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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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Related in: MedlinePlus

Suppression of the automiG silencing by hit compounds.Stably transfected automiG cells were grown for 24 h in the presence of CuSO4 and the indicated compounds. Fluorescence fold changes (A) were measured in triplicate experiments and GFP expression (B) was analyzed by western blot. Thin bars indicate the standard deviation. Red compound identifiers refer to compounds tested in HeLa cells (Fig. 8).
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pone-0074296-g006: Suppression of the automiG silencing by hit compounds.Stably transfected automiG cells were grown for 24 h in the presence of CuSO4 and the indicated compounds. Fluorescence fold changes (A) were measured in triplicate experiments and GFP expression (B) was analyzed by western blot. Thin bars indicate the standard deviation. Red compound identifiers refer to compounds tested in HeLa cells (Fig. 8).

Mentions: The 64 remaining molecules were clustered according to their structure in 22 chemical families (Table S2 and Fig. 5). One or 2 compounds of each family were re-tested in 96-well plates using a fluorescence plate reader in three independent trials (Fig. 6A). In addition, the effect of these molecules on GFP expression in automiG cells was directly assessed by western-blot (Fig. 6B). In both these assays, the effect of 2 compounds (D011-4150 and E711-0046) could not be reproduced. A third compound (PRE318) was associated to a significant fluorescence fold-change of automiG cells, but did not induce GFP expression in the western blot assay. Indeed, this compound becomes fluorescent when it was taken up by the cells (data not shown). At final, our pilot screen validated 29 molecules that strongly suppress the automiG silencing.


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)

Suppression of the automiG silencing by hit compounds.Stably transfected automiG cells were grown for 24 h in the presence of CuSO4 and the indicated compounds. Fluorescence fold changes (A) were measured in triplicate experiments and GFP expression (B) was analyzed by western blot. Thin bars indicate the standard deviation. Red compound identifiers refer to compounds tested in HeLa cells (Fig. 8).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0074296-g006: Suppression of the automiG silencing by hit compounds.Stably transfected automiG cells were grown for 24 h in the presence of CuSO4 and the indicated compounds. Fluorescence fold changes (A) were measured in triplicate experiments and GFP expression (B) was analyzed by western blot. Thin bars indicate the standard deviation. Red compound identifiers refer to compounds tested in HeLa cells (Fig. 8).
Mentions: The 64 remaining molecules were clustered according to their structure in 22 chemical families (Table S2 and Fig. 5). One or 2 compounds of each family were re-tested in 96-well plates using a fluorescence plate reader in three independent trials (Fig. 6A). In addition, the effect of these molecules on GFP expression in automiG cells was directly assessed by western-blot (Fig. 6B). In both these assays, the effect of 2 compounds (D011-4150 and E711-0046) could not be reproduced. A third compound (PRE318) was associated to a significant fluorescence fold-change of automiG cells, but did not induce GFP expression in the western blot assay. Indeed, this compound becomes fluorescent when it was taken up by the cells (data not shown). At final, our pilot screen validated 29 molecules that strongly suppress the automiG silencing.

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