Wednesday, December 13, 2017

DRSC screen contributes to research report on signaling and lifespan

Sung EJ, Ryuda M, Matsumoto H, Uryu O, Ochiai M, Cook ME, Yi NY, Wang H, Putney JW, Bird GS, Shears SB, Hayakawa Y. Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress. Proc Natl Acad Sci U S A. 2017 Dec 11. PMID: 29229844.

From the abstract: "A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. ... We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity."

Monday, December 11, 2017

RNAi and the sterile insect technique

Darrington M, Dalmay T, Morrison NI, Chapman T. Implementing the sterile insect technique with RNA interference - a review. Entomol Exp Appl. 2017 Sep;164(3):155-175. PMID: 29200471; PMCID: PMC5697603.

From the abstract: "We review RNA interference (RNAi) of insect pests and its potential for implementing sterile insect technique (SIT)-related control. The molecular mechanisms that support RNAi in pest species are reviewed in detail, drawing on literature from a range of species including Drosophila melanogaster Meigen and Homo sapiens L. ... RNAi represents a plausible, non-GM system for targeting populations of insects for control purposes, if RNAi effector molecules can be delivered environmentally (eRNAi). We consider studies of eRNAi from across several insect orders and review to what extent taxonomy, genetics, and differing methods of double-stranded (ds) RNA synthesis and delivery can influence the efficiency of gene knockdown. ..."

Monday, December 4, 2017

New report on qPCR design for RNAi knockdown effectiveness testing

Mainland RL, Lyons TA, Ruth MM, Kramer JM. Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines. BMC Res Notes. 2017 Nov 29;10(1):647. PMID: 29187229.

From the abstract: "... the strongest gene knockdown was detected when qPCR was performed using 5' primer sets in combination with mRNA-derived cDNA. Our results indicate that detection of undegraded mRNA cleavage fragments can result in underestimation of true knockdown efficiency for a RNAi construct. ..."

Wednesday, July 19, 2017

Short vs. long hairpins in fly RNAi

Bartoletti R, Capozzoli B, Moore J, Moran J, Shrawder B, Vivekanand P. Short hairpin RNA is more effective than long hairpin RNA in eliciting pointed loss-of-function phenotypes in Drosophila. Genesis. 2017 Jul;55(7). PMID: 28464429.

From the abstract: "Pointed (Pnt) is a transcriptional activator ... As mutations in pnt perturb the development of several tissues, we wanted to examine the effect and efficacy of using RNAi to target Pnt. We have expressed pnt RNAi in the eyes, oocyte, and heart cells using three different RNAi lines: Valium20, Valium10, and VDRC. Valium20 is distinct since it generates a short hairpin RNA (shRNA), while Valium10 and VDRC produce long hairpin dsRNA. We found that for each tissue examined Valium20 exhibited the strongest phenotype while the Valium10 and VDRC lines produced varying levels of severity and that the long hairpin RNA produced by the Valium10 and VDRC lines are unable to effectively knockdown pnt in embryonic tissues."

Monday, July 10, 2017

Methods papers on in vivo RNAi -- stem cell and germline studies

New methods papers on in vivo fly RNAi:

Riechmann V. In vivo RNAi in the Drosophila Follicular Epithelium: Analysis of Stem Cell Maintenance, Proliferation, and Differentiation. Methods Mol Biol. 2017;1622:185-206. doi: 10.1007/978-1-4939-7108-4_14. PubMed PMID: 28674810.

Blake AJ, Finger DS, Hardy VL, Ables ET. RNAi-Based Techniques for the Analysis of Gene Function in Drosophila Germline Stem Cells. Methods Mol Biol. 2017;1622:161-184. doi: 10.1007/978-1-4939-7108-4_13. PubMed PMID: 28674809.

Monday, June 19, 2017

Kinome RNAi screen reported in G3

Parsons LM, Grzeschik NA, Amaratunga K, Burke P, Quinn LM, Richardson HE. A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC and Crb Cell Polarity Genes in Epithelial Tissues. G3 (Bethesda). 2017 Jun 13. PMID: 28611255.

From the abstract: "... To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila 'cell polarity' eye and wing phenotypes. ... We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, JNK, and Notch signalling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways, not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras and lipid/Phospho-inositol-3-kinase (PI3K) signalling pathways. Additionally, we demonstrated that the 'nutrient sensing' kinases, Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. ..."

Monday, June 12, 2017

in vivo RNAi screen explores MANF ortholog DmManf

Lindström R, Lindholm P, Palgi M, Saarma M, Heino TI. In vivo screening reveals interactions between Drosophila Manf and genes involved in the mitochondria and the ubiquinone synthesis pathway. BMC Genet. 2017 Jun 2;18(1):52. PMID: 28578657; PMCID: PMC5455201.

From the abstract: "Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF) form an evolutionarily conserved family of neurotrophic factors. Orthologues for MANF/CDNF are the only neurotrophic factors as yet identified in invertebrates with conserved amino acid sequence. ... Here, we have employed the Drosophila genetics to screen for potential interaction partners of Drosophila Manf (DmManf) in vivo. ... In addition to the previously known functions of MANF/CDNF protein family, DmManf also interacts with mitochondria-related genes. Our data supports the functional importance of these evolutionarily significant proteins and provides new insights for the future studies."

Wednesday, May 17, 2017

RNAi screen focused on chaperone function in the fly eye

Raut S, Mallik B, Parichha A, V A, Sahi C, Kumar V. RNAi-Mediated Reverse Genetic Screen Identified Drosophila Chaperones Regulating Eye and Neuromuscular Junction Morphology. G3 (Bethesda). 2017 May 12. pii: g3.117.041632. PMID: 28500055.

From the abstract: "Accumulation of toxic proteins in neurons have been linked with the onset of neurodegenerative diseases ... Molecular chaperones help protein folding and resolubilization of unfolded proteins thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance largely remains unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C-Gal4 mediated RNAi knockdown revealed that about 50% of the chaperones are essential in Drosophila[.] Knocking down these genes in eyes revealed that about 30% of the essential chaperones are crucial for eye development. ..."

Monday, February 13, 2017

Genome-wide fly cell RNAi screen related to Wolbachia infection

White PM, Serbus LR, Debec A, Codina A, Bray W, Guichet A, Lokey RS, Sullivan W. Reliance of Wolbachia on High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen of Drosophila Cells. Genetics. 2017 Feb 3. pii: genetics.116.198903. PMID: 28159754.

From the abstract: "Wolbachia are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a Wolbachia-infected Drosophila cell line and genome-wide RNAi screening to identify host factors that influence Wolbachia titer. .. we identified 36 candidate genes that dramatically reduced Wolbachia titer and 41 that increased Wolbachia titer. ... knockdown of 7 genes in the host ubiquitin and proteolysis pathways significantly reduced Wolbachia titer. To test the in vivo relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of Wolbachia in the Drosophila oocyte ... Given Wolbachia lack essential amino acid biosynthetic pathways, the reliance of Wolbachia on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning Wolbachia with amino acids. In addition, the reliance of Wolbachia on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for Wolbachia's potent ability to prevent RNA virus replication."

Tuesday, January 24, 2017

RNAi and proteomics study identifies glial genes required for behavior

Ng FS, Sengupta S, Huang Y, Yu AM, You S, Roberts MA, Iyer LK, Yang Y, Jackson FR. TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior. Front Mol Neurosci. 2016 Dec 22;9:146. PMID: 28066175; PMCID: PMC5177635.

From the abstract: "... Glial astrocytes and glia-neuron signaling ... have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens ... Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). ... These results strongly support the idea that glia-neuron communication is vital for adult behavior."