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AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.

Carré C, Jacquier C, Bougé AL, de Chaumont F, Besnard-Guerin C, Thomassin H, Pidoux J, Da Silva B, Chalatsi E, Zahra S, Olivo-Marin JC, Munier-Lehmann H, Antoniewski C - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Drosophila Genetics and Epigenetics, Laboratory of Developmental Biology, CNRS UMR7622, Université Pierre et Marie Curie, Paris, France.

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.

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81 compounds induce strong fluorescence of automiG cells.The primary screen identified 46 compounds that induced a fluorescence fold-change >3.9 and 57 compound for which more that 500 fluorescent spots were counted from well imaging (see materials and methods). The merging of these two sets yield 81 compounds whose effects on automiG cells are presented. Each panel correspond to one image taken by the automated Nikon TE2000 inverted microscope. Note that out of the 81 compounds, 17 were discarded as false positives because they induced by themselves fluorescence (see Supporting Information Table S2, shaded identifiers).
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pone-0074296-g004: 81 compounds induce strong fluorescence of automiG cells.The primary screen identified 46 compounds that induced a fluorescence fold-change >3.9 and 57 compound for which more that 500 fluorescent spots were counted from well imaging (see materials and methods). The merging of these two sets yield 81 compounds whose effects on automiG cells are presented. Each panel correspond to one image taken by the automated Nikon TE2000 inverted microscope. Note that out of the 81 compounds, 17 were discarded as false positives because they induced by themselves fluorescence (see Supporting Information Table S2, shaded identifiers).

Mentions: S2R+ cells stably transfected with the automiG sensor were bathed in 96-well plate containing copper sulfate and 15,104 commercially available compounds. Two days later, induction of automiG expression was calculated as the ratio of fluorescence measured in individual wells to the mean fluorescence of negative controls included in each plate. Using an automated microscope we also acquired images of the wells. AutomiG induction level was calculated from these images using an in-house software that determines the number of fluorescent spots in one microscope field at 40×magnification (spot count). Fluorescence fold changes and spot counts were combined to identify compounds that strongly suppress automiG silencing. It is noteworthy that the positive GFP readout provided by the automiG sensor eliminated compounds that impair cell viability. We selected a set of 46 compounds with a fluorescence fold-change above 3.9 and a set of 57 compounds for which more than 500 fluorescent spots were counted from well imaging (Fig. 3). Both sets overlapped and merging them yielded a list of 81 hit compounds. Microscope images corresponding to these compounds were then examined one by one (Fig. 4). Out of 81, 17 compounds were discarded as false positives because they induced by themselves diffuse fluorescence of the culture medium and/or the formation of large fluorescent crystals (Table S2, shaded identifiers).


AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.

Carré C, Jacquier C, Bougé AL, de Chaumont F, Besnard-Guerin C, Thomassin H, Pidoux J, Da Silva B, Chalatsi E, Zahra S, Olivo-Marin JC, Munier-Lehmann H, Antoniewski C - PLoS ONE (2013)

81 compounds induce strong fluorescence of automiG cells.The primary screen identified 46 compounds that induced a fluorescence fold-change >3.9 and 57 compound for which more that 500 fluorescent spots were counted from well imaging (see materials and methods). The merging of these two sets yield 81 compounds whose effects on automiG cells are presented. Each panel correspond to one image taken by the automated Nikon TE2000 inverted microscope. Note that out of the 81 compounds, 17 were discarded as false positives because they induced by themselves fluorescence (see Supporting Information Table S2, shaded identifiers).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760873&req=5

pone-0074296-g004: 81 compounds induce strong fluorescence of automiG cells.The primary screen identified 46 compounds that induced a fluorescence fold-change >3.9 and 57 compound for which more that 500 fluorescent spots were counted from well imaging (see materials and methods). The merging of these two sets yield 81 compounds whose effects on automiG cells are presented. Each panel correspond to one image taken by the automated Nikon TE2000 inverted microscope. Note that out of the 81 compounds, 17 were discarded as false positives because they induced by themselves fluorescence (see Supporting Information Table S2, shaded identifiers).
Mentions: S2R+ cells stably transfected with the automiG sensor were bathed in 96-well plate containing copper sulfate and 15,104 commercially available compounds. Two days later, induction of automiG expression was calculated as the ratio of fluorescence measured in individual wells to the mean fluorescence of negative controls included in each plate. Using an automated microscope we also acquired images of the wells. AutomiG induction level was calculated from these images using an in-house software that determines the number of fluorescent spots in one microscope field at 40×magnification (spot count). Fluorescence fold changes and spot counts were combined to identify compounds that strongly suppress automiG silencing. It is noteworthy that the positive GFP readout provided by the automiG sensor eliminated compounds that impair cell viability. We selected a set of 46 compounds with a fluorescence fold-change above 3.9 and a set of 57 compounds for which more than 500 fluorescent spots were counted from well imaging (Fig. 3). Both sets overlapped and merging them yielded a list of 81 hit compounds. Microscope images corresponding to these compounds were then examined one by one (Fig. 4). Out of 81, 17 compounds were discarded as false positives because they induced by themselves diffuse fluorescence of the culture medium and/or the formation of large fluorescent crystals (Table S2, shaded identifiers).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Drosophila Genetics and Epigenetics, Laboratory of Developmental Biology, CNRS UMR7622, Université Pierre et Marie Curie, Paris, France.

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.

Show MeSH
Related in: MedlinePlus