Searching for novel cell cycle regulators in Trypanosoma brucei with an RNA interference screen

<b>Background</b><br /> The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators.<p></p> <b>Results</b><br /> Procyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators.<p></p> <b>Conclusion</b><br /> Our study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.<p></p&gt

Biased Genetic Screen Identifies Novel Genes Involved in Antiviral Defense

ABSTRACT RNA interference (RNAi) mediates potent antiviral response across kingdoms. In Caenorhabditis elegans nematodes, antiviral RNAi requires a virus sensor that is conserved in mammals and is amplified by secondary small interfering RNAs that are produced in a Dicer-independent manner. To better understand worm antiviral RNAi, I carried out a biased genetic screen, aiming to identify novel antiviral RNAi genes. To speed up the gene discovery process, the reporter worms used for this genetic screen were engineered to contain extra copies of 4 known antiviral RNAi genes. Therefore, genetic alleles derived from these 4 genes will be automatically rejected during screening process. Since a viral replicon transgene was used as reporter for loss of antiviral RNAi my genetic screen was expected to identify genes required for antiviral RNAi but not artificial double-stranded RNA (dsRNA) triggered RNAi. My genetic screen identified altogether 25 candidate alleles that appear to have derived from 13 candidate genes. Through genetic complementation tests and coding region sequencing I confirmed that 2 of the candidate genes are known antiviral RNAi genes rde-3 and rrf-1. Of the rest 11 candidate genes, 9 were found not to be required for classical RNAi. Interestingly, Orsay virus infection assay further suggested that 7 of these 9 candidate genes are dispensable for antiviral RNAi against Orsay virus. Since Orsay virus specifically infects intestine cells which have much weaker antiviral activity I believe that these 7 genes mainly function in non-intestine cells. Through whole genome sequencing I identified 2 of the candidate genes as rsd-6 and mut-16, 2 known genes required for artificial dsRNA triggered RNAi. Currently, rsd-6 and mut-16 are not known to play a role in antiviral RNAi. Thus, my genetic screen, for the first time, identified both rsd-6 and mut-16 as key components for antiviral RNAi

Transgene-assisted genetic screen identifies rsd-6 and novel genes as key components of antiviral RNA interference in Caenorhabditis elegans

© 2018 American Society for Microbiology. RNA interference (RNAi) is a widespread antiviral mechanism triggered by virus-produced double-stranded RNAs (dsRNAs). In Caenorhabditis elegans, antiviral RNAi involves a RIG-I-like RNA helicase, termed DRH-1 (dicer related RNA helicase 1), that is not required for classical RNAi triggered by artificial dsRNA. Currently, whether antiviral RNAi in C. elegans involves novel factors that are dispensable for classical RNAi remains an open question. To address this question, we designed and carried out a genetic screen that aims to identify novel genes involved in worm antiviral RNAi. By introducing extra copies of known antiviral RNAi genes into the reporter worms, we managed to reject alleles derived from 4 known antiviral RNAi genes, including the DRH-1 coding gene, during the screen. Our genetic screen altogether identified 25 alleles, which were assigned to 11 candidate genes and 2 known antiviral RNAi genes through genetic complementation tests. Using a mapping-by-sequencing strategy, we identified one of the candidate genes as rsd-6, a gene that helps maintain genome integrity through an endogenous gene-silencing pathway but was not known to be required for antiviral RNAi. More importantly, we found that two of the candidate genes are required for antiviral RNAi targeting Orsay virus, a natural viral pathogen of C. elegans, but dispensable for classical RNAi. Since drh-1 is so far the only antiviral RNAi gene not required for classical RNAi, we believe that our genetic screen led to identification of novel worm genes that may target virus-specific features to function in RNAi

Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway

BACKGROUND: Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. RESULTS: Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. CONCLUSIONS: Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens

Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs.

Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from human RIG-I can substitute for the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans, suggesting an analogous role for DRH-1 as an intracellular dsRNA sensor to initiate antiviral RNAi. Here, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in C. elegans Characterization of four distinct drh-1 mutants obtained from the screen revealed that DRH-1 did not function to initiate antiviral RNAi. We show that DRH-1 acted in a downstream step to enhance Dicer-dependent biogenesis of viral siRNAs in C. elegans As mammals produce Dicer-dependent viral siRNAs to target RNA viruses, our findings suggest a possible role for mammalian RLRs and interferon signaling in the biogenesis of viral siRNAs

Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis

Background: RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi is associated with off-target effects (OTEs), including microRNA (miRNA)-like OTEs. The contribution of reagent-specific OTEs to RNAi screen data sets can be significant. In addition, the post-screen validation process is time and labor intensive. Thus, the availability of robust approaches to identify candidate off-targeted transcripts would be beneficial. Results: Significant efforts have been made to eliminate false positive results attributable to sequence-specific OTEs associated with RNAi. These approaches have included improved algorithms for RNAi reagent design, incorporation of chemical modifications into siRNAs, and the use of various bioinformatics strategies to identify possible OTEs in screen results. Genome-wide Enrichment of Seed Sequence matches (GESS) was developed to identify potential off-targeted transcripts in large-scale screen data by seed-region analysis. Here, we introduce a user-friendly web application that provides researchers a relatively quick and easy way to perform GESS analysis on data from human or mouse cell-based screens using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), as well as for Drosophila screens using shRNAs. Online GESS relies on up-to-date transcript sequence annotations for human and mouse genes extracted from NCBI Reference Sequence (RefSeq) and Drosophila genes from FlyBase. The tool also accommodates analysis with user-provided reference sequence files. Conclusion: Online GESS provides a straightforward user interface for genome-wide seed region analysis for human, mouse and Drosophila RNAi screen data. With the tool, users can either use a built-in database or provide a database of transcripts for analysis. This makes it possible to analyze RNAi data from any organism for which the user can provide transcript sequences

Biosynthesis and enzymology of the Caenorhabditis elegans cuticle: identification and characterization of a novel serine protease inhibitor.

