Inducible RNA Interference Research Articles

A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain-containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2-green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum-derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis.

Two DYW Subclass PPR Proteins are Involved in RNA Editing of ccmFc and atp9 Transcripts in the Moss Physcomitrella patens: First Complete Set of PPR Editing Factors in Plant Mitochondria

The moss Physcomitrella patens has 11 RNA editing sites in mitochondrial transcripts. We previously identified six DYW subclass pentatricopeptide repeat (PPR) proteins as RNA editing factors for nine out of 11 sites. In this study, we identified two novel DYW subclass PPR proteins, PpPPR_65 and PpPPR_98, as RNA editing factors. Disruption of the PpPPR_65 gene resulted in a complete loss of RNA editing at two neighboring sites, ccmFc-C103 and ccmFc-C122, in the mitochondrial ccmFc transcript. To confirm this result, we further generated PpPPR_65 knockdown (KD) mutants by an inducible RNA interference (RNAi) system. The generated RNAi lines displayed reduced levels of RNA editing at both ccmFc-C103 and ccmFc-C122 sites. Next, we characterized the function of PpPPR_98 by constructing a KD mutant of PpPPR_98 expression. The KD mutant showed a 30% reduction in the level of atp9-C92 editing. When PpPPR_98 cDNA was introduced into the KD mutant, RNA editing levels were restored to the wild-type level. This indicates that PpPPR_98 is an editing factor for the atp9-C92 site. The recombinant PpPPR_98 protein bound to the upstream sequence of the editing site that was created by splicing of atp9 transcript. This suggests that atp9 RNA editing occurs after splicing of atp9 transcript. Our present and previous data provide the first evidence that all 11 known editing events require at least eight DYW subclass PPR proteins in the moss mitochondria.

AtCTF7 is required for establishment of sister chromatid cohesion and association of cohesin with chromatin during meiosis in Arabidopsis

BackgroundThe establishment of sister chromatid cohesion followed by its controlled release at the metaphase to anaphase transition is necessary for faithful segregation of chromosomes in mitosis and meiosis. Cohesion is established by the action of Ctf7/Eco1 on the cohesin complex during DNA replication following loading of cohesin onto chromatin by the Scc2-Scc4 complex. Ctf7 is also required for sister chromatid cohesion during repair of DNA double strand breaks. Ctf7 contains an acetyltransferase domain and a zinc finger motif and acetylates conserved lysine residues in the Smc3 subunit of cohesin. In Arabidopsis CTF7 is encoded by a single gene and mutations in AtCTF7 cause embryo lethality indicating that the gene is essential.ResultsTo study the function of Ctf7 in plants and to determine its role in sister chromatid cohesion, we constructed a conditional allele of AtCTF7 in Arabidopsis using an inducible RNA interference (RNAi) strategy, so as to avoid the embryo lethality caused by mutations in AtCTF7. We found that induction of RNAi against AtCTF7 caused severe inhibition and defects in growth during vegetative and reproductive stages as well as sterility. AtCTF7-RNAi plants displayed chromosome fragmentation and loss of sister chromatid cohesion during meiosis. Immunostaining for the cohesion subunit AtSCC3 showed a marked reduction in association of cohesin with chromatin during meiosis in AtCTF7-RNAi plants.ConclusionsWe find that AtCTF7 is essential for sister chromatid cohesion during meiosis in Arabidopsis and is required for association of cohesin with chromatin in prophase of meiosis.

Doxycycline-Dependent Inducible and Reversible RNA Interference Mediated by a Single Lentivirus Vector

RNA interference has been applied to the development of a method of silencing genes of interest. Short hairpin RNA (shRNA) expression vectors are useful in driving gene-silencing. Standard shRNA vectors produce a knockdown phenotype soon after transduction. An alternative strategy for conditional gene knockdown would be useful to investigate gene functions in a time-dependent manner. In this study, we developed an inducible gene knockdown system based on lentivirus-mediated gene transfer. A single lentivirus vector capable of inducible expression of a designed microRNA-based shRNA was generated using a tetracycline-dependent transactivation system. The lentiviral vector facilitated doxycycline-dependent inducible knockdown specific to the target gene. Withdrawal of doxycycline after transient treatment resulted in complete recovery of target gene expressions to normal levels. Thus the single lentiviral vector developed in this study should be a powerful tool for doxycycline-dependent inducible and reversible RNA interference in molecular genetic studies.

