Special Issue "RNA Interference"

Guest Editor
Prof. Dr. Jeremy C. Simpson
University College Dublin, School of Biology and Environmental Science, Science Center - West, Belfield, Dublin 4, Ireland
Website: http://www.ucd.ie/bioenvsci/ourstaff/academic/simpsonjeremy/
E-Mail: jeremy.simpson@ucd.ie

Submission

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. Papers will be published continuously (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are refereed through a peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Genes is an international peer-reviewed Open Access quarterly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 300 CHF (Swiss Francs). English correction and/or formatting fees of 250 CHF (Swiss Francs) will be charged in certain cases for those articles accepted for publication that require extensive additional formatting and/or English corrections.

• RNAi
• siRNA
• shRNA
• gene downregulation
• functional genomics
• cellular assays
• RNA therapeutics

Type of Paper: Article
Title: Intestine-specific RNAi in the C. elegans embryo reveals diffusion of effector molecules into other tissues
Authors: Tobias Wiesenfahrt ^1,* and Olaf Bossinger ^2,*
Affiliations: ¹ Department of Biochemistry and Molecular Biology, University of Calgary / 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1; E-Mail: twiesenf@ucalgary.ca; ² Molecular and Cellular Anatomy, Medical School, RWTH Aachen University / Wendlingweg 2, D-52074 Germany; E-Mails: olaf.bossinger@rwth-aachen.de
Abstract: RNAi (RNA-mediated interference) is an effective method to specifically down regulate gene function using double stranded RNA (dsRNA) to degrade the complementary mRNA. In C. elegans this effect is systemic. To study gene function in a tissue-specific manner we generated a transgenic strain, OLB11, that allows RNAi only in the intestine. We used an RNAi insensitive rde-1(ne219) mutant and expressed a wild-type copy of the rde-1 gene under control of the intestine specific elt-2 promoter. RNAi via feeding allows down regulation of gene function in the intestines of larvae and adults. To knock down gene function during embryonic development dsRNA has to be injected into the gonads of adult worms. In OLB11, RNAi against epithelial genes (let-413, dlg-1 and ajm-1) leads to knockdowns only in intestinal cells while gene functions in other epithelia seem unperturbed. RNAi against early maternally expressed genes did not induce any phenotypes in OLB11, probably due to stable maternal protein, as demonstrated by immunostaining against PAR-3. In contrast, intestine-specific RNAi against cyclin B3 (cyb-3), which is quickly synthesized and degraded during the cell-cycle, surprisingly leads to defects in non-intestinal cells also. As a possible mechanism the diffusion of effector molecules in the C. elegans embryo is discussed.

Type of Paper: Article
Title: siRNAs Trigger Efficient Silencing of a Parasitism Gene in Plant Parasitic Root-knot Nematodes
Authors: M.J. Arguel ^1,2,3, M.Jaouannet ^1,2,3, M. Magliano ^1,2,3, P. Abad ^1,2,3,* and M.N. Rosso ^1,2,3,4
Affiliations: ¹ INRA, UMR 1355 Institut Sophia Agrobiotech, Interactions Plantes-Nematodes, F-06903 Sophia Antipolis, France; E-Mail: abad@sophia.inra.fr
² Université de Nice Sophia Antipolis, F-06903 Sophia Antipolis, France
³ CNRS, UMR 7254 Institut Sophia Agrobiotech, Interactions Plantes-Nematodes, F-06903 Sophia Antipolis, France
⁴ INRA, UMR 1163 Biotechnologie des Champignons Filamenteux, Université Aix-Marseille, F-13288 Marseille, France
Abstract: Root-Knot Nematodes (RKN) are major polyphagous and cosmopolitan crop pests but the use of nematicides is strictly restricted due to environment and health concerns. The development of specific and environment friendly pest management strategies is expected from newly acquired RKN genomic data. Specific gene silencing by RNA interference (RNAi) is a powerful tool for reverse genomics that could allow the identification of parasitism genes essential for the success of infection. Regrettably, obtaining transgenic plants that induce gene-specific RNAi is time consuming and inappropriate for large scale screenings. On another hand, RNAi by soaking pre-parasitic juveniles with long double stranded RNA (dsRNA) is expensive and suspected to induce off-target effects. Interestingly, silencing of gene expressed in the amphids and in neurons has been shown by soaking nematodes with small interfering RNAs (siRNAs). In this study, we observed the transport of a labeled 21 base pair long dsRNA oligomer in the inner tissues of the nematode and we showed that neurotransmitters such as serotonin or glutamate in the soaking medium accelerated the accumulation of the oligomer into the esophagus, amphidial pounches and related neurons in the nematode. We optimized the siRNA strategy to knock-down the calreticulin gene Mi-CRT, a parasitism gene expressed in the esophageal glands of pre-parasitic and parasitic stages from the RKN Meloidogyne incognita. Efficient gene knock-down was demonstrated by quantification of Mi-CRT transcripts after treatment and infection assays. Our study provides the first evidence of siRNA-mediated silencing of a nematode parasitism gene. However, knock-down of Mi-CRT expression could not be detected in the parasitic stages, indicating that the silencing effect was not persistent after plant infection and that siRNA-mediated RNAi is best suited for functional analysis of genes involved in pre-parasitic stages or in the early steps of infection.

