a primary focus in the study of neurodegenerative diseases. Mounting evidence shows that glial cells exert both beneficial as well as detrimental effects in the pathology of several nervous system disorders, and modulation of glial activity is emerging as a viable therapeutic strategy for many diseases. Although glial cells are critical to the proper development and functioning of the nervous system, there is still relatively little known about the molecular mechanisms used by glial cells, how they exert their effects on neurons, and how glia and neurons communicate. Despite the relative simplicity and small size of the Drosophila nervous system, glial cell organization and function in flies shows a remarkable complexity similar to vertebrate glial cells. In this study I use Drosophila as a model organism to study cellular and molecular mechanisms of glial clearance of axonal debris after acute axotomy. In chapter two of this thesis, I characterize three distinct subtypes of glial cells in the adult brain; cell body glia which ensheath neuronal cell bodies in the cortex region of the brain, astrocyte like glial cells which bear striking morphological similarity to mammalian astrocytes and share common molecular components, and ensheathing glial cells which I show act as the primary phagocytic cell type in the neuropil region of the brain. In addition, I identify dCed-6, the ortholog of mammalian GULP, as a necessary component of the glial phagocytic machinery. In chapter three of this thesis, I perform a candidate based, in vivo, RNAi screen to identify novel genes involved in the glial engulfment of degenerating axon material. The Gal4/UAS system was used to drive UAS-RNAi for approximately 300 candidate genes with the glial specific repo-Gal4 driver. Two assays were used as a readout in this screen, clearance of axon material five days after injury, and Draper upregulation one day after maxillary palp or antennal injury. Overall, I identified 20 genes which, when knocked down specifically in glial cells, result in axon clearance defects after injury. Finally, in chapter four I identify Stat92E as a novel glial gene required for glial phagocytic function. I show that Stat92E regulates both basal and injury induced Draper expression. Injury-induced Draper expression is transcriptionally regulated through a Stat92E dependent non-canonical signaling mechanism whereby signaling through the Draper receptor activates Stat92E which in turn transcriptionally activates draper through a binding site located in the first intron of Draper. Draper represents only the second receptor known to positively regulate Stat92E transcriptional activity under normal physiological conditions.

The immune system is a complex network of interacting cells and tissues that is designed to protect the body from pathogens and other foreign substances. T cells are a major component of the immune system and consist of two distinct lineages distinguished by the expression of αβ or γδ T cell receptors (TCR). The Tec family kinase, Itk is an important mediator of signaling downstream of the TCR. Past studies on Itk has focused on how Itk regulates development, activation and differentiation of conventional αβ T cells and more recently how Itk regulates the development of innate-like αβ T cells. However, very little is known about the influence of Itk on γδ T cells. My studies show a previously unknown role for Itk in the development and function of γδ T cells. We report in the absence of Itk, γδ T cells were responsible for the spontaneously elevated levels of serum IgE and Itk-/- mice γδ T cells produced high levels of TH2 cytokines. Furthermore, there was an increase in γδ T cells specifically in the Vγ1.1+Vδ6.3+ (V6) subset that represents the dominant population of γδ NKT cells in Itk-/- mice. In addition, the V6 subset had increased expression of PLZF, a transcription factor normally required for αβ iNKT cell development. We further show that V6 cells develop and mature similar to αβ iNKT cells. Similar to defects previously seen in the terminal differentiation of Itk-/- αβ iNKT cell, V6 cells also had impaired maturation in the thymus in the absence of Itk. This data demonstrates a previously unknown role of Itk for the terminal maturation of V6 cells that has been shown to be the cell population that led to spontaneous dermatitis in mice. Given that drug companies have targeted Itk as a potential allergy drug due to Itk’s role in TH2 development and function, our data suggests that further studies on Itk are warranted.