RNAi Therapeutic Platforms for Lung Diseases

RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases.

Dicer-independent processing of short hairpin RNAs.

Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.

Longevity in the red flour beetle Tribolium castaneum is enhanced by broccoli and depends on nrf-2, jnk-1 and foxo-1 homologous genes

Diet is generally believed to affect the aging process. The effects of complex foods on life span can be investigated using simple models that produce rapid results and allow the identification of food-gene interactions. Here, we show that 1% lyophilized broccoli, added to flour as a dietary source, significantly increases the life span of the red flour beetle (Tribolium castaneum) under physiological conditions (32°C) and under heat stress (42°C). The beneficial effects of broccoli could also be reproduced by supplementing flour with the isothiocyanate sulforaphane at concentrations found in the broccoli-supplemented diet. We identified stress-resistant genes responsible for these effects on longevity by microinjecting pupae with double-stranded RNA to induce RNA interference (RNAi). The knockdown of transcripts encoding homologs of Nrf-2, Jnk-1 and Foxo-1 reduced the life span of beetles and abrogated the beneficial effects of broccoli, whereas the knockdown of Sirt-1 and Sirt-3 had no impact in either scenario. In conclusion, T. castaneum is a suitable model organism to investigate food-gene interactions that affect stress resistance and longevity, and RNAi can be used to identify functionally relevant genes. As a proof of principle, we have shown here that broccoli increases the longevity of beetles and mediates its effect through signaling pathways that include key stress-resistant factors such as Nrf-2, Jnk-1 and Foxo-1.

Intraperitoneal Administration of AAV9-shRNA Inhibits Target Gene Expression in the Dorsal Root Ganglia of Neonatal Mice

BackgroundThere is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA–expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA).ResultsIntraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA.ConclusionsAlthough we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.

Trachea-Derived Dpp Controls Adult Midgut Homeostasis in Drosophila

Homeostasis in adult tissues is maintained by resident stem cells and their progeny. Little is known about the regulation of tissue homeostasis by organ-organ interaction. Here we demonstrate that trachea-derived Decapentaplegic (Dpp), the main bone morphogenetic protein ligand in Drosophila, is essential for adult midgut homeostasis. We show that Dpp signaling is primarily activated in enterocytes (ECs). Depletion of Dpp signaling in ECs results in excess amounts of intestinal stem-cell-like cells and their progeny. Importantly, we find that Dpp is expressed specifically in tracheal cells that reach the intestinal cells through the visceral muscles. Depletion of dpp expression in tracheal cells phenocopies the Dpp loss-of-function defects in ECs. Our data demonstrate that the Drosophila trachea not only exchanges air for bodily needs but also produces a Dpp morphogen essential for neighboring tissue homeostasis. This work will provide important insights into the mechanisms of tissue homeostasis control by interorgan communication.

A Synthetic Lethal RNA Interference Screen Identifies Components of Genotoxic Stress Response As Targets for Anti-CCND1 Therapy in Mantle Cell Lymphoma.