Type of Paper: Article
Title: Regulation of Fibroblast Growth Factor-2 Expression and Cell Cycle Progression by an Endogenous Antisense RNA
Authors: Mark Baguma-Nibasheka, Leigh Ann MacFarlane and Paul R. Murphy
Affiliation: Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada; E-Mail: prmurphy@dal.ca
Abstract: Basic fibroblast growth factor (FGF-2) is a potent wide-spectrum mitogen whose overexpression is associated with immortalization and unregulated cell proliferation in many tumors. The FGF-2 gene locus is bi-directionally transcribed to produce FGF-2 mRNA from the “sense” strand and a cis-antisense RNA (NUDT6) from the NUDT6 gene on the “antisense” strand. The NUDT6 gene encodes a nudix motif protein of unknown function, while its mRNA has been implicated in the post-transcriptional regulation of FGF-2 expression. FGF-2 and NUDT6 are co-expressed in rat C6 glioma cells, and ectopic overexpression of NUDT6 suppresses cellular FGF-2 accumulation and cell cycle progression. However, the role of the endogenous antisense RNA in regulation of FGF-2 is unclear. In the present study, we employed siRNA-mediated gene knockdown to examine the role of the endogenous NUDT6 RNA in regulation of FGF-2 expression and cell cycle progression. Knockdown of either FGF-2 or NUDT6 mRNA was accompanied by a significant (>3 fold) increase in the complementary partner RNA. Similar reciprocal effects were observed at the protein level, indicating that these two transcripts are mutually regulatory. Remarkably knockdown of either FGF-2 or NUDT6 significantly reduced cell proliferation and inhibited S-phase re-entry following serum deprivation, implicating both FGF-2 and NUDT6 in the regulation of cell transformation and cell cycle progression.

Type of Paper: Review
Title: Combinatorial Use of RNAi with Mutations and Drugs and Examination of Multiple Subcellular Phenotypes to Identify Regulators of Protein Degradation in C. Elegans Muscle
Author: Nathaniel J. Szewczyk
Affiliation: MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Royal Derby Hospital, DE22 3DT, 01332 724615, UK; E-Mail: Nathaniel.Szewczyk@nottingham.ac.uk
Abstract: RNAi is a convenient, widely used  tool for screening for genes of interest. We have recently used this technology to screen 800 candidate genes for potential roles in regulating muscle protein degradation in vivo. To maximize confidence and assess reproducibility, we have only used previously validated RNAi constructs and have included time courses and replicates. To maximize mechanistic understanding, we have examined multiple sub-cellular phenotypes in multiple compartments in muscle. We have also tested knockdowns of putative regulators of degradation in the context of mutations or drugs that were previously shown to inhibit protein degradation by diverse mechanisms. Here we discuss how assaying multiple phenotypes, multiplexing RNAi screens with use of mutations and drugs, and use of bioinformatics can provide more data on rates of potential false positives and negatives as well as more mechanistic insight than simple RNAi screening.