Dengue viruses (DV) are re-emerging mosquito-borne pathogens for which four distinct lineages, grouped based on serology and referred to as serotypes 1-4 (DIV-D4V), have been described. Epidemiological data imply that re-infection with a "heterologous" serotype, i.e, one other than that to which the individual was originally exposed, enhances the risk for development of severe disease, dengue hemorrhagic fever (DHF). The hallmark of DHF is a transient capillary leakage syndrome of rapid onset, temporally associated with the resolution of fever and viremia. In its most grave form, the vascular permeability phenomenon in DHF may progress to dengue shock syndrome (DSS), which is often fatal in the absence of appropriate medical care. Despite the fulminant nature of vascular leakage during DHF/DSS, this phenomenon does not appear to be due to direct cytopathic effects of DV. Rather, inappropriate reactivation and/or regulation of dengue-specific memory are the prevailing theorized (immunopathological) etiologies. Traditional vaccine development techniques have proven insufficient for DV, since any vaccine must offer complete protection against all four serotypes to avoid enhanced pathology on natural viral challenge. Understanding the underlying mechanisms that contribute to dengue disease, particularly the development of dengue-specific memory, is therefore of critical importance. Dengue immunopathology and the specific aspects of immunological memory that determine disease severity are heatedly debated. Previous research in our lab has suggested that T cell responses contribute to the severity of dengue illness. Clinical data indicate enhanced immune activation in more grave cases of DV infection, and serotype cross-reactive T cells from multiple individuals are present after both primary and secondary dengue infections. However, little is known about the conditions under which T cells are primed and dengue-specific memory is generated. Dendritic cells (DCs) are bone marrow-derived cells that play a central role in directing activity within the immune system. DCs shape quantitative and qualitative aspects of adaptive immunity, and therefore the intrinsic characteristics of host memory to a pathogen. DCs are essential in generating primary immune responses, due to their particular effectiveness in stimulating naïve T cells. DCs also play important roles in the reactivation of memory to an infectious agent, and as reservoirs for the dissemination of invading microorganisms. Exposure to pathogens or their products initiates a series of phenotypic and functional changes in DCs, termed maturation. DC maturation involves a coordinated response of immunomodulatory surface molecule elaboration and cytokine production, culminating in antigen presentation to, and co-stimulation of, T cells specific for the invading agent. The DC response is ostensibly tailored to facilitate effective elimination by regulating effective downstream interactions of the DC with T cells. A number of viruses have evolved to infect DCs and alter their functional behavior, facilitating their own survival within the host, and the herd. DV readily infects DCs both in primary cell cultures and in vivo. However, reports on the effects of DV infection on DC maturation vary both with regard to some of the cytokines produced, and the phenotypes of infected versus bystander cells. Although DCs appear to be activated following DV exposure, responses on the single-cell level appear to depend on the infection state of the cell, hypothetically driven by intracellular virus-mediated effects. Therefore, downstream responses to these divergent populations - i.e., actively infected cells versus uninfected bystander cells - are likely to be the consequence of at least two modes of DC behavior. Because DCs play a pivotal role in adaptive immune development, and because the resulting memory response appears to be critical in affecting disease pathology after heterologous DV re-infection, I sought to explore the phenomena of DC maturation in response to dengue exposure, and to begin to answer the question of how active infection alters the functional capabilities of DCs. Notably, primary dengue infection is generally well-controlled with minimal pathology. Therefore, this thesis addresses the hypothesis that DV infection of DCs results in cellular activation and stimulation of antiviral immunity, despite virus-mediated alteration of DC maturation. In order to address this hypothesis, I examined both DV infection-dependent and independent effects on DC functional responses including surface molecule regulation secretory activity, and CD4 T cell allostimulatory