AbstractAbstract 2430 Background.Almost all cases of mantle cell lymphoma (MCL) harbor the t(11:14) chromosomal translocation resulting in overexpression of the cell cycle regulatory protein cyclin D1 (CCND1), which promotes cell proliferation and poor survival. Targeting CCND1 in vitro and clinically, however, is not sufficient to cause tumor cell death, suggesting that additional mechanisms compensate for MCL growth and survival. Unraveling these additional signals will help identify novel targets for rational combination therapies in MCL. Previously, we developed a novel functional genomics tool using an inducible RNA interference (RNAi) library, which can simultaneously assess the role of thousands of genes in cell viability in tumor cell lines. Here we apply the inducible RNAi screen to identify synthetic lethal interactions with CCND1 in MCL. The screen uncovered several components of the DNA damage response as potential new combination targets for anti-CCND1 therapy in mantle cell lymphoma. Methods.The RNAi library was previously constructed using a retroviral vector that inducibly expresses small-hairpin RNA (shRNA). Each shRNA vector contains a unique 60-mer bar code that can hybridize to a corresponding complementary sequence spotted on a custom Agilent oligonucleotide microarray. To perform a synthetic lethal RNAi screen for CCND1, we first established a stable MCL line (UPN-1) that express an inducible CCND1 or control shRNA, transduced these lines with the pooled shRNA library, selected for transduced cells with puromycin, and induced shRNA expression for eight days. Genomic DNA containing bar code sequences was then amplified by PCR, fluorescently labeled, and hybridized onto microarrays. Each screen was repeated four times to enable statistical analysis. Candidate shRNAs obtained from the screen were validated for synergistic killing of MCL cells when combined with CCND1 knockdown. We evaluated genotoxic stress response triggered by DNA damage following CCND1 inactivation in MCL lines by Western blots. DNA damage and repair were assessed by comet assays and immuno-fluorescent staining of DNA repair proteins including phospho-H2AX, RAD51 and 53BP1. Results.The RNAi screen uncovers multiple shRNAs targeting RIPK1, RIPK3, NEMO, and TAK1, which sensitize MCL cells to CCND1 inhibition. RIPK1, NEMO, and TAK1 have been shown to play an essential role in cells undergoing genotoxic stress by linking DNA damage-induced ATM activation and NF-kB activity. We demonstrated that silencing CCND1 in the MCL cell lines UPN-1, JEKO-1, Z138, and Granta-519 up-regulates RIPK1 mRNA and protein expression, in addition to increased phosphorylation of DNA damage response proteins such as ATM, CHEK1/2 and H2AX. We observed a two-fold increase of DNA damage levels in CCND1 knockdown cells as assessed by comet assays. We also detected cell cycle-independent increase of DNA double strand break (DSB) foci in CCND1 knockdown cells by staining with fluorescently labeled anti-phospho-H2AX antibody.Knockdown of RIPK1 in MCL lines (UPN-1 and JEKO-1) resulted in apoptotic cell death and these RIPK1 shRNA-transduced cells are hypersensitive to irradiation and DNA damaging agents, indicating RIPK1 plays a protective role against DNA damage-induced apoptosis. The survival role of RIPK1 in MCL cells may correlate with the DNA repair function as demonstrated by the inability of RIPK1 knockdown cells to efficiently resolve etoposide-induced DNA DSB foci over time. Furthermore, we also found that RIPK1 knockdown cells failed to down-regulate the G2/M cell cycle checkpoint protein CDC25B and to up-regulate ATM phosphorylation and Ku86 protein expression in response to genotoxic stress. Blocking these RIPK1-dependent responses could sensitize MCL cells to CCND1 knockdown-induced DNA damage. Similar analyses of the other hits from the RNAi screen are on going. Conclusions.There are few viable treatment options for mantle cell lymphoma. We have identified the receptor interacting protein kinase 1 (RIPK1), of the DNA damage response pathway, as a potential therapeutic target whose downregulation sensitizes MCL cells to anti-CCND1 treatment, possibly by promoting insurmountable genotoxic stress. Successful implementation of our functional genetic screens for genes that sensitize MCL cells to anti-CCND1 treatment could define novel targets suitable for effective combination therapies. Disclosures:No relevant conflicts of interest to declare.

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.

Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.

RNA Interference Induction by Long Hairpin dsRNAs Expressed from Chromosomal DNA of Bombyx mori Cells

RNA Interference Induction by Long Hairpin dsRNAs Expressed from Chromosomal DNA of Bombyx mori Cells

Proteomics and Metabolomics of Arabidopsis Responses to Perturbation of Glucosinolate Biosynthesis

To understand plant molecular networks of glucosinolate metabolism, perturbation of aliphatic glucosinolate biosynthesis was established using inducible RNA interference (RNAi) in Arabidopsis. Two RNAi lines were chosen for examining global protein and metabolite changes using complementary proteomics and metabolomics approaches. Proteins involved in metabolism including photosynthesis and hormone metabolism, protein binding, energy, stress, and defense showed marked responses to glucosinolate perturbation. In parallel, metabolomics revealed major changes in the levels of amino acids, carbohydrates, peptides, and hormones. The metabolomics data were correlated with the proteomics results and revealed intimate molecular connections between cellular pathways/processes and glucosinolate metabolism. This study has provided an unprecedented view of the molecular networks of glucosinolate metabolism and laid a foundation towards rationale glucosinolate engineering for enhanced defense and quality.