The nematode Caenorhabditis elegans represents an excellent model in which to examine nematode gene expression and function. A completed genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of nematode genes. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. Many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the previously uncharacterized bli-5 locus in C. elegans. The mutant phenotype, spatial expression pattern and the over-expression phenotype of the BLI-5 protease inhibitor and their relevance to collagen biosynthesis are discussed

Protein interaction network topology uncovers melanogenesis regulatory network components within functional genomics datasets

<p>Abstract</p> <p>Background</p> <p>RNA-mediated interference (RNAi)-based functional genomics is a systems-level approach to identify novel genes that control biological phenotypes. Existing computational approaches can identify individual genes from RNAi datasets that regulate a given biological process. However, currently available methods cannot identify which RNAi screen "hits" are novel components of well-characterized biological pathways known to regulate the interrogated phenotype. In this study, we describe a method to identify genes from RNAi datasets that are novel components of known biological pathways. We experimentally validate our approach in the context of a recently completed RNAi screen to identify novel regulators of melanogenesis.</p> <p>Results</p> <p>In this study, we utilize a PPI network topology-based approach to identify targets within our RNAi dataset that may be components of known melanogenesis regulatory pathways. Our computational approach identifies a set of screen targets that cluster topologically in a human PPI network with the known pigment regulator Endothelin receptor type B (EDNRB). Validation studies reveal that these genes impact pigment production and EDNRB signaling in pigmented melanoma cells (MNT-1) and normal melanocytes.</p> <p>Conclusions</p> <p>We present an approach that identifies novel components of well-characterized biological pathways from functional genomics datasets that could not have been identified by existing statistical and computational approaches.</p

Large-Scale RNAi Screen to Identify Genes Involved in Axon Guidance in Caenorhabditis elegans

Diese Arbeit wurde durchgeführt, um Gene, die axonale Wegfindung im Nervensystem von Caenorhabditis elegans steuern, zu identifizieren. C. elegans stellt aufgrund seiner einzigartigen physiologischen Eigenschaften ein gutes Modellsystem für das Studium einer Vielzahl biologischer Prozesse dar. Das Nervensystem von C. elegans ist einfach strukturiert und umfasst 302 Neuronen. Diese Neuronen bilden stereotype Netzwerke mit ihren anterior-posterior und dorsal-ventral verlaufenden axonalen Fortsätzen aus. In dieser Arbeit nutzen wir die kürzlich beschriebene Methode der RNA Interferenz (RNAi) im Wurm zur Identifikation von neuen Genen der axonalen Wegfindung. Allerdings ist das Nervensystem von C. elegans resistent gegen systemische RNAi und Transport von doppelsträngigen RNA Molekülen in benachbarte nicht-neuronale Zellen veranlasst keine neuronale RNAi. Aus diesem Grund begannen wir mit der Identifizierung von C. elegans Mutanten, die eine erhöhte Empfindlichkeit für RNAi im Nervensystem aufweisen. Eine chemische Mutagenese wurde durchgeführt, gefolgt von einem Screen nach Mutanten mit effizienter RNAi im Nervensystem. Eine der Mutanten (nre-1, für neuronal RNAi efficient) zeigte starke Suppression der Genexpression im Nervensystem nach RNAi durch Füttern. Wir nutzten die nre-1 supersensitive Mutante für einen revers genetischen Screen zur Idenfizierung von Genen der axonalen Wegfindung in C. elegans. Um die Fortsätze der Nervenzellen sichtbar zu machen, wurde ein transgener Stamm im nre-1 Hintergrund erzeugt, in dem ein Teil der Inter- und Motoneurone durch gelb fluoreszierendes Protein (YFP) markiert ist. Dieser Stamm wurde für einen Screen von 2416 Genen auf Chromosom I verwendet. Dazu wurde eine library von Bakterienklonen, die einem bestimmten Gen entsprechende dsRNA exprimieren, an C. elegans verfüttert. Der Screen führte zur Identifizierung von 57 Kandidatengenen, die penetrante axonale Wegfindungsdefekte in Motoneuron-Kommissuren und Axonen des Ventralstrangs in C. elegans zur Folge haben. Die identifizierten Gene sind involviert in eine Vielzahl von biologischen Prozessen wie DNA-Metabolismus, Translation, Transkription und Signaltransduktion. Einige kodieren für Zelloberflächenmoleküle und Zytoskelettkomponenten. Zusätzlich zu neuen Genen konnten im Screen Gene identifiziert werden, die in andere biologische Prozesse involviert sind, aber bis jetzt nicht mit axonaler Wegfindung in Verbindung gebracht wurden. Beispielsweise führt Verlust von pry-1, einem Axin Homolog in C. elegans, zu axonalen Defekten. Axin ist ein assoziierter Faktor des ß-Catenin Komplexes und damit ein negativer Regulator in Wnt vermittelter Signaltransduktion. Weitere Studien an anderen, in diesem Screen identifizierten Kandidatengenen wie z.B. neuen Rezeptoren, Signalmolekülen, Kinasen und Transkriptionsfaktoren können uns in Zukunft einen weiteren Einblick in die molekularen Mechanismen der axonalen Wegfindung geben