priming. DCs derived from human peripheral blood monocytes were readily infected with multiple strains of DV. DV infection of DCs derived from separate donors was dose-dependent, with substantial variability in DC susceptibility to infection. Exposure to live DV activated surface molecule expression in DCs, similar to the effects of defined maturation stimuli including a combination of TNF-α and IFN-α, or LPS. In addition, UV-inactivated DV induced expression of cell surface molecules, albeit to a lesser extent than did live virus demonstrating inherent stimulatory properties of DV particles. Using intracellular staining for DV envelope (E) protein, I detected increased surface molecule expression on both infected DCs and uninfected bystander DCs from the same culture, as compared to mock-infected DCs. These data indicate that activation was not prevented in cells undergoing active viral replication. However, the degree of surface molecule induction depended on the infection state of the cell. Infected DCs had enhanced PD-L2 and MHC II expression relative to uninfected bystander cells, while PD-L1, CD80, CD86, and MHC I expression were suppressed with active infection. Therefore, intracellular DV replication altered the process of cell surface molecule regulation within these cells. DV infection of DCs also resulted in the secretion of a broad array of cytokines and chernokines. These included the antiviral cytokine IFN-α, inflammatory cytokines TNF-α, IL-6, and IL-1α, and inflammatory chemokines IP10, MCP-1, MIP-1α, and RANTES. DV infection did not induce DC production of the IL-12 p70 heterodimer, and secretion of the immunosuppressive cytokine IL-10 was low in most experiments. Similar to the results seen with surface molecule induction, UV inactivation of DV reduced, but did not eliminate, cytokine and chemokine responses. At the single-cell level, TNF-α and IP10 production profiles of infected DCs and uninfected bystander DCs were distinct. DV infection in DCs reduced production of IP10, but stimulated TNF-α as compared to uninfected bystander cells in the same culture. Blocking experiments demonstrated that IFN-α/β produced by DCs in response to infection actively inhibited viral protein expression and drove IP10, but not TNF-α, production. DV infection of DCs did not consistently suppress DC stimulation of allogeneic CD4 T cell proliferation. In cases where infection enhanced DC stimulatory function, T cell proliferation was less pronounced than that induced by DCs activated with exogenous TNF-α plus IFN-α. Increasing multiplicity of infection (MOI) of DCs with DV resulted in increasing DC infection rates, but a statistically significant trend at the highest MOIs for decreased T cell alloproliferation, suggesting that direct infection of DCs reduces their CD4 T cell priming function. MOI-dependent reduction in DC stimulatory function depended on replication-competent virus. Increased MOIs during DV infection of DCs did not cause an elevation in detectable IL-10 in supernatants derived from T-DC co-cultures. In addition, increased DV MOI of DCs was not associated with increased levels of either IL-13 or IFN-γ in supernatants from T-DC co-culture, suggesting that actively infected DC do not skew CD4 T cells towards a specific Th phenotype. These data demonstrate that DV infection induces functional maturation of DCs that is modified by the presence of virus through both IFN-dependent and independent mechanisms. However, the allostimulatory phenotype of DCs was not universally enhanced, nor was it skewed towards antiviral (Th1)-type responses. These data suggest a model whereby dengue infection during primary illness results in controlled immune stimulation through activation of bystander DCs, and the generation of mixed Th-type responses. Direct DV infection of DCs appears to attenuate activation of, and potentially clearance by, antiviral mechanisms. During secondary infection, reduced IP10 production and enhanced TNF-α secretion by infected cells coupled with MHC I downregulation and enhanced PD-L2 expression, would subvert both Th1 CD4 T cell recruitment and result in CD8 T cell suppression and death. Furthermore, DV-specific effects on DCs would allow for continued viral replication in the absence of effective clearance. These DV-mediated effects would modify T cell memory responses to infected DC, and potentially facilitate the expansion of pathologic T cell subsets. Contributing to this pathological cascade, antibody-dependent enhancement of infection in monocytic cells and macrophages would shift antigen presentation and cytokine production paradigms, increasing the risk of DHF.