Identification of chemosensory receptor genes in Manduca sexta and knockdown by RNA interference

BackgroundInsects detect environmental chemicals via a large and rapidly evolving family of chemosensory receptor proteins. Although our understanding of the molecular genetic basis for Drosophila chemoreception has increased enormously in the last decade, similar understanding in other insects remains limited. The tobacco hornworm, Manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. It is also a major agricultural pest on solanaceous crops. However, little sequence information and lack of genetic tools has prevented molecular genetic analysis in this species. The ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in M. sexta would be greatly beneficial.ResultsHere, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. We also used dsRNA feeding as a method to induce RNA interference in larval chemosensory tissues.ConclusionsWe report identification of new chemosensory receptor genes including 17 novel odorant receptors and one novel gustatory receptor. Further, we demonstrate that systemic RNA interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. Together, our results further the development of M. sexta as a model for functional analysis of insect chemosensation.

Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC

The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy.

Antiviral Effects of Small Interfering RNA Simultaneously Inducing RNA Interference and Type 1 Interferon in Coxsackievirus Myocarditis

Antiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5'-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5' end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases.

An Inducible RNA Interference System in Physcomitrella patens Reveals a Dominant Role of Augmin in Phragmoplast Microtubule Generation

Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.

Oxidative stress responses in Caenorhabditis elegans with reduced mrck‐1 expression

MRCK‐1 (myotonic dystrophy kinase‐related Cdc42 binding kinase) acts downstream of the Rho GTPase CDC‐42 to determine cell polarity and is critical for C. elegans embryonic development. Given the conserved GXXXXCGKTC redox‐sensitive motif in the phosphoryl‐binding loop of CDC‐42, a role for MRCK‐1 in cellular responses to reactive oxygen species (ROS) is being explored. An RNA interference (RNAi) construct of 190 bp targeting exon 5 of the mrck‐1 gene on chromosome V was cloned into the RNAi expression vector pL4440 to enable production of double‐stranded RNA. Mrck‐1 RNAi induction in worm strains with oxidative stress responsive green fluorescent protein (GFP)‐ promoter fusions, including hsp‐16.2, sod‐3 and gst‐4, enables the measurement of oxidative stress induced GFP expression. Ongoing experiments include exposing worm strains to temperature, ultra‐violet (UV) light and chemical stressors to elucidate a possible phenotype related to diminished MRCK‐1 function under oxidative stress conditions. This work was supported by the Henson Undergraduate Research Grant program.

Development of RNAi tools for studying Leishmania (Viannia) and their application to the study of the Leishmania flagellum

RNA interference (RNAi) is a powerful tool for genetic analysis. While many Leishmania species lost the RNAi pathway deep in evolution, species within the Leishmania subgenus Viannia retain RNAi pathway genes. Correspondingly, introduction of constructs expressing dsRNA in L. (V.) braziliensis resulted in substantial down regulation of all target mRNAs tested. We are optimizing and developing protocols that will facilitate the use of RNAi and applied them to the study of the Leishmania flagellum. We developed vectors allowing rapid generation of stem‐loop (StL) constructs using Gateway (Invitrogen®) technology, and are working on methods for inducible RNAi and maximization of RNAi activity. The Leishmania promastigote flagellum contains paraflagellar rod (PFR); RNAi of genes encoding two major proteins PFR1 or PFR2 yielded parasites lacking the PFR and exhibiting motility defects. We probed the role of the intraflagellar transport pathway (IFT) by targeting four genes implicated in anterograde or retrograde trafficking. We were unable to recover transfectants bearing the respective StL constructs. The same StL constructs were transfected into an Argonaute null mutant which lacks a functional RNAi pathway. All IFT StL constructs were now successfully introduced into the Argonaute mutant. These data suggest that the IFT pathway is essential for the viability of L. braziliensis promastigotes.