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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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Suppression of RNA interference by hit compounds.S2R+ stably transfected with the automiG-Δ1–Δ2 construct were incubated for 24 h with the indicated compounds and GFP dsRNA. GFP expression was then induced by CuSO4 and cells were grown for further 24 h. Fluorescence relative to DMSO controls (A) was measured just before (white and dark grey bars) and 24 h after copper induction (black bars). GFP expression levels (B) were analyzed by western blot 24 h after copper induction. Asterisks point to compounds that inhibit RNAi. Note that compounds C614–5795 to C788–0877 triggered GFP expression before copper induction; although they were accordingly associated to high level of GFP, they were not marked as RNAi inhibitors. Red compound identifiers refer to compounds tested in HeLa cells (Fig. 8).
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pone-0074296-g007: Suppression of RNA interference by hit compounds.S2R+ stably transfected with the automiG-Δ1–Δ2 construct were incubated for 24 h with the indicated compounds and GFP dsRNA. GFP expression was then induced by CuSO4 and cells were grown for further 24 h. Fluorescence relative to DMSO controls (A) was measured just before (white and dark grey bars) and 24 h after copper induction (black bars). GFP expression levels (B) were analyzed by western blot 24 h after copper induction. Asterisks point to compounds that inhibit RNAi. Note that compounds C614–5795 to C788–0877 triggered GFP expression before copper induction; although they were accordingly associated to high level of GFP, they were not marked as RNAi inhibitors. Red compound identifiers refer to compounds tested in HeLa cells (Fig. 8).

Mentions: As Ago2 is the effector of the RNAi pathway, we also tested whether the compounds identified using the automiG sensor inhibit RNA interference triggered by long dsRNAs. To this aim, automiG-Δ1–Δ2 S2R+ cells were bathed for 24 h with both chemicals and dsRNA targeting the GFP coding sequence. Although the cells were grown in the absence of CuSO4 during this period of time, 8 of 32 compounds caused a significant increase of fluorescence relative to the DMSO controls (Fig. 7A, grey bars). This was expected from compound PRE318 that emits fluorescence when it is taken up into S2R+ cells (see above). The 7 other compounds may be contaminated with heavy divalent cations that would induce the expression of the GFP protein before copper induction. After additional 24 h in the presence of CuSO4, fluorescence relative to DMSO controls was stable or even decreased in cells treated by a majority of the compounds (Fig. 7A, black bars). In contrast, strong increase in fluorescence as well as a high level of GFP was detected for 5 compounds (Fig. 7, stars), indicating that they interfered with GFP RNAi.


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 RNA interference by hit compounds.S2R+ stably transfected with the automiG-Δ1–Δ2 construct were incubated for 24 h with the indicated compounds and GFP dsRNA. GFP expression was then induced by CuSO4 and cells were grown for further 24 h. Fluorescence relative to DMSO controls (A) was measured just before (white and dark grey bars) and 24 h after copper induction (black bars). GFP expression levels (B) were analyzed by western blot 24 h after copper induction. Asterisks point to compounds that inhibit RNAi. Note that compounds C614–5795 to C788–0877 triggered GFP expression before copper induction; although they were accordingly associated to high level of GFP, they were not marked as RNAi inhibitors. 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-g007: Suppression of RNA interference by hit compounds.S2R+ stably transfected with the automiG-Δ1–Δ2 construct were incubated for 24 h with the indicated compounds and GFP dsRNA. GFP expression was then induced by CuSO4 and cells were grown for further 24 h. Fluorescence relative to DMSO controls (A) was measured just before (white and dark grey bars) and 24 h after copper induction (black bars). GFP expression levels (B) were analyzed by western blot 24 h after copper induction. Asterisks point to compounds that inhibit RNAi. Note that compounds C614–5795 to C788–0877 triggered GFP expression before copper induction; although they were accordingly associated to high level of GFP, they were not marked as RNAi inhibitors. Red compound identifiers refer to compounds tested in HeLa cells (Fig. 8).
Mentions: As Ago2 is the effector of the RNAi pathway, we also tested whether the compounds identified using the automiG sensor inhibit RNA interference triggered by long dsRNAs. To this aim, automiG-Δ1–Δ2 S2R+ cells were bathed for 24 h with both chemicals and dsRNA targeting the GFP coding sequence. Although the cells were grown in the absence of CuSO4 during this period of time, 8 of 32 compounds caused a significant increase of fluorescence relative to the DMSO controls (Fig. 7A, grey bars). This was expected from compound PRE318 that emits fluorescence when it is taken up into S2R+ cells (see above). The 7 other compounds may be contaminated with heavy divalent cations that would induce the expression of the GFP protein before copper induction. After additional 24 h in the presence of CuSO4, fluorescence relative to DMSO controls was stable or even decreased in cells treated by a majority of the compounds (Fig. 7A, black bars). In contrast, strong increase in fluorescence as well as a high level of GFP was detected for 5 compounds (Fig. 7, stars), indicating that they interfered with GFP RNAi.

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