Carrier mediated nutrient import is vital for cell and tissue homeostasis. Structural insights of carrier mediated transport, particularly the human glucose transporter GLUT1, are essential for understanding the mechanisms of human metabolic disease, and provide model systems for cellular processes as a whole. GLUT1 function and expression is characterized by a complexity unexplained by the current hypotheses for carrier-mediated sugar transport (9). It is possible that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae(RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1) to characterize its functional properties. Identical protein sequences generated different kinetic parameters when expressed in RE700A yeast, erythrocytes, and HEK293 cells. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific. Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 sugar export site. Paradoxically, very low concentrations of these inhibitors produce a modest stimulation of sugar transport (16). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, intracellular binding sites for e1 ligands CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of exit site ligands determine binding site affinity and cooperativity. Our findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of GLUT1 quaternary structure and evaluate the major determinants of ligand binding affinity and cooperativity. Cytochalasin B (CB) inhibits GLUT1 substrate transport at or near the endofacial sugar binding site. N-bromosuccinamide analysis combined with 3H-CB photolabeling implicates the region between Trp388 and Trp412 in ligand binding. Although its structure has been modeled(5), the specific residues comprising the sugar binding site are unknown. A series of alanine point mutants were made, and mutant protein 2-deoxy glucose transport was tested in the presence of increasing [CB]. Arg126Ala and Cys421Ala GLUT1 mutations altered CB affinity but were determined not to be in the e1 site. The Arg400Ala mutation decreased binding affinity for CB, and may comprise part of the e1 binding site. Because point mutations were individually insufficient to abrogate CB binding, Trp388 to Trp412 chimeras were made. GLUT1/GLUT4388-412/GLUT1 and GLUT1/GLUT5388-412/GLUT1 chimeras showed moderately less sensitivity to CB inhibition of transport; these amino acids likely comprise regions determinant of CB binding affinity. Furthermore GLUT1/GLUT5388-412/GLUT1 shows enhancement of 2-DG uptake at 50nM CB, but an overall dose response indistinguishable from WT GLUT1. A multisite fit of the data suggested GLUT1/GLUT5388-412/GLUT1 chimera possesses strong first site affinity for CB but slight negative second-site cooperativity. We conclude that point mutants were insufficient to abrogate CB binding and that the Trp388 to Trp412 sequence is necessary for CB binding affinity but is not the sole determinant of inhibition of 2 deoxyglucose uptake by CB. We discuss these results with their implications for structure-function sequence localization of the CB binding site, and by extension, the e1 sugar binding site.

From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome. The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology. To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome.

Studies have demonstrated that under conditions of chronic metabolic stress, GLUT1-mediated sugar transport is upregulated at the blood-brain barrier by a number of mechanisms. Although acute metabolic stress has also been shown to increase GLUT1-mediated transport, the mechanisms underlying this regulation remain unclear. This work attempts to explain how GLUT1-mediated sugar uptake is increased during acute metabolic stress, as well as explore the factors involved in this modulation of sugar transport in blood-brain barrier endothelial cells. Glucose depletion, KCN and FCCP were applied to brain microvascular endothelial cell line bEnd.3 in order to induce acute metabolic stress by ATP depletion. Kinetic sugar uptake measurements in combination with qPCR, whole cell lysate western blots, and cell-surface biotinylation were employed to probe for changes in GLUT1-mediated sugar uptake, GLUT1 expression levels, and GLUT1 localization during metabolic stress. Finally, the role of AMP-activated kinase (AMPK) in the bEnd.3 cell response to acute stress was examined using the specific AMPK activator AICAR and inhibitor Compound C. The data presented in this thesis supports the following two conclusions: 1. GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress is increased 3-7 fold due to translocation of intracellular GLUT1 to the plasma membrane, with no change in expression of total GLUT1 protein, and 2. AMPK plays a direct role in modulating increases in GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress by regulating trafficking of GLUT1 to the plasma membrane.

In the Drosophila germ line, PIWI-interacting RNAs (piRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. We examined the genetic requirements for the biogenesis and function of piRNAs in both female and male germ line. We found that piRNAs function through the PIWI, rather than the AGO, family Argonaute proteins, and the production of piRNAs requires neither microRNA (miRNA) nor small interfering RNA (siRNA) pathway machinery. These findings allowed the discovery of the third conserved small RNA silencing pathway, which is distinct from both the miRNA and RNAi pathways in its mechanisms of biogenesis and function. We also found piRNAs in flies are modified. We determined that the chemical structure of the 3´-terminal modification is a 2´-O-methyl group, and also demonstrated that the same modification occurs on the 3´ termini of siRNAs in flies. Furthermore, we identified the RNA methyltransferase Drosophila Hen1, which catalyzes 2´-O-methylation on both siRNAs and piRNAs. Our data suggest that 2´-O-methylation by Hen1 is the final step of biogenesis of both the siRNA pathway and piRNA pathway. Studies from the Hannon Lab and the Siomi Lab suggest a ping-pong amplification loop for piRNA biogenesis and function in the Drosophila germline. In this model, an antisense piRNA, bound to Aubergine or Piwi, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. We isolated the loss-of-function mutations in ago3, allowing a direct genetic test of this model. We found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. Moreover, we also discovered a second Ago3-independent piRNA pathway in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line.