Van den Brande S, Gijbels M, Wynant N, Santos D, Mingels L, Gansemans Y, Van Nieuwerburgh F, Vanden Broeck J. The presence of extracellular microRNAs in the media of cultured Drosophila cells. Sci Rep. 2018 Nov 23;8(1):17312. PMID: 30470777.
Abstract: "While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. In the present study, we demonstrate the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed our qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals."
Monday, November 26, 2018
Monday, November 19, 2018
RNAi screen used to identify host cell factors required for baculovirus entry
Hodgson JJ, Buchon N, Blissard GW. Identification of insect genes involved in baculovirus AcMNPV entry into insect cells. Virology. 2018 Nov 13;527:1-11. doi: 10.1016/j.virol.2018.10.022. PMID: 30445201.
Abstract: "The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a model enveloped DNA virus that infects and replicates in lepidopteran insect cells, and can efficiently enter a wide variety of non-host cells. Budded virions of AcMNPV enter cells by endocytosis and traffic to the nucleus where the virus initiates gene expression and genome replication. While trafficking of nucleocapsids by actin propulsion has been studied in detail, other important components of trafficking during entry remain poorly understood. We used a recombinant AcMNPV virus expressing an EGFP reporter in combination with an RNAi screen in Drosophila DL1 cells, to identify host proteins involved in AcMNPV entry. The RNAi screen targeted 86 genes involved in vesicular trafficking, including genes coding for VPS and ESCRT proteins, Rab GTPases, Exocyst proteins, and Clathrin adaptor proteins. We identified 24 genes required for efficient virus entry and reporter expression, and 4 genes that appear to restrict virus entry. "
Abstract: "The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a model enveloped DNA virus that infects and replicates in lepidopteran insect cells, and can efficiently enter a wide variety of non-host cells. Budded virions of AcMNPV enter cells by endocytosis and traffic to the nucleus where the virus initiates gene expression and genome replication. While trafficking of nucleocapsids by actin propulsion has been studied in detail, other important components of trafficking during entry remain poorly understood. We used a recombinant AcMNPV virus expressing an EGFP reporter in combination with an RNAi screen in Drosophila DL1 cells, to identify host proteins involved in AcMNPV entry. The RNAi screen targeted 86 genes involved in vesicular trafficking, including genes coding for VPS and ESCRT proteins, Rab GTPases, Exocyst proteins, and Clathrin adaptor proteins. We identified 24 genes required for efficient virus entry and reporter expression, and 4 genes that appear to restrict virus entry. "
Genome-wide screen for factors related to Wolbachia interaction with dipteran host cells
Grobler Y, Yun CY, Kahler DJ, Bergman CM, Lee H, Oliver B, Lehmann R. Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation. PLoS Pathog. 2018 Nov 13;14(11):e1007445. doi: 10.1371/journal.ppat.1007445. PMID: 30422992.
From the abstract: "Wolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. ... We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. ... This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship."
From the abstract: "Wolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. ... We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. ... This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship."
Thursday, November 15, 2018
Thinking about size: in vivo RNAi screen for neuronal genes related to energy homeostasis
Trinh I, Gluscencova OB, Boulianne GL. An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion. Mol Metab. 2018 Oct 19. pii: S2212-8778(18)30828-7. doi:10.1016/j.molmet.2018.10.006. PMID: 30389349.
From the abstract: "... Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, to identify genes/pathways that function in the nervous system to regulate energy balance. ... We performed an in vivo RNAi screen in Drosophila neurons and assayed for obese or lean phenotypes by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects. ... We identified 24 genes including 20 genes that have not been previously associated with energy homeostasis. One identified hit, Diacylglycerol kinase (Dgk), has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. ... Altogether, we have identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila."
From the abstract: "... Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, to identify genes/pathways that function in the nervous system to regulate energy balance. ... We performed an in vivo RNAi screen in Drosophila neurons and assayed for obese or lean phenotypes by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects. ... We identified 24 genes including 20 genes that have not been previously associated with energy homeostasis. One identified hit, Diacylglycerol kinase (Dgk), has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. ... Altogether, we have identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila."
Where am I? in vivo RNAi screen looks at larval chordotonal organs
Hassan A, Timerman Y, Hamdan R, Sela N, Avetisyan A, Halachmi N, Salzberg A. An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs. G3 (Bethesda). 2018 May 31;8(6):1871-1884. doi: 10.1534/g3.118.200218. PubMed PMID: 29678948; PubMed Central PMCID: PMC5982817.
From the abstract: "The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. ... Here we describe an RNAi screen that focused on the ChO's accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The 'cell elongation' group included the transcription factors Delilah and Stripe ... Other genes found to affect the pattern of ChO cell elongation ... represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties."
From the abstract: "The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. ... Here we describe an RNAi screen that focused on the ChO's accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The 'cell elongation' group included the transcription factors Delilah and Stripe ... Other genes found to affect the pattern of ChO cell elongation ... represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties."
Subscribe to:
Posts (Atom)
Posts
