title: AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.
abstract: Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.
authors.affiliation: Drosophila Genetics and Epigenetics, Laboratory of Developmental Biology, CNRS UMR7622, Université Pierre et Marie Curie, Paris, France.
authors.forename: Clément
authors.initials: C
authors.lastname: Carré
authors.forename: Caroline
authors.initials: C
authors.lastname: Jacquier
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Bougé
authors.forename: Fabrice
authors.initials: F
authors.lastname: de Chaumont
authors.forename: Corinne
authors.initials: C
authors.lastname: Besnard-Guerin
authors.forename: Hélène
authors.initials: H
authors.lastname: Thomassin
authors.forename: Josette
authors.initials: J
authors.lastname: Pidoux
authors.forename: Bruno
authors.initials: B
authors.lastname: Da Silva
authors.forename: Eleftheria
authors.initials: E
authors.lastname: Chalatsi
authors.forename: Sarah
authors.initials: S
authors.lastname: Zahra
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Olivo-Marin
authors.forename: Hélène
authors.initials: H
authors.lastname: Munier-Lehmann
authors.forename: Christophe
authors.initials: C
authors.lastname: Antoniewski
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics; D015374:Biosensing Techniques; D002460:Cell Line; D004330:Drosophila; D029721:Drosophila Proteins / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034622:RNA Interference
chemical_list: C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D035683:MicroRNAs; D049452:Green Fluorescent Proteins
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